WO2011069527A1 - Treatment scheme for idd and other automimmune diseases - Google Patents

Treatment scheme for idd and other automimmune diseases Download PDF

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WO2011069527A1
WO2011069527A1 PCT/EP2009/008801 EP2009008801W WO2011069527A1 WO 2011069527 A1 WO2011069527 A1 WO 2011069527A1 EP 2009008801 W EP2009008801 W EP 2009008801W WO 2011069527 A1 WO2011069527 A1 WO 2011069527A1
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bcg
cells
treatment
combination
active agents
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French (fr)
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Ulrich Freiherr Von Arnim
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Ulrich Freiherr Von Arnim
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention is directed to a combination of active agents comprising an immunopotentiator and heparin.
  • the present invention is further directed to the use of a combination of active agents for the treatment of diabetes-type I.
  • Diabetes-type I is caused by the destruction of the cells in the pancreas which produce insulin.
  • This destruction of cells in the pancreas has the character of an auto-immune disease as the body's-immune system, which is supposed to prevent attacks by infections, instead attacks the body's own cells.
  • type-l diabetes mellitus has been recognized as a major financial problem for public health, affecting both developed and developing countries.
  • Impaired fasting plasma glucose has been previously associated with endothelial dysfunction, higher levels of inflammatory markers and increased risk of complications and cardiovascular events.
  • a lengthy investigation of the immunology of type-l Diabetes has revealed that all diabetics carry either DR3 and DR4 or both.
  • all express hibernating viruses in their islet cells which feed the autoaggressive T-cell response against islet cells and causes insulin dependent diabetes.
  • the BCG vaccine contains a life but weakened form of Mycobacterium bovis, which is the bacterium that causes tuberculosis.
  • the vaccine has been named BCG because a strain of bacterium known as Bacillus Callmette-Guerine is used.
  • mice have used BCG to kill the immuno-cells which are responsible for the destruction of the insulin producing cells in the pancreas.
  • these studies are limited to experimental animal models.
  • the present inventors have surprisingly found that a combination of active agents comprising an immunopotentiator/immunomodulator selected from the group consisting of BCG , Freud's adjuvant, peptides, leukotrienes, interleukins; and, and heparin is very effective against diabetes type I.
  • Freud's adjuvant is used as an immunopotentiator/immunomodulator, it can be complete or incomplete Freud's adjuvant.
  • a particularly preferred peptide is Cyclosporin A.
  • interleukins are used as immunopotentiator/immunomodulator
  • particulary preferred interleukins are IL4, IL6 and IL9.
  • Heparin is a highly-sulfated glycosaminoglycan which is frequently used as anticoagulant. Heparin has the highest negative charge intensity of any known biological molecule. Heparin is a natural anticoagulant produced by basophiles and mast cells.
  • the present inventors have surprisingly found, that by the combination of an immunopotentiator/immunomodulator selected from the group consisting of BCG , Freud's adjuvant, peptides, leukotrienes, interleukins; with heparin, very potent treatment against diabetes type I becomes possible.
  • the immunopotentiator/immunomodulator is BCG.
  • the heparin has a weight average molecular weight of equal or less than 10.000 Da.
  • the present inventors have found that it is advantageous to use heparin having a weight average molecular weight of equal or less than 10.000 Da in the combination of active agents according to the present invention because heparin of higher molecular weight is less effective.
  • the heparin has a weight average molecular weight of 2000 to 10.000 Da.
  • the present inventors have found out, that particularly good results can be achieved when the combination of active agents additionally comprises a guanine analogue.
  • Guanine analogues are the most commonly used antiviral agent for the treatment of diseases like herpes zoster and genital herpes.
  • the present inventors have surprisingly found that the inclusion of these guanine analogues in the combination of active agents increases the anti diabetes type I effect.
  • the guanine analogue included in the combination of active agents is selected from the group consisting of famciclovir, valaciclovir, adcyclovir and penciclovir.
  • the guanine analogue is famciclovir.
  • a particularly effective combination of active agents additionally comprises a protease inhibitor.
  • the protease inhibitor is a serine protease inhibitor.
  • a particularly preferred protease inhibitor is alpha-1-antitrypsin.
  • Alpha- 1-antitrypsin is a glycoprotein generally known as serum trypsin inhibitor. It is a serine protease inhibitor inhibiting a wide variety of proteases. It protects tissues from enzymes of inflammatory cells, especially elastases.
  • active agents comprising BCG as immunopotentiator, low molecular weight heparin (heparin having a weight average molecular weight of equal or less than 10.000 Da)., famciclovir as a guanine analogue and alpha-1-antitrypsin as a protease inhibitor.
  • the combination of active agents comprises BCG in amount of 1 to 10 x 10 8 , in particular 4 to 6 x 10 8 bacteria.
  • the combination of active agents comprises heparin, in particular heparin having a weight average molecular weight of equal or less 10000 Da, in an amount of 0.1 to 10, in particular 3 to 6 IU (international units) per kilogram bodyweight.
  • Heparin typically has an activity of approximately 100 IU/mg. Accordingly, the combination of active agents preferably comprises heparin, in particular heparin having a weight average molecular weight of equal or less 10000 Da, in an amount of 0.001 to 0.1 mg, in particular 0.03 to 0.06 mg.
  • the combination of active agents comprises heparin, in particular heparin having a mass average molecular weight of equal or less then 10000 Da, in an amount of less than 300 IU. This amount can be and preferably is independent from the body weight.
  • the combination of active agents comprises famciclovir in an amount of 10 to 1000, in particular 50 to 500 milligram.
  • the combination of active agents comprises alpha- 1-antitrypsin in an amount of 10 to 1000, in particular 100 to 250 mg per kilogram body weight.
  • the present invention is in particular directed to the combination of active agents described above for the treatment of diabetes type I. Further, the present invention is in particular directed to the use of the combination of active agents described above for the preparation of a medicament for the treatment of diabetes type I.
  • a patient initially receives a physical examination including laboratory testing such as HbAlc, blood sedimentation rate (ESR), blood sugar levels, creatinine, liver parameters and blood pressure.
  • laboratory testing such as HbAlc, blood sedimentation rate (ESR), blood sugar levels, creatinine, liver parameters and blood pressure.
  • ESR blood sedimentation rate
  • the patient receives laboratory testing for Islet cell antibodies, insulin antibodies, tyrosine phosphatase, glutameldecarboxylase, HLA- DQ2 ⁇ DQ3 and a leucocyte transformation test [LTT](immunology).
  • Islet cell Ag 512 a recombinant human Ag that was isolated from an islet cDNA expression library by screening with human insulin- dependent diabetes mellitus sera. Specificity of reaction with diabetic leukocytes was demonstrated initially by LTT with a small number of diabetic and normal leukocyte samples. To permit quantitative and rapid serum testing, ICA512 was purified and adapted to an LTT format. In this way, a sensitivity of 98% with diabetic Leukocytes has been measured with a panel of 15,000 Leukocytes.
  • DNA sequencing of ICA512-3 a cDNA that contains a 1644 bp open reading frame, suggests that it codes for a transmembrane protein having a single membrane- spanning segment and a cytoplasmic domain that is closely related to the first intracellular (catalytic) domain of the T cell protein tyrosine phosphatase, CD45.
  • Northern blot analysis of poly(A)+ RNAs from several human tissues indicates that ICA512 mRNA is expressed in brain and pancreas.
  • the patient receives the first dosage ofBacillus Calmette Guerin Connaught (BCG).
  • BCG Bacillus Calmette Guerin Connaught
  • the first dosage for an adult or child over 12 years of age is 500 million units of BCG.
  • the BCG is injected intracutaneously.
  • the BCG for intracutaneous injection is prepared as follows:
  • the BCG is dissolved in 0.9 ml of physiological solution for injection and mixed with 5 IU of Fragmin D per Kg of body weight to a maximum of 300 IU.
  • the so-prepared mixture is injected intracutaneously into the patient.
  • the area of injection can be anywhere on the body. However, the best location for injection is the deltoid area of the upper arm.
  • the intracutaneous BCG injections are repeated every three weeks.
  • the results of the LTT are essential. The LTT has to be performed prior to each subsequent injection. The reason for the wide dosage range described in the claims (100 to 800 million units of BCG) is determined by the individual patient's response to the therapy measured by the LTT.
  • the treatment protocol for children under the age of 12 is as follows:
  • Laboratory testing such as HbAlc, blood sedimentation rate (ESR), blood sugar levels, creatinine, liver parameters and blood pressure.
  • ESR blood sedimentation rate
  • the patient receives laboratory testing for Islet cell antibodies, insulin antibodies, tyrosine phosphatase, glutameldecarboxylase, HLA-DQ2 ⁇ DQ3 and a leucocyte transformation test [LTT] (immunology).
  • Islet cell Ag 512 a recombinant human Ag that was isolated from an islet cDNA expression library by screening with human insulin- dependent diabetes mellitus sera. Specificity of reaction with diabetic leukocytes was demonstrated initially by LTT with a small number of diabetic and normal leukocyte samples. To permit quantitative and rapid serum testing, ICA512 was purified and adapted to an LTT format. In this way, a sensitivity of 98% with diabetic Leukocytes has been measured with a panel of 15,000 Leukocytes.
  • DNA sequencing of ICA512-3 a cDNA that contains a 1644 bp open reading frame, suggests that it codes for a transmembrane protein having a single membrane- spanning segment and a cytoplasmic domain that is closely related to the first intracellular (catalytic) domain of the T cell protein tyrosine phosphatase, CD45.
  • Northern blot analysis of poly(A)+ RNAs from several human tissues indicates that ICA512 mRNA is expressed in brain and pancreas.
  • the initial BCG dosage for the age group 2-5 years is 100 million units injected intracutaneously mixed as follows: The BCG is dissolved in 0.9 ml of physiological solution for injection and mixed with 5 IU of Fragmin D per Kg of body weight.
  • the results of the LTT are essential.
  • the LTT has to be performed prior to each subsequent injection.
  • the reason for the wide dosage range described in the claims (100 to 800 million units of BCG) is determined by the individual patient's response to the therapy measured by the LTT.
  • the initial dosage of BCG is 250 million units.
  • the initial BCG dosage for the age group 2-5 years is 100 million units injected intracutaneously mixed as follows: The BCG is dissolved in 0.9 ml of physiological solution for injection and mixed with 5 IU of Fragmin D per Kg of body weight to a maximum of weight to a maximum of 300 IU of Fragmin D.
  • Fragmin D is one of the low molecular weight heparins because it proved, according to our studies, to be the most effective product. However, there are other heparins available which could also be used and ultimately they will lead to the same treatment success.
  • the LTT results are measured according to their pathophysiological migration patterns. In other words we are measuring how T Cells are migrating and interacting with Islet Cells. Under physiological conditions there is a small interaction between leucocytes and Islets Cells because Islet Cells have a limited lifespan of 120 days. Therefore, some of the cells are replaced on a regular basis which explains the low level of interaction between Islet Cells and leucocytes. Under Diabetic conditions the Islet Cells are being destroyed at an increased rate and via the LTT, we measure at what rate Islet Cells arebeing destroyed and by what type of T Cells or leucocytes. The aforesaid treatment with BCG reduces this destruction slowly if the dosage is administered correctly. At the same time, if the dosage is correct, the level of side effects is reduced.
  • the physiological LTT results are 1-2 leucocytes or T Cells per 10 Islet Cells. Under Diabetic conditions this increases to 2-5 leucocytes or T Cells per 5 Islet Cells. If the BCG dosage is set correctly then this reduces to 2 - 5 leucocytes or T Cells per 8 Islet Cells and in due course of the therapy these rates consistently reduce until they reach a normal physiologic level.
  • the aforesaid treatment with BCG is administered every 3 weeks for 9 to 12 months or until 3 consecutive LTT tests, each 3 weeks apart, have returned normal physiological results.
  • famicyclovir In addition to the aforesaid BCG therapy we have added famicyclovir to the treatment protocol. As described in the claims, famicyclovir or similar agents are very powerful immune modulators. Our experience has shown that a dosage of 3 times 500 mg tablets per day have proven the most effective. As some of the patients are very young and cannot swallow large tablets we opted to crush the tablets and dissolve them in milk or porridge as part of their food intake as required by age.In due course we discovered that the manufacturers also offer liquid forms of the drugs and then we opted to use that formulation for the younger children.
  • alpha anti trypsin which is a unique interleukin and cytokine activator.
  • AAT alpha anti trypsin
  • JKD human serum sources
  • the adequate dosage is measured via a laboratory test determining serum AAT levels.
  • a fourth potential active agent is Imiquimode, a well-established immune modulator and Langheran's Cell activator.
  • Imiquimode a well-established immune modulator and Langheran's Cell activator.
  • the overall treatment time could be reduced to 1-3 months with a treatment success rate of above 9 out of 10 patients.
  • the present invention includes inter the following aspects:
  • the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da. Further the invention is also directed to a medical composition kit suitable for such an administration scheme.
  • the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 10 8 bacteria, in particular 4 to 6 x 10 8 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks.
  • the invention is also directed to a medical composition kit suitable for such an administration scheme.
  • the invention includes an administration scheme for treating, diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 10 8 bacteria, in particular 4 to 6 x 10 8 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks, additionally comprising the step of administering famciclovir.
  • the invention is also directed to a medical composition kit suitable for such an administration scheme.
  • the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 10 8 bacteria, in particular 4 to 6 x 10 8 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks, additionally comprising the step of administering famciclovir, wherein the dosage of administration for famciclovir is 3 times 10 to 1000, in particular 50 to 500 mg, per day.
  • the invention is also directed to a medical composition kit suitable for such an administration scheme.
  • the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 10 8 bacteria, in particular 4 to 6 x 10 8 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks, additionally comprising the step of administering famciclovir, wherein the dosage of administration for famciclovir is 3 times 10 to 1000, in particular 50 to 500 mg, per day.
  • the invention is also directed to a medical composition kit suitable for such an administration scheme.
  • the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 10 8 bacteria, in particular 4 to 6 x 10 8 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks, additionally comprising the step of administering famciclovir, wherein the dosage of administration for famciclovir is 3 times 10 to 1000, in particular 50 to 500 mg, per day, additionally comprising the step of administering alpha-1-antitrypsin.
  • the invention is also directed to a medical composition kit suitable for such an administration scheme.
  • the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 10 8 bacteria, in particular 4 to 6 x 10 8 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks, additionally comprising the step of administering famciclovir, wherein the dosage of administration for famciclovir is 3 times 10 to 1000, in particular 50 to 500 mg, per day, additionally comprising the step of administering alpha-1-antitrypsin, wherein the dosage of administration for alpha-1-antitrypsin is 10 to 1000, in particular 100 to 250 mg, per kg bodyweight once a week.
  • the invention is also directed to a medical composition
  • the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 10 8 bacteria, in particular 4 to 6 x 10 8 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks, additionally comprising the step of administering famciclovir, wherein the dosage of administration for famciclovir is 3 times 10 to 1000, in particular 50 to 500 mg, per day, additionally comprising the step of administering alpha-1-antitrypsin, wherein the dosage of administration for alpha-1-antitrypsin is 10 to 1000, in particular 100 to 250 mg, per kg bodyweight once a week, additionally comprising the step of administering Imiquimode.
  • the invention is also directed to a medical composition kit suitable for such an administration
  • the determining parameters were Blood sugar levels, reduction in insulin dosage, HbAlc, OGTT, food intake and safety of BCG, Famiciclovir and Fragmin-D, in insulindependent subjects.
  • the study was a double blind, crossover clinical trial with a three-weekly single dose of BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught) Famiciclovir (3x500mg/day) and Fragmin-D (Heparin with mass average molecular wheight of less than 10000 Da) (5 IU/kg to a maximum of 300 IU once per day) in 6,000 type-1 diabetic subjects.
  • BCG l-8xl0 8 Bacillus Calmette Guerin Connaught
  • Famiciclovir 3x500mg/day
  • Fragmin-D Heparin with mass average molecular wheight of less than 10000 Da
  • Famiciclovir and Fragmin-D produced a statistically significant treatment effect that eliminates the need for insulin in 8,385 out of 10,000 patients.
  • type-1 diabetes mellitus and increased longevity has been recognized as a major financial problem for public health, affecting both developed and developing countries.
  • Impaired fasting plasma glucose has been previously associated with endothelial dysfunction, higher levels of inflammatory markers and increased risk of complications and cardiovascular events.
  • a lengthy investigation of the immunology of type-1 Diabetes has revealed that all diabetics carry either DR3 and DR4 or both.
  • all express hibernating viruses in their islet cells which feed the auto aggressive T-cell response against islet cells and causes insulin dependent diabetes.
  • the aim of this clinical trial was to study the efficacy of the treatment with BCG, Famiciclovir and Fragmin-D.
  • BCG was the most effective in-vivo Tumor necrosis factor stimulant.
  • Famiciclovir was the most effective in vivo interferon gamma stimulants in a group of insulin dependent diabetic, hypertensive and non-hypertensive subjects.
  • Low molecular weight heparin such as Fragmin-D, was the most effective in-vivo surface lipoprotein modulator permitting hibernating virus, bacteria or other foreign sub- immune response stimulating antigens to be expressed in an immune response stimulating form.
  • a randomized, double blind, cross-over, placebo-controlled, clinical trial was designed to assess the effects of BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught) Famiciclovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) in 6,000 type-1 diabetic subjects during 12 months) on the Homeostasis Model Assessment (HOMA) index, lipid profile, prothrombotic state, oxidative stress and plasma levels of inflammatory markers and Hbalc levels.
  • the participants were recruited from the "German Clinical Trial Subjects Pool". Subjects who fulfill selection criteria received permanent educational, nutritional and exercise support during their participation in the study. After a 15 days-run-in period with tight Diabetes control and life-style recommendations, the patients who have a treatment compliance equal or greater than 80% were randomly assigned to one of the treatment groups.
  • BCG immunization 1 day after Cy treatment almost completely prevented insulitis in the islets of Cy-treated mice.
  • Cy treatment reduced the endogenous production of antiGAD67 antibody
  • BCG vaccination 1 day after Cy treatment restored the production of antiGAD67 antibody of IgGl isotype.
  • the comprehensive effect of BCG vaccination on cytokine production in Cy-treated mice was to increase IL-4 production and change the IL-4/tFN-gamma ratio in both serum and supernatant of spleen cell cultures.
  • BCG-induced protection was associated with increased splenic CD4 + CD45 RB high T cells. Taken together, these results indicate that BCG treatment counteracts the effect of Cy on autoimmune process in IDDM.
  • BCG immunization sequentially induced the production of TNF- ⁇ alpha ⁇ , IFN- ⁇ gamma> and IL-4 by splenocytes, increased the expression of Fashigh (Apo-l/CD95), Fas ligand (FasL, CD95L) and TNF receptor (TNFR) on T cells leading to T cell apoptosis.
  • Fashigh Apo-l/CD95
  • FasL, CD95L Fas ligand
  • TNFR TNF receptor
  • mice In mice, the patterns of cytokine production after infection or immunization with mycobacteria are dependent on many factors, such as the route, the nature of mycobacteria and the mouse strains used (14,15). Both TNF- ⁇ alpha ⁇ and IFN- ⁇ gamma ⁇ are the major Thl cytokines produced early after mycobacteria infection, and this is followed by Th2-type cytokine production (16,77). The production of IL-4 after BCG immunization in syngeneic islet transplanted NOD mice occurs late and is maintained (12). IFN- ⁇ gamma ⁇ has been found to induce apoptosis of activated CD4 T cells in mice infected with mycobacteria (18).
  • TNF- ⁇ alpha ⁇ has been shown to induce apoptosis in mature T cells (21) and in diabetogenic T cells of diabetic NOD mice (22). TNF- ⁇ alpha ⁇ suppresses spontaneous diabetes in NOD mice when given late but not early during the development of disease (23-25). Therefore, Thl-like cytokines might be a primary factor for CFA or BCG-induced down-regulation of destructive autoimmunity by activation-induced cell death (AICD) of diabetogenic T cells .
  • AICD activation-induced cell death
  • Fas-FasL pathway is well recognized as an efficient way to induce the apoptosis of activated Thl and cytotoxic CD8 T cells (26-28).
  • TNF- ⁇ alpha ⁇ induces T cell apoptosis through TNFR, which plays a pivotal role in maintaining immune privilege of the eye through FasL-induced cell death promoted by TNF- ⁇ alpha ⁇ (29).
  • TNFR TNFR
  • splenocytes isolated from both BCG and saline-treated diabetic mice were transferred to NOD.SCID mice.
  • Histological examination showed that splenocytes from BCG-immunized diabetic mice induced much less insulitis in recipient NOD.SCID mice than those from the saline-treated group (data not shown). Therefore splenocytes from BCGimmunized diabetic NOD mice had a reduced ability to transfer disease.
  • Fig. 1 shows the BCG immunization of diabetic NOD mice down-regulates the diabetogenic potential of splenocytes.
  • NOD.SCID mice were injected i.v. with 12 x 10 6 splenocytes from diabetic NOD mice immunized with saline or BCG 6 days (BCG-6d) or fl2 days (BCG-12d) prior to transfer.
  • BCG-6d BCG 6 days
  • BCG-12d fl2 days
  • BCG immunization decreases the proportion of T cells by inducing apoptosis.
  • BCG immunization significantly decreased the proportions of CD4, CD8 and CD45RBIow T cells and increased CDllb positive macrophages in a time course study.
  • the difference between BCG-6d and BCG-12d groups in CDB and CD45RBIow T cells are also significant (Fig. 2A).
  • CD45RBIow CD4 T cells are considered diabetogenic Thl cells in diabetic NOD mice. Therefore, the reduction of both CD4SRBIow CD4 and CD8 T cells in BCG-immunized NOD mice indicates the down-regulation of diabetogenic Thl cells.
  • Fig. 2 shows the decreased proportion of T cells and increased apoptotic T cells in BCG-immunized diabetic NOD mice.
  • splenocytes (106) were directly stained with FITC or PE-conjugated anti-CDllb, CD4 or CD8 mAb alone or anti-CD4, CD8 and CD45RB mAbs in combination. Results are representative of three experiments, and are presented as the mean (%) ⁇ SEM of three mice.
  • splenocytes from saline or BCG-treated (BCG-6d and BCG-12d) groups were cultured in medium for 24 h and then stained for TUNEL positive CD4 and CD8 T cells.
  • the numbers shown in each representative dot plot graph are the mean (%) ⁇ SEM for TUNEL positive CD4 or CD8 T cells, representative of two separate experiments. *P ⁇ 0.05-0.001 compared with saline group; ⁇ dagger ⁇ P ⁇ 0.05-0.02 compared with BCG-12d group.
  • TNF- ⁇ alpha ⁇ , IFN- ⁇ gamma ⁇ and IL-4 Intracellular and secreted TNF- ⁇ alpha ⁇ , IFN- ⁇ gamma ⁇ and IL-4 in BCG- Immunized diabetic NOD mice were analyzed by intracellular cytokine staining and ELISA, respectively.
  • intracellular cytokine expression revealed that in comparison with the saline group, the total number of TNF- ⁇ alpha ⁇ or IFN- ⁇ gamma ⁇ positive splenocytes (macrophages plus CD4 T cells) was significantly increased in the BCG-12d group. But the highest number of TNF- ⁇ alpha ⁇ positive cells was found in the BCC-6d group.
  • CD4 T cells of BCC-6d group and macrophages of BCG-12d group had a significant increase of TNF- ⁇ alpha ⁇ expression.
  • a significantly high expression of IFN- ⁇ gamma ⁇ was observed only in the BCG-12d group.
  • the major source of IFN- ⁇ gamma ⁇ in the BCG-12d group was macrophages, and it correlated with the highest proportion of macrophages and lowest proportion of T cells in this group.
  • IL-4 expression was increased to a higher level in the BCG-12d group.
  • the patterns of cytokines secreted into supernatant were similar to the patterns of cytokine positive splenocytes, except for TNF- ⁇ alpha ⁇ in the saline group (Fig. 3B).
  • Splenocytes from the saline group produced as much TNF- ⁇ alpha ⁇ as the BCG-6d group, when cultured with BCG but not medium alone (data not shown) for 3 days.
  • the peak of TNF- ⁇ alpha ⁇ production is earlier than that of IFN- ⁇ gamma ⁇ , and T cells are the major source.
  • the level of IL-4 remained high in diabetic mice 15 days after BCG-immunization (data not shown).
  • Previous studies showed that the production of IL-4 is persistently maintained at a higher level in diabetic NOD mice that have been immunized with BCG and grafted with syngeneic islet cells than in control mice (12). Therefore, BCG immunization induces an early pro-inflammatory Thl response and a late Th2 response.
  • Fig. 3 shows the changes of cytokine pattern in diabetic NOD mice immunized with BCG.
  • diabetic NOD mice were injected (i.p.) with neutralizing mAb to IFN- ⁇ gamma ⁇ or IL-4 during BCG priming. Isotype-matched rat IgG and saline were used as controls.
  • the incidence of diabetes in NOD.SCID mice transferred with splenocytes from anti-IL-4 mAb (Fig. 4A) or anti-IFN- ⁇ gamma ⁇ mAbtreated mice (Fig. 4B) was examined. There was no significant difference in the incidence of diabetes between anti-IL-4 mAb-treated and control groups.
  • anti-IFN- ⁇ gamma ⁇ mAb treatment partially abolished the effect of BCG on the impairment of diabetogenic T cells.
  • Fig. 4 shows the systemic administration of neutralizing mAb to IFN- ⁇ gamma ⁇ but not IL-4 during BCG immunization restores the diabetogenic potential of splenocytes.
  • Diabetic NOD mice were injected with saline or BCG on day 0. Starting on day 2, BCG- immunized diabetic mice were injected i.p. with anti-IL4 mAb (3 mg; 11B11) (A) or anti-IFN- ⁇ gamma ⁇ mAb (1,6 mg; R46A2) (B) every 2 days for a total of five injections. Rat IgC and saline were used as controls.
  • mice were injected i.p. with saline or BCG (300 pg). Two days later, mice were injected i.p. with 1.6 mg of rat IgG or anti-IFN- ⁇ gamma ⁇ mAb every other day for 10 days.
  • b Splenocytes were double stained for CD45RBIow or TUNEL positive CD4 T cells. Results are expressed as percent positive cells in splenocytes and CD4 T cells, respectively, and are from two separate experiments.
  • c Splenocytes were incubated with BCG (100 pg/ml) for 4 days. Response was measured 16 h after addition of [3H]TdR and expressed as mean ⁇ Delta ⁇ cpm ⁇ SD of triplicate cultures.
  • *P 0.0090 and NS
  • ⁇ dagger ⁇ P 0.0036 and 0.0152 for vs saline and IFN- ⁇ gamma ⁇ mAb groups, respectively
  • ⁇ ddagger ⁇ NS for vs IFN- ⁇ gamma ⁇ mAb group.
  • Fig. 5 shows the administration of IFN- ⁇ gamma ⁇ and/or TNF- ⁇ alpha ⁇ down-regulates diabetogenic T cells through induction of apoptosis.
  • Splenocytes were prepared 1 day after the last injection and stained for CD4 and CD8 T cells (A).
  • splenocytes were incubated for 24 h in medium and them double stained for TUNEL positive CD4 or CD8 T cells (B). Results are expressed as the mean (%) ⁇ SEM for positive cells in splenocytes, CD4 or CD8 T cells. *P ⁇ 0.05-0.001 compared with saline group.
  • IFN- ⁇ gamma ⁇ and/or TNF- ⁇ alpha ⁇ incubation enhances apoptosis and Fas/FasL expression of diabetogenic T cells.
  • Thl cytokine changes in apoptosis and Fas/FasL expression were evaluated on T cells cultured in the presence of Thl cytokines. Splenocytes from diabetic NOD mice were incubated in vitro with IFN- ⁇ gamma ⁇ and/or TNF- ⁇ alpha ⁇ for 2 days, then double stained for TUNEL positive CD4 or CD8 T cells.
  • Figure 7(A) shows concentration-dependent induction of T cell apoptosis by IFN- ⁇ gamma ⁇ and/or TNF- ⁇ alpha ⁇ .
  • a significant increase in apoptosis of CD4 T cells was found using the higher concentration of IFN- ⁇ gamma ⁇ (100, 500 ng/ml) or TNF- ⁇ alpha ⁇ (5,25 ng/ml) alone or in combination.
  • Increased apoptosis in CD8 T cells was only found significant at the highest concentration of IFN ⁇ gamma ⁇ (500 ng/ml) and TNF- ⁇ alpha ⁇ (25 ng/ml) in combination.
  • Fas/FasL expression Parallel increases in Fas/FasL expression were observed on both CD4 and CD8 T cells incubated with the highest concentration of IFN- ⁇ gamma ⁇ , TNF- ⁇ alpha ⁇ alone or in combination (Fig.7B).
  • a significant increase in Fas/FasL expression on CD4 T cells was found in all three cytokine-treated groups.
  • CD8 T cells a significant increase in Fas/FasL expression was only found in the presence of TNF- ⁇ alpha ⁇ alone or in combination with IFN- ⁇ gamma ⁇ .
  • T cell apoptosis and Fas/FasL expression found in splenocyte culture in the presence of IFN- ⁇ gamma ⁇ and/or TNF- ⁇ alpha ⁇ further suggests the primary role of Thl cytokines in BCG-induced immune regulation in diabetic NOD mice.
  • Fig. 6 shows the IFN- ⁇ gamma ⁇ and/or TNF- ⁇ alpha ⁇ incubation induces T cell apoptosis and Fas/FasL expression.
  • Splenocytes from diabetic NOD mice were incubated with medium alone, 10/0.5, 100/5 and 500/25 ng/ml of IFN- ⁇ gamma ⁇ / TNF- ⁇ alpha ⁇ alone or in combination for 48 h.
  • Apoptotic T cells were determined by double staining with TUNEL and CD4 or CD8 mAb (A).
  • Fas/FasL expression splenocytes were collected from cultures in the presence of 500/25 ng/ml of IFN- ⁇ gamma ⁇ and/or TNF- ⁇ alpha ⁇ (B). Results are expressed as the mean (%) ⁇ SEM for TUNEL, Fas or FasL positive CD4 and CD8 T cells, and are from three to five separate experiments. *P ⁇ 0.05-0.01 compared with medium control.
  • Fas and TNF pathways are involved in BCG-induced apoptosis of diabetogenic T cells.
  • BCG immunization up-regulates the expression of Fas/FasL and TNFR, which may lead to the apoptosis of diabetogenic T cells.
  • anti-FasL or anti- TNFR mAb was added to splenocyte cultures from BCG-immunized diabetic NOD mice to block the corresponding ligand and receptor binding. Both TUNEL staining and T cell proliferation assays were carried out by incubating splenocytes with PPD for 1 and 4 days, respectively.
  • Fig. 7 shows the in vitro blocking FasL or TNFR1 with neutralizing mAb reduces BCG- induced T cell apoptosis and increases T cell proliferative response.
  • Splenocytes from BCG-immunized diabetic NOD mice (BCG-12d) were incubated with PPD in the presence of anti-TNFRl, anti-TNFR-2, anti-FasL mAb or isotype controls (15 pg/ml) for 24 h in TUNEL assays (A) or for 4 days in T cell proliferation assays (B).
  • Results are expressed as the mean (%) ⁇ SEM for TUNEL positive T cells (CD4 plus CD8 T cells) from three experiments, or the mean cpm ⁇ SD of triplicate cultures from a representative of three experiments. *P ⁇ 0.02-0.01 compared with corresponding isotype controls.
  • CFA or BCG mycobacterial preparation
  • CFA or BCG mycobacterial preparation
  • It may involve the induction of regulatory cells, cytokine switch and T cell apoptosis (12,22,31).
  • Diabetic NOD mice have a dominant population of diabetogenic T effector cells that can adoptively transfer disease in non-diabetic recipients [for review, see (32)].
  • down-regulation of diabetogenic T cells by BCG may also involve induction of T cell anergy, peripheral deletion and/or induction of regulatory cells.
  • CD45RBIow CD4 T cells are IFN- ⁇ gamma ⁇ -secreting Thl cells in longlived immunity to mycobacterium (35).
  • soluble FasL has been found to induce apoptosis in CD4+CD45RBIow 'memory' cells (36).
  • BCG has been shown to induce a strong Thl response with secretion of TNF- ⁇ alpha ⁇ and IFN- ⁇ gamma ⁇ both in mouse and human (39,40).
  • a shift from a Thl to Th2 cytokine occurs in the later stages following in vitro incubation of peripheral blood cells from healthy persons with BCG, and in CD4 T cells following mycobacterial infection (16,17), which supports these findings.
  • IL-4 production occurs later and is maintained even for a few months after BCG-induced protection of syngeneic islet grafts when IFN- ⁇ gamma ⁇ production is no longer significant (12).
  • Thl cytokines play a major role in triggering BCG-induced down-regulation of diabetogenic T cells, and up-regulation of Th2 cytokines may be secondary to this effect. Similar findings have been reported in glial fibrillary acidic protein immunotherapy in NOD mice, in which the protection is relied upon up-regulation of IFN- ⁇ gamma ⁇ production (41). It is speculated that BCG down-regulates both induction and effector phases of diabetogenic T cells by induction of Thl apoptosis and activation of Th2 cells through IL-10 production.
  • TNF- ⁇ alpha ⁇ a pro-inflammatory factor
  • T cells are also the producer of TNF- ⁇ alpha ⁇ (42,43).
  • macrophages become a major source of IFN- ⁇ gamma ⁇ in BCG-treated diabetic NOD mice, which might result from the apoptosis of IFN- ⁇ gamma ⁇ -producing T cells and the increased number of activated macrophages.
  • IFN- ⁇ gamma ⁇ has been investigated in IFN- ⁇ gamma ⁇ KO mice (20), in mycobacterium and other microbe infection models (18,45). IFN- ⁇ gamma ⁇ has also been shown to inhibit the development of diabetes in NOD mice (19). Whether CFA or BCG directly affects islets through cytokines need to be clarified. CFA reverses established diabetes by eliminating TNF ⁇ alpha ⁇ sensitive diabetogenic T cells and promoting the regeneration of endogenous islet ⁇ cells (22). EExpression of TNF- ⁇ alpha ⁇ in the islets also suppresses spontaneous diabetes by preventing the development of islet specific T cells (46).
  • TNF- ⁇ alpha ⁇ prevents the recurrence of diabetes in NOD mice by reducing CD4 and CD8 T cells and Thl cytokine production in local islet grafts and in splenocytes (47).
  • the synergistic effects of IFN- ⁇ gamma ⁇ and TNF- ⁇ alpha ⁇ have been shown to reduce insulitis (24). They reported that IFN- ⁇ gamma ⁇ and TNF- ⁇ alpha ⁇ may synergistically contribute to the apoptosis of diabetogenic T cells in BCG-immunized diabetic NOD mice.
  • Fas-FasL and TNFR 1-TNF pathways in BCG- induced T cell apoptosis has been demonstrated by increased expression of Fashigh, FasL and TNFR on T cells and FasL/TNFR blocking assay.
  • T cells that express high level of Fas are particularly sensitive to apoptosis.
  • the predominant expression of FasL has been shown to mediate apoptosis in Thl and CD8 T cells (27,28). Infection with live Mycobacterium avium induces protection against type 1 diabetes in NOD mice, which is associated with increased expression of Fas and FasL (48).
  • BCG immunotherapy in diabetic NOD mice is mediated by the early up- regulation of TNF ⁇ alpha ⁇ and IFN- ⁇ gamma ⁇ production.
  • TNF- ⁇ alpha ⁇ and IFN- ⁇ gamma ⁇ triggers the apoptosis of diabetogenic T cells through both Fas-FasL and TNF-TNF 1. pathways.
  • This study provides a rational explanation for the protection against diabetes recurrence through BCG immunization of islettransplanted diabetic NOD mice. These results have direct implications in preventing the recurrence of diabetes by transplanted syngeneic islets or ⁇ cells generated through stem cell technology.
  • BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught) Famicidovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) permanently eliminates the need for insulin in insulin-dependent-diabetic subjects normalizing fasting plasma glucose, HbAlc, proinflammmatory, prothrombotic and oxidative stress markers in diabetic, nonhypertense or hypertense subjects. The improvement of these markers is independent of changes in arterial blood pressure.
  • BCG l-8xl0 8 Bacillus Calmette Guerin Connaught
  • Famiciclovir 3x500mg/day
  • Fragmin-D 5 IU/kg to a maximum of 300 IU once per day
  • Famiciclovir 3x500mg/day
  • Fragmin-D 5 IU/kg to a maximum of 300 IU once per day
  • Fig. 8 shows the randomized, double blind, placebo-controlled, cross-over clinical trial.
  • Fig. 8 is a study design - the protocol included Fragmin D (5 IU/kg to a maximum of 300 IU once per day) as an addition.
  • Treatment A BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) for 12 months and then switch to placebo treatment for another 12 months
  • Treatment B Placebo tablets administered identical to treatment A,.
  • Group 1 received the treatment A during the first 52 weeks and then the treatment B during the last 52 weeks.
  • Group 2 received the treatment B during the first 52 weeks and then the treatment A during the last 52 weeks.
  • - Patient requiring treatment with immunosuppressive agents for any circumstance.
  • - Patient who requires a major surgical procedure during the next 12 months, after enrollment (Abdominal or thoracic surgery, vascular, neurosurgery, urologic or gynecologic surgical procedure).
  • a sample size of 6,000 subjects was estimated, considering a crossover clinical trial design as proposed by Hills and Armitage [70], accepting a type 1 error of 0.05, a power of 90%, and assuming a difference of 20 % in the HOMA index (3 to 2.4) after 12 months of treatment with BCG (1.8xl0 8 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) and a maximum standard deviation of 1.5.
  • the final size of the sample, adjusted for a drop-out of 8%, is 6,600 subjects (3,300 in each group).
  • the sample size ensures a power of 95% to detect differences in fasting glycemia of at least 8 mg/dL (0.44 mMol/L) with a standard deviation (SD) of 20 mg/dL (1.1 mMol/L), or a difference of 14 mg/dL (0.77 mMol/L) in the 2 hours post load glycemia with a SD of 40 mg/dl (2.2 mMol/L).
  • SD standard deviation
  • Visit B included measurements of blood pressure, anthropometric parameters, and electrocardiogram.
  • a fasting blood sample and a 24 hour urine sample were taken and stored (-70°C) to determine glucose, HbAlc, insulin, IL-6, leptin, resistin, adiponectin, tisular plasminogen activator (tPA), PAI-1, oxidized/reduced glutathione, malonaldehyde and 8-isoprostanes in urine.
  • tPA tisular plasminogen activator
  • Weight were measured with the patient standing and then registered after rounding it to the nearest 200 grams. The weight scale were calibrated to 0 before each measurement.
  • Heart rate number of beats per minute were measured in the radial artery.
  • Blood pressure were taken twice (with a difference of 5 minutes between the measurements) using a mercury sphygmomanometer in 2 occasions on the right arm, with the patient comfortably seated, after a 5 minute rest.
  • Systolic blood pressure (SBP) were determined by the first audible sound (Korotkoff phase 1).
  • Diastolic blood pressure (DBP) were registered when the sound disappears (Korotkoff phase 5).
  • the patient should not have smoked 30 minutes prior to the blood pressure measurement.
  • the pneumatic arm cuff must cover 2/3 of the upperarm's length; its inferior border must be 2-3 cm over the antecubital space; the cuff was slowly deflated.
  • MBP mean blood pressure
  • Hip circumference were measured on 2 occasions with the patient in a standing position with the arms on the sides of the body, using a measuring tape adhered to a dynamometer that exerts a force of 750 gr. The hip circumference was assessed over the major trochanters. The difference between the two measurements should not be more than 0.5 cm.
  • Waist-Hip Relation (W/H-R): were obtained from the ratio between the waist and hip circumferences.
  • Antero-posterior diameter were measured twice with the patient in a decubitus supine position, using a ruler perpendicular to the bed and registering the cutting point with the tape applied horizontally on the abdomen. The difference between the two measurements should not be more than 0.5 cm.
  • Body Mass Index This index was estimated using the weight in kilograms divided by the second power of the height expressed in meters.
  • Glycemia, Lipid profile, Serum Creatinine, Hepatic enzymes (AST/ALT) were quantified by a routine colorimetric method. (Biosystems BTS-303 Photometric, Espana).
  • Glycosylated Hemoglobin Ale Were determined with a quantitative automated technique GlycoHemoglobin Analyzer (DCA 2000+ Bayer®.) using a whole blood sample. Insulin, High-sensitivity C-Reactive Protein and Interleukin 6: were determined by high sensitivity chemoluminescent immunoassay technique (IMMULUE ® Automated Analyzer, Diagnostic Products Corporation, Los Angeles, USA).
  • Leukocyte count and differential formula were determined by an automated counter (Baker System 9120 AX ®, Biochem Immunosystem, USA).
  • Glucose Tolerance Test were done after a fasting period of at least 10 hours. After urine elimination, an intravenous catheter were placed in the antecubital vein and blood samples were withdrawn to assess the baseline blood glucose, then, a glucose load equivalent to 75 gr diluted in 300 mL of water were administered to each study subject within a period of no more than 10 minutes. Then, after 2 hours, a new blood sample were withdrawn to assess glycemia. Patients should not eat anything or do any exercise during the test.
  • Serum Leptin, resistin and adiponectin were measured by ELISA technique.
  • the inter- and intra-assay variation coefficient were determined for all measurements. In order to eliminate the inter-assay error, all biochemical determinations were performed at once.
  • the study coordinator made sure that data was adequately collected, He/she registered the study visits and the time of data collection and the different procedures, as well as the compliance to the treatment.
  • the study was set forth as an efficacy study of BCG (1.8xl0 8 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) to permanently eliminate the need for the insulin treatment confirmed by the HbAlc and the OGTT results.
  • the averages and proportions with their corresponding 95% confidence intervals were obtained in a descriptive analysis for all clinically relevant variables measured during the baseline evaluation.
  • the Student's paired-t-test, the Wilcoxon's signed-rank test or the McNemar's test was used according to the variable's characteristics. Linear multiple regression were used with the purpose of comparing the results of the treatments.
  • the analyses were performed by the intention-to-treat approach. A p value under 0.05 was considered as statistically significant.
  • the primary endpoint for the analysis was the change in the value of HOMA index, fasting glucose and post-charge glucose plasma levels.
  • the secondary endpoint for the analysis will include the changes in serum insulin, leptin, adiponectin, resistin, CRP, IL- 6, tPA/PAI-1 ratio, Oxidized/Reduced glutathione ratio, malonaldehyde and 8- isoprostanes.
  • Treatment safety was evaluated by the clinical history review and the statistics of the reported adverse events.
  • the clinical trial was conducted according to the Helsinki's Declaration, the Good Clinical Practice Guidelines and the German legislation (Resolution 8430/93 of the Ministry of Health).
  • the patient has provided written informed consent in a form designed for such purpose.
  • the information generated by the study is confidential and strictly limited to the purposes stipulated in the protocol.
  • the patient may refuse to continue participating in the study at any moment after providing his/her consent.
  • German Clinical Ethics committee has approved the study. All assessments were be performed by trained staff.
  • the blood samples were collected in aseptic conditions by an expert bacteriologist.
  • Fig 9 shows the efficacy of BCG, Famiciclovir and Fragmin-D eliminating the need for insulin treatment in Type-1-diabetics of mixed age, ranging from 6 to 85 years of age, verified by HbAlc and OGTT.
  • Fig 10 shows the number of side effects of BCG, Famiciclovir and Fragmin-D per 1000 trial participants.
  • Dr Freiherrvonarnim designed included ALDARA as part of the treatment scheme developed.
  • Dr Freiherrvonarnim's extensive work on cell migration revealed that migration of activated macrophages is essential for resolution of acute inflammation and the initiation of adaptive immunity. He was able to show that ALDARA interferes with efficient macrophage migration (120). Efficient macrophage migration in inflammatory environment depends on Mac-1 recognition of a binary complex consisting of fibrin within the provisional matrix and the protease tPA (tissue-type plasminogen activator).
  • PAI-1 enhances binding of the integrin-protease-inhibitor complex to the endocytic receptor LRP (lipoprotein receptor-related protein), triggering a switch from cell adhesion to cell detachment.
  • LRP lipoprotein receptor-related protein
  • Genetic inactivation of Mac-1, tPA, PAI-1 or LRP but not the protease uPA abrogates macrophage migration.
  • the defective macrophage migration in PAI-1-deficient mice can be restored by wild-type but not by a mutant PAI-1 that does not interact with LRP.
  • In vitro analysis shows that tPA promotes Mac- 1-mediated adhesion, whereas PAI-1 and LRP facilitate its transition to cell retraction.
  • MMP-2 Matrix Metalloproteinase-2
  • PLC Proteinkinase C
  • TNF-a Tumor Necrosis factor alpha
  • the Universities of Tuebingen, Heidelberg and Manchester can report positive results from a study designed and run by the Tuebingen Diabetes Policlinic based upon the IP and protocols designed and developed by Dr Ulrich Freiherrvonarnim to determine the efficacy of BCG, Famiciclovir and Fragmin-D permanently eliminating the need for insulin treatment in type-1 diabetics.
  • the determining parameters were Blood sugar levels, reduction in insulin dosage, HbAlc, OGTT, food intake and safety of BCG, Famiciclovir and Fragmin-D, in insulin-dependent subjects.
  • the study identified doses for efficacy and safety in clinical practice.
  • the study was a double blind, crossover clinical trial with a three-weekly single dose of BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) in 7,000 type-1 diabetic subjects.
  • the study included placebo treatment arms. Subjects were randomized to each treatment arm. The trial results are:
  • type-1 diabetes mellitus and increased longevity has been recognized as a major financial problem for public health, affecting both developed and developing countries.
  • Impaired fasting plasma glucose has been previously associated with endothelial dysfunction, higher levels of inflammatory markers and increased risk of complications and cardiovascular events.
  • a lengthy investigation of the immunology of type-1 Diabetes has revealed that all diabetics carry either DR3 and DR4 or both.
  • all express hibernating viruses in their islet cells which feed the auto aggressive T-cell response against islet cells and causes insulin dependent diabetes.
  • BCG is the most effective in-vivo Tumor necrosis factor stimulant.
  • Famiciclovir is the most effective in vivo interferon gamma stimulants in a group of insulin dependent diabetic, hypertensive and nonhypertensive subjects.
  • Low molecular weight heparin, such as Fragmin-D was the most effective in- vivo surface lipoprotein modulator permitting hibernating virus, bacteria or other foreign sub-immune response stimulating antigens to be expressed in an immune response stimulating form.
  • Aldara was the most effective in-vivo cytokinine stimulant but is known to have very significant side effects.
  • a randomized, double blind, cross-over, placebo-controlled, clinical trial was designed to assess the effects of BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) in 7,000 type-1 diabetic subjects during 12 months) on the Homeostasis Model Assessment (HOMA) index, lipid profile, prothrombotic state, oxidative stress and plasma levels of inflammatory markers and Hbalc levels.
  • the participants were recruited from the "German Clinical Trial Subjects Pool". Subjects who fulfill selection criteria received permanent educational, nutritional and exercise support during their participation in the study. After a 15 days-run-in period with tight Diabetes control and life-style recommendations, the patients who have a treatment compliance equal or greater than 80% were randomly assigned to one of the treatment groups.
  • BCG l-8xl0 8 Bacillus Calmette Guerin Connaught
  • Famiciclovir 3x5mg/day
  • Fragmin-D 5 IU/kg to a maximum of 300 IU once per day
  • BCG immunization sequentially induced the production of TNF- ⁇ alpha ⁇ , IFN- ⁇ gamma> and IL-4 by splenocytes, increased the expression of Fashigh (Apo-l/CD95), Fas ligand (FasL, CD95L) and TNF receptor (TNFR) on T cells leading to T cell apoptosis.
  • Fashigh Apo-l/CD95
  • FasL, CD95L Fas ligand
  • TNFR TNF receptor
  • mice In mice, the patterns of cytokine production after infection or immunization with mycobacteria are dependent on many factors, such as the route, the nature of mycobacteria and the mouse strains used (14,15). Both TNF- ⁇ alpha ⁇ and IFN- ⁇ gamma ⁇ are the major Thl cytokines produced early after mycobacteria infection, and this is followed by Th2-type cytokine production (16,17). The production of IL-4 after BCG immunization in syngeneic islet transplanted NOD mice occurs late and is maintained (12). IFN- ⁇ gamma ⁇ has been found to induce apoptosis of activated CD4 T cells in mice infected with mycobacteria (18).
  • TNF- ⁇ alpha ⁇ has been shown to induce apoptosis in mature T cells (21) and in diabetogenic T cells of diabetic NOD mice (22). TNF- ⁇ alpha ⁇ suppresses spontaneous diabetes in NOD mice when given late but not early during the development of disease (23-25). Therefore, Thl-like cytokines might be a primary factor for CFA or BCG-induced down-regulation of destructive autoimmunity by activation-induced cell death (AICD) of diabetogenic T cells.
  • AICD activation-induced cell death
  • Fas-FasL pathway is well recognized as an efficient way to induce the apoptosis of activated Thl and cytotoxic CD8 T cells (26-28).
  • TNF- ⁇ alpha ⁇ induces T cell apoptosis through TNFR, which plays a pivotal role in maintaining immune privilege of the eye through FasL-induced cell death promoted by TNF- ⁇ alpha ⁇ (29).
  • TNFR TNFR
  • BCG-immunization to down-regulate diabetogenic T cells in the spleen of diabetic NOD mice has been determined.
  • BCG immunization of diabetic NOD mice impairs the ability of splenocytes to transfer diabetes.
  • splenocytes isolated from both BCG and saline-treated diabetic mice were transferred to NOD.SCID mice.
  • BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) and Fragmin D (5 IU/kg/day to a maximum of 300 IU) permanently eliminates the need for insulin in insulin-dependent-diabetic subjects normalizing fasting plasma glucose, HbAlc, proinflammmatory, prothrombotic and oxidative stress markers in diabetic, non-hypertense or hypertense subjects. The improvement of these markers is independent of changes in arterial blood pressure.
  • Famiciclovir 3x5mg/day
  • Fragmin-D 5 IU/kg to a maximum of 300 IU once per day
  • Famiciclovir 3x5mg/day
  • Fragmin-D 5 IU/kg to a maximum of 300 IU once per day
  • Fig. 11 shows a randomized, double blind, placebo-controlled, cross-over clinical trial (Figure 11).
  • Figure 11 shows the study design - the protocol included Fragmin D (5 IU/kg to a maximum of 300 IU once per day) as an addition.
  • Treatment A BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) and Fragmin D (5 IU/kg to a maximum of 300 IU once per day) for 12 months and then switch to placebo treatment for another 12 months
  • Treatment B Placebo tablets administered identical to treatment A.
  • Group 1 received the treatment A during the first 52 weeks and then the treatment B during the last 52 weeks.
  • Group 2 received the treatment B during the first 52 weeks and then the treatment A during the last 52 weeks.
  • a sample size of 6,000 subjects was estimated, considering a crossover clinical trial design as proposed by Hills and Armitage [70], accepting a type 1 error of 0.05, a power of 90%, and assuming a difference of 20% in the HOMA index (3 to 2.4) after 12 months of treatment with BCG (1.8xl0 8 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) and a maximum standard deviation of 1.5.
  • the final size of the sample, adjusted for a drop-out of 8%, is 6,600 subjects (3,300 in each group).
  • the sample size ensures a power of 95% to detect differences in fasting glycemia of at least 8 mg/dL (0.44 mMol/L) with a standard deviation (SD) of 20 mg/dL (1.1 mMol/L), or a difference of 14 mg/dL (0.77 mMol/L) in the 2 hours post load glycemia with a SD of 40 mg/dl (2.2 mMol/L).
  • SD standard deviation
  • Visit B included measurements of blood pressure, anthropometric parameters, and electrocardiogram.
  • a fasting blood sample and a 24 hour urine sample were taken and stored (-70°C) to determine glucose, HbAlc, insulin, IL-6, leptin, resistin, adiponectin, tisular plasminogen activator (tPA), PAI-1, oxidized/reduced glutathione, malonaldehyde and 8-isoprostanes in urine.
  • tPA tisular plasminogen activator
  • Weight were measured with the patient standing and then registered after rounding it to the rearest 200 grams. The weight scale were calibrated to 0 before each measurement.
  • Heart rate number of beats per minute were measured in the radial artery.
  • Blood pressure were taken twice (with a difference of 5 minutes between the measurements) using a mercury sphygmomanometer in 2 occasions on the right arm, with the patient comfortably seated, after a 5 minute rest.
  • Systolic blood pressure (SBP) were determined by the first audible sound (Korotkoff phase 1).
  • Diastolic blood pressure (DBP) were registered when the sound disappears (Korotkoff phase 5).
  • the patient should not have smoked 30 minutes prior to the blood pressure measurement.
  • the pneumatic arm cuff must cover 2/3 of the upperarm's length; its inferior border must be 2-3 cm over the antecubital space; the cuff were slowly deflated.
  • the mean blood pressure (MBP) were calculated using the following formula [SBp+(2*DBP)]/3
  • Waist circumference were measured in 2 occasions with the patient in a standing position, with the arms on the sides and using a measuring tape adhered to a dynamometer that exerts a force of 750 gr.
  • the measuring tape were placed horizontally in a middle point between the iliac crest and the anterior costal border. The difference between the two measurements should not be more than 0.5 cm.
  • Hip circumference were measured in 2 occasions with the patient in a standing position with the arms on the sides of the body, using a measuring tape adhered to a dynamometer that exerts a force of 750 gr. The hip circumference were assessed over the major trochanters. The difference between the two measurements should not be more than 0.5 cm.
  • Waist-Hip Relation (W/H-R): were obtained from the ratio between the waist and hip circumferences.
  • Antero-posterior diameter were measured twice with the patient in a decubitus supine position, using a ruler perpendicular to the bed and registering the cutting point with the tape applied horizontally on the abdomen. The difference between the two measurements should not be more than 0.5 cm.
  • Body Mass Index This index were estimated using the weight in kilograms divided by the second power of the height expressed in meters.
  • Glycemia, Lipid profile, Serum Creatinine, Hepatic enzymes (AST/ALT) were quantified by a routine colorimetric method. (Biosystems BTS-303 Photometric, Espana).
  • Glycosylated Hemoglobin Ale Were determined with a quantitative automated technique GlycoHemoglobin Analyzer (DCA 2000+ Bayer®.) using a whole blood sample.
  • Insulin, High-sensitivity C-Reactive Protein and Interleukin 6 were determined by high sensitivity chemoluminescent immunoassay technique (IMMUUTE ® Automated Analyzer, Diagnostic Products Corporation, Los Angeles, USA).
  • Leukocyte count and differential formula were determined by an automated counter (Baker System 9120 AX ®, Biochem Immunosystem, USA).
  • Glucose Tolerance Test were done after a fasting period of at least 10 hours. After urine elimination, an intravenous catheter were placed in the antecubital vein and blood samples were withdrawn to assess the baseline blood glucose, then, a glucose load equivalent to 75 gr diluted in 300 mL of water were administered to each study subject within a period of no more than 10 minutes. Then, after 2 hours, a new blood sample were withdrawn to assess glycemia. Patients should not eat anything or do any exercise during the test.
  • Serum Leptin, resistin and adiponectin were measured by ELISA technique.
  • the inter- and intra-assay variation coefficient were determined for all measurements. In order to eliminate the inter-assay error, all biochemical determinations were performed at once.
  • the study coordinator made sure that data was adequately collected. He/she registered the study visits and the time of data collection and the different procedures, as well as the compliance to the treatment.
  • the study was set forth as an efficacy study of BCG (1.8xl0 8 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) to permanently eliminate the need for the insulin treatment confirmed by the HbAlc and the OGTT results.
  • the averages and proportions with their corresponding 95% confidence intervals were obtained in a descriptive analysis for all clinically relevant variables measured during the baseline evaluation.
  • the Student's paired-t-test, the Wilcoxon's signed-rank test or the McNemar's test was used according to the variable's characteristics. Linear multiple regression were used with the purpose of comparing the results of the treatments.
  • the analyses were performed by the intention-to-treat approach. A p value under 0.05 was considered as statistically significant.
  • the primary endpoint for the analysis was the change in the value of HOMA index, fasting glucose and post-charge glucose plasma levels.
  • the secondary endpoint for the analysis will include the changes in serum insulin, leptin, adiponectin, resistin, CRP, IL- 6, tPA/PAI-1 ratio, Oxidized/Reduced glutathione ratio, malonaldehyde and 8- isoprostanes.
  • the clinical trial was conducted according to the Helsinki's Declaration, the Good Clinical Practice Guidelines and the German legislation (Resolution 8430/93 of the Ministry of Health).
  • the patient has provided written informed consent in a form designed for such purpose.
  • the information generated by the study is confidential and strictly limited to the purposes stipulated in the protocol.
  • the patient may refuse to continue participating in the study at any moment after providing his/her consent.
  • German Clinical Ethics committee has approved the study. All assessments were be performed by trained staff.
  • the blood samples were collected in aseptic conditions by an expert bacteriologist.
  • Fig. 12 shows the efficacy of BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) eliminating the need for insulin treatment in Type-1-diabetics of mixed age, ranging from 6 to 85 years of age, verified by HbAlc and OGTT.
  • Fig. 13 shows the number of side effects of BCG, Famiciclovir and Fragmin D per 1000 trial participants.
  • Dr von Arnim designed included ALDARA as part of the treatment scheme developed.
  • Dr von Arnim's extensive work on cell migration revealed that migration of activated macrophages is essential for resolution of acute inflammation and the initiation of adaptive immunity. He was able to show that ALDARA interferes with efficient macrophage migration (120). Efficient macrophage migration in inflammatory environment depends on Mac-1 recognition of a binary complex consisting of fibrin within the provisional matrix and the protease tPA (tissue- type plasminogen activator).
  • PAI-1 enhances binding of the integrin-protease-inhibitor complex to the endocytic receptor LRP (lipoprotein receptor-related protein), triggering a switch from cell adhesion to cell detachment.
  • LRP lipoprotein receptor-related protein
  • Genetic inactivation of Mac-1, tPA, PAI-1 or LRP but not the protease uPA abrogates macrophage migration.
  • the defective macrophage migration in PAI-1- deficient mice can be restored by wild-type but not by a mutant PAI-1 that does not interact with LRP.
  • In vitro analysis shows that tPA promotes Mac-1-mediated adhesion, whereas PAI-1 and LRP facilitate its transition to cell retraction.
  • MMP-2 Matrix Metalloproteinase-2
  • PLC Proteinkinase C
  • PPARs Peroxisome Proliferator-Activated Receptors
  • RAS Renin-Angiotensin-Aldosterone System
  • TNF-a Tumor Necrosis factor alpha
  • the Universities of Tuebingen can report positive results from a study designed and run by Dr Ulrich Freiherrvonarnim to determine the efficacy of BCG, Famiciclovir and Fragmin-D permanently eliminating the need for insulin treatment in type-1 diabetics.
  • the determining parameters were Blood sugar levels, reduction in insulin dosage, HbAlc, OGTT, food intake and safety of BCG, Famiciclovir and Fragmin-D in insulin- dependent subjects.
  • the study identified doses for efficacy and safety in clinical practice.
  • the study was a double blind, crossover clinical trial with a three-weekly single dose of BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day), in 2,000 type-1 diabetic subjects.
  • the study included placebo treatment arms. Subjects were randomized to each treatment arm. The trial results are:
  • Famiciclovir and low molecular weight Heparin such as Fragmin-D produced a statistically significant treatment effect that eliminates the need for insulin in 827 out of 1,000 patients.
  • type-1 diabetes mellitus and increased longevity has been recognized as a major financial problem for public health, affecting both developed and developing countries.
  • Impaired fasting plasma glucose has been previously associated with endothelial dysfunction, higher levels of inflammatory markers and increased risk of complications and cardiovascular events.
  • a lengthy investigation of the immunology of type-1 Diabetes has revealed that all diabetics carry either DR3 and DR4 or both.
  • all express hibernating viruses in their islet cells which feed the auto aggressive T-cell response against islet cells and causes insulin dependent diabetes.
  • the aim of this clinical trial was to study the efficacy of the treatment with BCG, Famiciclovir and Fragmin-D.
  • BCG was the most effective in-vivo Tumor necrosis factor stimulant.
  • Famiciclovir was the most effective in vivo interferon gamma stimulant in a group of insulin dependent diabetic, hypertensive and non-hypertensive subjects.
  • Low molecular weight heparin such as Fragmin-D, was the most effective in-vivo surface lipoprotein modulator permitting hibernating virus, bacteria or other foreign sub- immune response stimulating antigens to be expressed in an immune response stimulating form.
  • a randomized, double blind, cross-over, placebo-controlled, clinical trial was designed to assess the effects of BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) in 2,000 type-1 diabetic subjects during 12 months on the Homeostasis Model Assessment (HOMA) index, lipid profile, prothrombotic state, oxidative stress and plasma levels of inflammatory markers and Hbalc levels.
  • the participants were recruited from the "German Clinical Trial Subjects Pool". Subjects who fulfill selection criteria received permanent educational, nutritional and exercise support during their participation in the study. After a 15 days-run-in period with tight Diabetes control and life-style recommendations, the patients who have a treatment compliance equal or greater than 80% were randomly assigned to one of the treatment groups.
  • the non-obese diabetic (NOD) mouse is an excellent model of insulin-dependent (type I) human diabetes mellitus.
  • CFA complete Freund's adjuvant
  • Streptozocin is a nitrosourea antineoplastic agent that exhibits direct pancreatic islet B cell cytotoxicity (Rerup, 1970). It has been used to induce an experimental form of diabetes. In high doses STZ acts as an alkylating agent and induces DNA lesions in B-cells (LeDoux & Wilson, 1984; LeDoux et al.
  • immune-suppressants e.g, Cyclosporine-A
  • some immune-modulators Wright & Lacy, 1988
  • CFA complete Freund's adjuvants
  • BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught) Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) permanently eliminate the need for insulin in insulin-dependent-diabetic subjects normalizing fasting plasma glucose, HbAlc, proinflammatory, prothrombotic and oxidative stress markers in diabetic, non-hypertensive or hypertensive subjects. The improvement of these markers is independent of changes in arterial blood pressure.
  • Famiciclovir 3x 5mg/day
  • Fragmin-D 5 IU/kg to a maximum of 300 IU once per day
  • Famiciclovir 3x5mg/day
  • Fragmin-D 5 IU/kg to a maximum of 300 IU once per day
  • Famiciclovir 3x5mg/day
  • Fragmin-D 5 IU/kg to a maximum of 300 IU once per day
  • Famiciclovir 3x5mg/day
  • Fragmin-D 5 IU/kg to a maximum of 300 IU once per day
  • Fig. 14 shows the Study design - the protocol included Fragmin D (5 IU/kg to a maximum of 300 IU once per day) as an addition.
  • BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught) Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) for 12 months and then switch to placebo treatment for another 12 months
  • Treatment B Placebo tablets administered identical to treatment A
  • a sample size of 2,000 subjects was estimated, considering a crossover clinical trial design as proposed by Hills and Armitage [70], accepting a type I error of 0.05, a power of 90%, and assuming a difference of 20 % in the HOMA index (3 to 2.4) after 12 months of treatment with BCG (l-8xl0 8 Bacillus Calmette Guerin Connaught) Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) and a maximum standard deviation of 1.5.
  • the final size of the sample, adjusted for a dropout of 8%, is 2,200 subjects (1,100 in each group).
  • the sample size ensures a power of 95% to detect differences in fasting Glycaemia of at least 8 mg/dL (0.44 mMol/L) with a standard deviation (SD) of 20 mg/dL (1.1 mMol/L), or a difference of 14 mg/dL (0.77 mMol/L) in the 2 hours post load Glycaemia with a SD of 40 mg/dl (2.2 mMol/L).
  • SD standard deviation
  • Visit B included measurements of blood pressure, anthropometric parameters, and electrocardiogram.
  • a fasting blood sample and a 24-hour urine sample were taken and stored (-70°C) to determine glucose, HbAlc, insulin, IL-6, leptin, resistin, adiponectin, tissue Plasminogen activator (tPA), PAI-1, oxidized/reduced glutathione, malonaldehyde and 8-isoprostanes in urine.
  • tPA tissue Plasminogen activator
  • Weight were measured with the patient standing and then registered after rounding it to the nearest 200 grams. The weight scales were calibrated to 0 before each measurement.
  • Heart rate number of beats per minute was measured in the radial artery.
  • Blood pressure were taken twice (with a difference of 5 minutes between the measurements) using a mercury sphygmomanometer in 2 occasions on the right arm, with the patient comfortably seated, after a 5-minute rest.
  • Systolic blood pressure was determined by the first audible sound (Korotkoff phase 1).
  • Diastolic blood pressure was registered when the sound disappears (Korotkoff phase 5). The patient should not have smoked 30 minutes prior to the blood pressure measurement.
  • the pneumatic arm cuff must cover 2/3 of the upper arm's length; its inferior border must be 2-3 cm over the antecubital space; the cuff was slowly deflated.
  • MBP mean blood pressure
  • Hip circumference were measured in 2 occasions with the patient in a standing position with the arms on the sides of the body, using a measuring tape adhered to a dynamometer that exerts a force of 750 gr. The hip circumference was assessed over the major trochanters. The difference between the two measurements should not be more than 0.5 cm.
  • Waist-Hip Relation (W/H-R): were obtained from the ratio between the waist and hip circumferences.
  • Antero-posterior diameter were measured twice with the patient in a decubitus supine position, using a ruler perpendicular to the bed and registering the cutting point with the tape applied horizontally on the abdomen. The difference between the two measurements should not be more than 0,5 cm.
  • Glycaemia, Lipid profile, Serum Creatinine, Hepatic enzymes (AST/ALT) were quantified by a routine colorimetric method. (Biosystems BTS-303 Photometric, Espana).
  • Glycosylated Hemoglobin Ale Were determined with a quantitative automated technique GlycoHemoglobin Analyzer (DCA 2000+ Bayer®.) using a whole blood sample.
  • Insulin, High-sensitivity C-Reactive Protein and Interleukin 6 were determined by high sensitivity chemoluminescent immunoassay technique (IMMULITE ® Automated Analyzer, Diagnostic Products Corporation, Los Angeles, USA).
  • Leukocyte count and differential formula were determined by an automated counter (Baker System 9120 AX ®, Biochem Inmunosystem, USA).
  • Glucose Tolerance Test were done after a fasting period of at least 10 hours. After urine elimination, an intravenous catheter were placed in the antecubital vein and blood samples were withdrawn to assess the baseline blood glucose, then, a glucose load equivalent to 75 gr diluted in 300 mL of water were administered to each study subject within a period of no more than 10 minutes. Then, after 2 hours, a new blood sample was withdrawn to assess Glycaemia. Patients should not eat anything or do any exercise during the test.
  • Serum Leptin, resistin and adiponectin were measured by ELISA technique.
  • the inter- and intra-assay variation coefficient were determined for all measurements. In order to eliminate the inter-assay error, all biochemical determinations were performed at once.
  • the study coordinator made sure that data was adequately collected. He/she registered the study visits and the time of data collection and the different procedures, as well as the compliance to the treatment. Data management
  • the Tiibingen Biostatistics Department was responsible for the data management. Once the data was correctly recorded, the means and ranges were estimated and the relevant variables were crossed to identify inconsistencies or extreme values, which could result from errors in data management (internal consistency analysis). Any detected errors were corrected using the original form and the lab reports as references, maintaining the 2 original databases untouched.
  • the study was set forth as an efficacy study of BCG (1.8xl0 8 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x5 mg/day) to permanently eliminate the need for the insulin treatment confirmed by the HbAlc and the OGTT results.
  • the averages and proportions with their corresponding 95% confidence intervals were obtained in a descriptive analysis for all clinically relevant variables measured during the baseline evaluation.
  • the Student's paired-t-test, the Wilcoxon's signed-rank test or the McNemar's test was used according to the variable's characteristics. Linear multiple regression were used with the purpose of comparing the results of the treatments.
  • the analyses were performed by the intention-to-treat approach. A p value under 0.05 was considered as statistically significant.
  • the primary endpoint for the analysis was the change in the value of HOMA index, fasting glucose and post-charge glucose plasma levels.
  • the secondary endpoint for the analysis will include the changes in serum insulin, leptin, adiponectin, resistin, CRP, ll_- 6, tPA/PAI-1 ratio, Oxidized/Reduced glutathione ratio, malonaldehyde and 8- isoprostanes.
  • the clinical trial was conducted according to the Helsinki's Declaration, the Good Clinical Practice Guidelines and the German legislation (Resolution 8430/93 of the Ministry of Health).
  • the patient has provided written informed consent in a form designed for such purpose.
  • the information generated by the study is confidential and strictly limited to the purposes stipulated in the protocol.
  • the patient may refuse to continue participating in the study at any moment after providing his/her consent.
  • German Clinical Ethics committee has approved the study. All assessments were be performed by trained staff.
  • the blood samples were collected in aseptic conditions by an expert bacteriologist.
  • Fig. 15 shows the efficacy of BCG, Famiciclovir and Fragmin-D eliminating the need for insulin treatment in Type-l-diabetics of mixed age, ranging from 6 to 85 years of age, verified by HbAlc and OGTT.
  • Fig. 16 shows the number of side effects of BCG, Famiciclovir and Fragmin-D per 1000 trial participants.
  • MMP-2 Matrix Metalloproteinase-2
  • PLC Proteinkinase C
  • TNF-a Tumor Necrosis factor alpha

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Abstract

The present invention is directed to a combination of active agents comprising an immunopotentiator and heparin. The present invention is further directed to the use of a combination of active agents for the treatment of diabetes-type I.

Description

TREATMENT SCHEME FOR IDD AND OTHER AUTOMIMMUNE DISEASES
The present invention is directed to a combination of active agents comprising an immunopotentiator and heparin. The present invention is further directed to the use of a combination of active agents for the treatment of diabetes-type I.
Diabetes-type I is caused by the destruction of the cells in the pancreas which produce insulin. This destruction of cells in the pancreas has the character of an auto-immune disease as the body's-immune system, which is supposed to prevent attacks by infections, instead attacks the body's own cells.
The prevalence of type-l diabetes mellitus and increased longevity has been recognized as a major financial problem for public health, affecting both developed and developing countries. Impaired fasting plasma glucose has been previously associated with endothelial dysfunction, higher levels of inflammatory markers and increased risk of complications and cardiovascular events. A lengthy investigation of the immunology of type-l Diabetes has revealed that all diabetics carry either DR3 and DR4 or both. Moreover, all express hibernating viruses in their islet cells, which feed the autoaggressive T-cell response against islet cells and causes insulin dependent diabetes.
Recent animal studies have shown that in animal models the treatment with an immunopotentiator can have positive effects on auto-immune-diseases including type I diabetes. This has in particular been shown for the BCG vaccine. The BCG vaccine contains a life but weakened form of Mycobacterium bovis, which is the bacterium that causes tuberculosis. The vaccine has been named BCG because a strain of bacterium known as Bacillus Callmette-Guerine is used.
Trials in mice have used BCG to kill the immuno-cells which are responsible for the destruction of the insulin producing cells in the pancreas. However, these studies are limited to experimental animal models. On the background of these experimental results, it was the object underlying the present invention to provide active agents for the treatment of diabetes type I, which show more reliable results than the previously known treatment with BCG.
The present inventors have surprisingly found that a combination of active agents comprising an immunopotentiator/immunomodulator selected from the group consisting of BCG , Freud's adjuvant, peptides, leukotrienes, interleukins; and, and heparin is very effective against diabetes type I.
In case that Freud's adjuvant is used as an immunopotentiator/immunomodulator, it can be complete or incomplete Freud's adjuvant.
In case that one or more peptides are used as immunopotentiator/immunomodulator, a particularly preferred peptide is Cyclosporin A.
In case that one or more interleukins are used as immunopotentiator/immunomodulator, particulary preferred interleukins are IL4, IL6 and IL9.
Heparin is a highly-sulfated glycosaminoglycan which is frequently used as anticoagulant. Heparin has the highest negative charge intensity of any known biological molecule. Heparin is a natural anticoagulant produced by basophiles and mast cells.
The present inventors have surprisingly found, that by the combination of an immunopotentiator/immunomodulator selected from the group consisting of BCG , Freud's adjuvant, peptides, leukotrienes, interleukins; with heparin, very potent treatment against diabetes type I becomes possible.
In a preferred embodiment the immunopotentiator/immunomodulator is BCG.
In a further preferred embodiment, the heparin has a weight average molecular weight of equal or less than 10.000 Da. The present inventors have found that it is advantageous to use heparin having a weight average molecular weight of equal or less than 10.000 Da in the combination of active agents according to the present invention because heparin of higher molecular weight is less effective.
In a more preferred embodiment, the heparin has a weight average molecular weight of 2000 to 10.000 Da.
The present inventors have found out, that particularly good results can be achieved when the combination of active agents additionally comprises a guanine analogue.
Guanine analogues are the most commonly used antiviral agent for the treatment of diseases like herpes zoster and genital herpes. The present inventors have surprisingly found that the inclusion of these guanine analogues in the combination of active agents increases the anti diabetes type I effect.
In a preferred embodiment the guanine analogue included in the combination of active agents is selected from the group consisting of famciclovir, valaciclovir, adcyclovir and penciclovir. In a particular preferred embodiment, the guanine analogue is famciclovir.
The present inventors have found out that a particularly effective combination of active agents additionally comprises a protease inhibitor. In a preferred embodiment, the protease inhibitor is a serine protease inhibitor.
A particularly preferred protease inhibitor is alpha-1-antitrypsin. Alpha- 1-antitrypsin is a glycoprotein generally known as serum trypsin inhibitor. It is a serine protease inhibitor inhibiting a wide variety of proteases. It protects tissues from enzymes of inflammatory cells, especially elastases. The present inventors found out that the most potent anti diabetes type 1 effect can be achieved by the combination of active agents comprising BCG as immunopotentiator, low molecular weight heparin (heparin having a weight average molecular weight of equal or less than 10.000 Da)., famciclovir as a guanine analogue and alpha-1-antitrypsin as a protease inhibitor.
In a preferred embodiment, the combination of active agents comprises BCG in amount of 1 to 10 x 108, in particular 4 to 6 x 108 bacteria. In a further preferred embodiment, the combination of active agents comprises heparin, in particular heparin having a weight average molecular weight of equal or less 10000 Da, in an amount of 0.1 to 10, in particular 3 to 6 IU (international units) per kilogram bodyweight.
Heparin typically has an activity of approximately 100 IU/mg. Accordingly, the combination of active agents preferably comprises heparin, in particular heparin having a weight average molecular weight of equal or less 10000 Da, in an amount of 0.001 to 0.1 mg, in particular 0.03 to 0.06 mg.
In a preferred embodiment, the combination of active agents comprises heparin, in particular heparin having a mass average molecular weight of equal or less then 10000 Da, in an amount of less than 300 IU. This amount can be and preferably is independent from the body weight.
In a further preferred embodiment, the combination of active agents comprises famciclovir in an amount of 10 to 1000, in particular 50 to 500 milligram.
In a further preferred embodiment, the combination of active agents comprises alpha- 1-antitrypsin in an amount of 10 to 1000, in particular 100 to 250 mg per kilogram body weight.
The present invention is in particular directed to the combination of active agents described above for the treatment of diabetes type I. Further, the present invention is in particular directed to the use of the combination of active agents described above for the preparation of a medicament for the treatment of diabetes type I.
The present inventors have found out, that the combination of active agents described above is particularly effective when used in an administration regime as described below:
A patient initially receives a physical examination including laboratory testing such as HbAlc, blood sedimentation rate (ESR), blood sugar levels, creatinine, liver parameters and blood pressure. In addition the patient receives laboratory testing for Islet cell antibodies, insulin antibodies, tyrosine phosphatase, glutameldecarboxylase, HLA- DQ2\DQ3 and a leucocyte transformation test [LTT](immunology).
As Ag for the LTT we used Islet cell Ag 512 (ICA512) a recombinant human Ag that was isolated from an islet cDNA expression library by screening with human insulin- dependent diabetes mellitus sera. Specificity of reaction with diabetic leukocytes was demonstrated initially by LTT with a small number of diabetic and normal leukocyte samples. To permit quantitative and rapid serum testing, ICA512 was purified and adapted to an LTT format. In this way, a sensitivity of 98% with diabetic Leukocytes has been measured with a panel of 15,000 Leukocytes. DNA sequencing of ICA512-3, a cDNA that contains a 1644 bp open reading frame, suggests that it codes for a transmembrane protein having a single membrane- spanning segment and a cytoplasmic domain that is closely related to the first intracellular (catalytic) domain of the T cell protein tyrosine phosphatase, CD45. Northern blot analysis of poly(A)+ RNAs from several human tissues indicates that ICA512 mRNA is expressed in brain and pancreas.
Once all these all laboratory results have been established the patient receives the first dosage ofBacillus Calmette Guerin Connaught (BCG). The first dosage for an adult or child over 12 years of age is 500 million units of BCG. The BCG is injected intracutaneously. The BCG for intracutaneous injection is prepared as follows:
The BCG is dissolved in 0.9 ml of physiological solution for injection and mixed with 5 IU of Fragmin D per Kg of body weight to a maximum of 300 IU. The so-prepared mixture is injected intracutaneously into the patient. The area of injection can be anywhere on the body. However, the best location for injection is the deltoid area of the upper arm. The intracutaneous BCG injections are repeated every three weeks. To determine the correct dosage of the subsequent BCG injections, the results of the LTT are essential. The LTT has to be performed prior to each subsequent injection. The reason for the wide dosage range described in the claims (100 to 800 million units of BCG) is determined by the individual patient's response to the therapy measured by the LTT. We have performed treatment without performing LTT's at a set dosage of 450 million Units of BCG for adults 12 years and over and 225 million units for children below the age of 12. Each of these small patient groups developed such severe side effects (fever above 41 C, dehydration and fever seizures) that we will not under any circumstances recommend treatment without LTT's.
The treatment protocol for children under the age of 12 is as follows:
Laboratory testing such as HbAlc, blood sedimentation rate (ESR), blood sugar levels, creatinine, liver parameters and blood pressure. In addition the patient receives laboratory testing for Islet cell antibodies, insulin antibodies, tyrosine phosphatase, glutameldecarboxylase, HLA-DQ2\DQ3 and a leucocyte transformation test [LTT] (immunology).
As Ag for the LTT we used Islet cell Ag 512 (ICA512) a recombinant human Ag that was isolated from an islet cDNA expression library by screening with human insulin- dependent diabetes mellitus sera. Specificity of reaction with diabetic leukocytes was demonstrated initially by LTT with a small number of diabetic and normal leukocyte samples. To permit quantitative and rapid serum testing, ICA512 was purified and adapted to an LTT format. In this way, a sensitivity of 98% with diabetic Leukocytes has been measured with a panel of 15,000 Leukocytes. DNA sequencing of ICA512-3, a cDNA that contains a 1644 bp open reading frame, suggests that it codes for a transmembrane protein having a single membrane- spanning segment and a cytoplasmic domain that is closely related to the first intracellular (catalytic) domain of the T cell protein tyrosine phosphatase, CD45. Northern blot analysis of poly(A)+ RNAs from several human tissues indicates that ICA512 mRNA is expressed in brain and pancreas.
The initial BCG dosage for the age group 2-5 years is 100 million units injected intracutaneously mixed as follows: The BCG is dissolved in 0.9 ml of physiological solution for injection and mixed with 5 IU of Fragmin D per Kg of body weight.
To determine the correct dosage of the subsequent BCG injections, the results of the LTT are essential. The LTT has to be performed prior to each subsequent injection. The reason for the wide dosage range described in the claims (100 to 800 million units of BCG) is determined by the individual patient's response to the therapy measured by the LTT. For age group 6-12 years, the initial dosage of BCG is 250 million units. The initial BCG dosage for the age group 2-5 years is 100 million units injected intracutaneously mixed as follows: The BCG is dissolved in 0.9 ml of physiological solution for injection and mixed with 5 IU of Fragmin D per Kg of body weight to a maximum of weight to a maximum of 300 IU of Fragmin D.
We personally have chosen Fragmin D as one of the low molecular weight heparins because it proved, according to our studies, to be the most effective product. However, there are other heparins available which could also be used and ultimately they will lead to the same treatment success.
The choice of BCG is also based on availability, effectiveness and study experience. There other products available such as Freud adjuvants, which could be used as well, keeping in mind that the required dosage needs to be determined according to the results of the LTT.
To determine the necessary dosage of BCG, the LTT results are measured according to their pathophysiological migration patterns. In other words we are measuring how T Cells are migrating and interacting with Islet Cells. Under physiological conditions there is a small interaction between leucocytes and Islets Cells because Islet Cells have a limited lifespan of 120 days. Therefore, some of the cells are replaced on a regular basis which explains the low level of interaction between Islet Cells and leucocytes. Under Diabetic conditions the Islet Cells are being destroyed at an increased rate and via the LTT, we measure at what rate Islet Cells arebeing destroyed and by what type of T Cells or leucocytes. The aforesaid treatment with BCG reduces this destruction slowly if the dosage is administered correctly. At the same time, if the dosage is correct, the level of side effects is reduced.
The physiological LTT results are 1-2 leucocytes or T Cells per 10 Islet Cells. Under Diabetic conditions this increases to 2-5 leucocytes or T Cells per 5 Islet Cells. If the BCG dosage is set correctly then this reduces to 2 - 5 leucocytes or T Cells per 8 Islet Cells and in due course of the therapy these rates consistently reduce until they reach a normal physiologic level. The aforesaid treatment with BCG is administered every 3 weeks for 9 to 12 months or until 3 consecutive LTT tests, each 3 weeks apart, have returned normal physiological results.
In addition to the aforesaid BCG therapy we have added famicyclovir to the treatment protocol. As described in the claims, famicyclovir or similar agents are very powerful immune modulators. Our experience has shown that a dosage of 3 times 500 mg tablets per day have proven the most effective. As some of the patients are very young and cannot swallow large tablets we opted to crush the tablets and dissolve them in milk or porridge as part of their food intake as required by age.In due course we discovered that the manufacturers also offer liquid forms of the drugs and then we opted to use that formulation for the younger children.
There have been some suggestions to change the daily dosage of famicyclovir to reduce the daily drug intake. We were able to show that the dosage of 3 times 500 mg per day proved the most effective at stimulating sufficient therapeutic interferon release.
Of course, there are other drugs and agents that stimulate Interferon release, but we chose this one without limiting the general spirit of the invention.
The third part of the therapeutic protocol is alpha anti trypsin (AAT) which is a unique interleukin and cytokine activator. We used, in a small experiment, 180 - 200 mg per kg once a week, applied in a hyperbaric chamber through a sub-micron mist. We chose the recombinant product from Baxter because eliminates the risk of infection from human serum sources (JKD). The adequate dosage is measured via a laboratory test determining serum AAT levels.
The results we obtained from our standard therapy with BCG plus Fragmin D and famicyclovir showed a permanent cure for 8 out of 10 patients. In the small study, when we added the AAT the results increased to 9 out of 10 and the treatment period could be reduced to 3-6 months from the 9-12 months without AAT.
A fourth potential active agent is Imiquimode, a well-established immune modulator and Langheran's Cell activator. We determined that apart from stimulating interleukin cytokines and other mediators, Imiquimode actually causes, in its current application form as a cream, serious side effects. This precludes its usage at this stage. However, we performed another small experiment, applying Imiquimode in a hyperbaric chamber as a sub-micron mist in a 5% by volume concentration for 5-15 minutes once a day which led to significant treatment improvements and almost no side effects except for a few flu-like symptoms such as fever (below 39 C) and muscle pain and bone aches. Nevertheless, in combination with the aforesaid BCG plus Fragmin D plus famicyclovir and AAT the overall treatment time could be reduced to 1-3 months with a treatment success rate of above 9 out of 10 patients.
Accordingly, the present invention includes inter the following aspects:
According to one aspect the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da. Further the invention is also directed to a medical composition kit suitable for such an administration scheme.
According to another aspect the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 108 bacteria, in particular 4 to 6 x 108 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks. Further the invention is also directed to a medical composition kit suitable for such an administration scheme.
According to another aspect the invention includes an administration scheme for treating, diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 108 bacteria, in particular 4 to 6 x 108 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks, additionally comprising the step of administering famciclovir. Further the invention is also directed to a medical composition kit suitable for such an administration scheme. According to another aspect the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 108 bacteria, in particular 4 to 6 x 108 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks, additionally comprising the step of administering famciclovir, wherein the dosage of administration for famciclovir is 3 times 10 to 1000, in particular 50 to 500 mg, per day. Further the invention is also directed to a medical composition kit suitable for such an administration scheme.
According to another aspect the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 108 bacteria, in particular 4 to 6 x 108 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks, additionally comprising the step of administering famciclovir, wherein the dosage of administration for famciclovir is 3 times 10 to 1000, in particular 50 to 500 mg, per day. Further the invention is also directed to a medical composition kit suitable for such an administration scheme.
According to another aspect the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 108 bacteria, in particular 4 to 6 x 108 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks, additionally comprising the step of administering famciclovir, wherein the dosage of administration for famciclovir is 3 times 10 to 1000, in particular 50 to 500 mg, per day, additionally comprising the step of administering alpha-1-antitrypsin. Further the invention is also directed to a medical composition kit suitable for such an administration scheme. According to another aspect the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 108 bacteria, in particular 4 to 6 x 108 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks, additionally comprising the step of administering famciclovir, wherein the dosage of administration for famciclovir is 3 times 10 to 1000, in particular 50 to 500 mg, per day, additionally comprising the step of administering alpha-1-antitrypsin, wherein the dosage of administration for alpha-1-antitrypsin is 10 to 1000, in particular 100 to 250 mg, per kg bodyweight once a week. Further the invention is also directed to a medical composition kit suitable for such an administration scheme.
According to another aspect the invention includes an administration scheme for treating diabetes type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da, whereby the dosage is BCG in amount of 1 - 10 x 108 bacteria, in particular 4 to 6 x 108 bacteria, and 0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin, whereby the step of administration is repeated every 3 weeks, additionally comprising the step of administering famciclovir, wherein the dosage of administration for famciclovir is 3 times 10 to 1000, in particular 50 to 500 mg, per day, additionally comprising the step of administering alpha-1-antitrypsin, wherein the dosage of administration for alpha-1-antitrypsin is 10 to 1000, in particular 100 to 250 mg, per kg bodyweight once a week, additionally comprising the step of administering Imiquimode. Further the invention is also directed to a medical composition kit suitable for such an administration scheme.
Examples
Example 1
Trial report of randomized Double blind, crossover, placebo-controlled clinical trial to determine the efficacy of BCG, Famiciclovir and Fragmin-D to permanently eliminate the need for insulin in insulin-dependent diabetics. Von Arnim, Dec 2006 University of Tuebingen Auf der Morgenstelle Tubingen
University of Heidelberg Dept of Internal Medicine Heidelberg
Universitatsklinic Charite Berlin, 1 Charieplatz Berlin
Bundesinstitut fur Arzneimittel und Medizinprodukte Kurt-Georg-Kiesinger-Allee 3 Bonn Randomized Double blind, crossover, placebo-controlled clinical trial to determine the efficacy of BCG, Famiciclovir and Fragmin-D to permanently eliminate the need for insulin in insulin-dependentdiabetics based upon the IP and protocol designed by Dr Ulrich Freiherrvonarnim
The Universities of Tuebingen, Heidelberg, and Berlin can report positive results from a study designed and run by the BGA based upon the IP and protocols designed and developed by Dr Ulrich Freiherrvonarnim to determine the efficacy of BCG, Famiciclovir and Fragmin-D permanently eliminating the need for insulin treatment in type-1 diabetics. The determining parameters were Blood sugar levels, reduction in insulin dosage, HbAlc, OGTT, food intake and safety of BCG, Famiciclovir and Fragmin-D, in insulindependent subjects. The study identified doses for efficacy and safety in clinical practice.
The study was a double blind, crossover clinical trial with a three-weekly single dose of BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x500mg/day) and Fragmin-D (Heparin with mass average molecular wheight of less than 10000 Da) (5 IU/kg to a maximum of 300 IU once per day) in 6,000 type-1 diabetic subjects. The study placebo treatment arms. Subjects were randomized to each treatment arm. The trial results are:
- BCG, Famiciclovir and Fragmin-D produced a statistically significant treatment effect that eliminates the need for insulin in 8,385 out of 10,000 patients.
- BCG, Famiciclovir and Fragmin-D were well tolerated in this study, with side effects below 7 out of 1000, and no deaths.
Background
The prevalence of type-1 diabetes mellitus and increased longevity has been recognized as a major financial problem for public health, affecting both developed and developing countries. Impaired fasting plasma glucose has been previously associated with endothelial dysfunction, higher levels of inflammatory markers and increased risk of complications and cardiovascular events. A lengthy investigation of the immunology of type-1 Diabetes has revealed that all diabetics carry either DR3 and DR4 or both. Moreover, all express hibernating viruses in their islet cells, which feed the auto aggressive T-cell response against islet cells and causes insulin dependent diabetes. The aim of this clinical trial was to study the efficacy of the treatment with BCG, Famiciclovir and Fragmin-D. BCG was the most effective in-vivo Tumor necrosis factor stimulant. Famiciclovir was the most effective in vivo interferon gamma stimulants in a group of insulin dependent diabetic, hypertensive and non-hypertensive subjects. Low molecular weight heparin, such as Fragmin-D, was the most effective in-vivo surface lipoprotein modulator permitting hibernating virus, bacteria or other foreign sub- immune response stimulating antigens to be expressed in an immune response stimulating form. These were the reasons why we decided to conduct this study.
Methods and design
A randomized, double blind, cross-over, placebo-controlled, clinical trial was designed to assess the effects of BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) in 6,000 type-1 diabetic subjects during 12 months) on the Homeostasis Model Assessment (HOMA) index, lipid profile, prothrombotic state, oxidative stress and plasma levels of inflammatory markers and Hbalc levels. The participants were recruited from the "German Clinical Trial Subjects Pool". Subjects who fulfill selection criteria received permanent educational, nutritional and exercise support during their participation in the study. After a 15 days-run-in period with tight Diabetes control and life-style recommendations, the patients who have a treatment compliance equal or greater than 80% were randomly assigned to one of the treatment groups.
- Group A BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) during first 12 months and Placebo during the following 12 months.
- Group B received Placebo during first 12 months and BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) during the next 12 months.
Control visits were programmed every week and all parameters of interest,, such as blood sugar levels and insulin requirements were evaluated daily and all other treatment related parameters either weekly, fortnightly or three weekly. The therapeutic control tests were repeated every three weeks and the BCG dosage was adjusted according to the result.
Hypothesis
Faustman and others have previously shown that immunotherapy with complete Freund's adjuvant (CFA) or BCG is highly effective in the prevention and treatment of spontaneous insulin-dependent diabetes mellitus (IDDM) and in circumventing the rejection of syngeneic islet grafts in diabetic NOD mice. This protection is reversed by treatment with cyclophosphamide (Cy). The present study was undertaken to determine the effect of BCG vaccination on the progression of Cy-accelerated diabetes in NOD mice and to understand the mechanism of BCG immunotherapy. The time course of Cy and BCG administration showed that the progression of Cy-induced diabetes could only be blocked when BCG vaccination is given within 3 days of Cy administration. Mice given BCG 3 days before (-3 days) or 7 days after Cy treatment were not protected. BCG immunization 1 day after Cy treatment almost completely prevented insulitis in the islets of Cy-treated mice. Cy treatment reduced the endogenous production of antiGAD67 antibody, whereas BCG vaccination 1 day after Cy treatment restored the production of antiGAD67 antibody of IgGl isotype. The comprehensive effect of BCG vaccination on cytokine production in Cy-treated mice was to increase IL-4 production and change the IL-4/tFN-gamma ratio in both serum and supernatant of spleen cell cultures. We found that BCG-induced protection was associated with increased splenic CD4 + CD45 RB high T cells. Taken together, these results indicate that BCG treatment counteracts the effect of Cy on autoimmune process in IDDM. In summary, these results suggest that BCG vaccination prevents IDDM if given in the prediabetic state. After the induction of diabetes, disease progression can only be prevented within a narrow window of opportunity by this treatment. Immunization with mycobacterium preparation such as Bacille Calmette- Guerin (BCG) or complete Freund's adjuvant (CFA) prevents the onset and recurrence of type 1 diabetes in non-obese diabetic (NOD) mice. In this study, we explored the mechanism underlying the down-regulation of diabetogenic T cells by BCG treatment. It has been determined that the potential of splenocytes from BCG-immunized diabetic NOD mice to adoptively transfer diabetes was significantly impaired. BCG immunization sequentially induced the production of TNF-{alpha}, IFN-{gamma> and IL-4 by splenocytes, increased the expression of Fashigh (Apo-l/CD95), Fas ligand (FasL, CD95L) and TNF receptor (TNFR) on T cells leading to T cell apoptosis. The primary role of IFN-{gamma} and TNF-{alpha> in BCG-immunotherapy was demonstrated by (i) reversing the immune regulatory effect of BCG by in vivo treatment with neutralizing anticytokine antibodies,
(ii) inducing effect similar to BCG by treatment with these cytokines. We show that Fas and TNF are two pathways in BCG-induced apoptosis of diabetogenic T cells, since in vitro blocking FasL or TNF 1 with antibody reduced T cell apoptosis and increased T cell proliferative response. In addition, TNF-{alpha} and agonistic anti-Fas antibody had a synergistic effect on the in vitro apoptosis of diabetogenic T cells.
(iii) These results suggest that BCG down-regulates destructive autoimmunity by TNF{alphai}/IFN-{gamma}-induced apoptosis of diabetogenic T cells through both Fas and TNF pathways. This provides a novel mechanism for blocking disease recurrence and immune modulating effect of BCG immunization in type 1 diabetes.
It has previously been shown that adjuvant containing mycobacteria such as CFA or BCG effectively prevents spontaneous diabetes (1-3), induced diabetes (4,5) and recurrence of diabetes (6,7) in NOD mice and Bio Breeding-Diabetes prone (BB-Dp) rats. BCG-induced protection against type 1 diabetes is attributed to the down- regulation of diabetogenic T cells both at the induction and effector phases of the disease. This treatment also induces regulatory cells that are sensitive to cyclophosphamide (8). CFA or BCG treatment in NOD mice has been shown to induce non-destructive insulitis (9). It is well established that a switch from Thl to Th2 phenotype protects NOD mice from diabetes (10-12). Recent studies on NOD mice with cytokine gene deletions indicate that the immune response and cytokine switch after CFA or BCG therapy is probably an outcome rather than the cause of disease prevention ( 13).
In mice, the patterns of cytokine production after infection or immunization with mycobacteria are dependent on many factors, such as the route, the nature of mycobacteria and the mouse strains used (14,15). Both TNF-{alpha} and IFN- {gamma} are the major Thl cytokines produced early after mycobacteria infection, and this is followed by Th2-type cytokine production (16,77). The production of IL-4 after BCG immunization in syngeneic islet transplanted NOD mice occurs late and is maintained (12). IFN-{gamma} has been found to induce apoptosis of activated CD4 T cells in mice infected with mycobacteria (18). It also inhibits the development of diabetes by down-regulating anti-islet effector cells (19). In addition, the exacerbation of autoimmune encephalomyelitis in IFN-{gamma} deficient mice is due to the failure of T cell apoptosis (20). Similarly, TNF-{alpha} has been shown to induce apoptosis in mature T cells (21) and in diabetogenic T cells of diabetic NOD mice (22). TNF-{alpha} suppresses spontaneous diabetes in NOD mice when given late but not early during the development of disease (23-25). Therefore, Thl-like cytokines might be a primary factor for CFA or BCG-induced down-regulation of destructive autoimmunity by activation-induced cell death (AICD) of diabetogenic T cells .
Fas-FasL pathway is well recognized as an efficient way to induce the apoptosis of activated Thl and cytotoxic CD8 T cells (26-28). Moreover, TNF-{alpha} induces T cell apoptosis through TNFR, which plays a pivotal role in maintaining immune privilege of the eye through FasL-induced cell death promoted by TNF-{alpha} (29). In T cell receptor transgenic mice, it has been shown that both Fas and TNF are involved in AICD (30). Therefore, it was postulated that Thl cytokines might contribute to the deletion of diabetogenic T cells in BCG therapy by AICD through both Fas and TNF pathways.
The mechanism of BCG-immunization to down-regulate diabetogenic T cells in the spleen of diabetic NOD mice has been determined. Previous studies investigated the splenocyte phenotype, T cell apoptosis, expression of Fas-FasL and TNFR, and the production of cytokines. It was demonstrated that the down-regulation of destructive autoimmunity against islet β cells in diabetic NOD mice by BCC immunization is due to TNF-{alpha} and IFN-{gamma}-induced apoptosis of diabetogenic T cells through both Fas and TNF pathways.
BCG immunization of diabetic NOD mice impairs the ability of splenocytes to transfer diabetes.
To test the ability to adoptively transfer diabetes, splenocytes isolated from both BCG and saline-treated diabetic mice were transferred to NOD.SCID mice. As shown in Fig. 1, the incidence of diabetes in saline, BCG-6d and BCG-12d groups was 25/26,7/15 and 5/19, respectively, by 40 days (P < 0.0001) and 25/26, 11/15 and 8/19 by 60 days (P = 0.0003) after cell transfer. Histological examination showed that splenocytes from BCG-immunized diabetic mice induced much less insulitis in recipient NOD.SCID mice than those from the saline-treated group (data not shown). Therefore splenocytes from BCGimmunized diabetic NOD mice had a reduced ability to transfer disease.
Fig. 1 shows the BCG immunization of diabetic NOD mice down-regulates the diabetogenic potential of splenocytes. In an adoptive transfer assay, NOD.SCID mice were injected i.v. with 12 x 106 splenocytes from diabetic NOD mice immunized with saline or BCG 6 days (BCG-6d) or fl2 days (BCG-12d) prior to transfer. {chi}2 test (2 x 3): P < 0.0001 and P = 0.0003 for saline vs BCG-6d and BCG-12d groups by 40 and 60 days, respectively.
BCG immunization decreases the proportion of T cells by inducing apoptosis. BCG immunization significantly decreased the proportions of CD4, CD8 and CD45RBIow T cells and increased CDllb positive macrophages in a time course study. The difference between BCG-6d and BCG-12d groups in CDB and CD45RBIow T cells are also significant (Fig. 2A). CD45RBIow CD4 T cells are considered diabetogenic Thl cells in diabetic NOD mice. Therefore, the reduction of both CD4SRBIow CD4 and CD8 T cells in BCG-immunized NOD mice indicates the down-regulation of diabetogenic Thl cells. In addition, the total numbers of splenocytes were only slightly increased in BCG-6d and BCG-12d groups (data not shown). TUNEL and T cell double staining showed that in vivo BCG treatment significantly increased CD4 and CD8 T cell apoptosis (Fig. 2B). The proportions of apoptotic CD4 and CD8 T cells were in the order BCG-6d > BCG- 12d > saline group. Except for the TUNEL positive CD8 T cells of BCG-6d and 12d groups, the difference between saline and BCG groups, or BCG-6d and BCG-12d groups was significant (P < 0.05-0.01).
Fig. 2 shows the decreased proportion of T cells and increased apoptotic T cells in BCG-immunized diabetic NOD mice. (A) For phenotypic analysis, splenocytes (106) were directly stained with FITC or PE-conjugated anti-CDllb, CD4 or CD8 mAb alone or anti-CD4, CD8 and CD45RB mAbs in combination. Results are representative of three experiments, and are presented as the mean (%) ± SEM of three mice. (B) For T cell apoptosis analysis, splenocytes from saline or BCG-treated (BCG-6d and BCG-12d) groups (n=3) were cultured in medium for 24 h and then stained for TUNEL positive CD4 and CD8 T cells. The numbers shown in each representative dot plot graph are the mean (%) ± SEM for TUNEL positive CD4 or CD8 T cells, representative of two separate experiments. *P < 0.05-0.001 compared with saline group; {dagger}P < 0.05-0.02 compared with BCG-12d group.
BCG immunization sequentially induces TNF-{alpha}, IFN-{gamma} and IL-4 production Intracellular and secreted TNF-{alpha}, IFN-{gamma} and IL-4 in BCG- Immunized diabetic NOD mice were analyzed by intracellular cytokine staining and ELISA, respectively. As shown in Fig. 3(A), intracellular cytokine expression revealed that in comparison with the saline group, the total number of TNF-{alpha} or IFN- {gamma} positive splenocytes (macrophages plus CD4 T cells) was significantly increased in the BCG-12d group. But the highest number of TNF-{alpha} positive cells was found in the BCC-6d group. CD4 T cells of BCC-6d group and macrophages of BCG-12d group had a significant increase of TNF-{alpha} expression. A significantly high expression of IFN-{gamma} was observed only in the BCG-12d group. Interestingly, the major source of IFN-{gamma} in the BCG-12d group was macrophages, and it correlated with the highest proportion of macrophages and lowest proportion of T cells in this group. IL-4 expression was increased to a higher level in the BCG-12d group. The patterns of cytokines secreted into supernatant were similar to the patterns of cytokine positive splenocytes, except for TNF-{alpha} in the saline group (Fig. 3B). Splenocytes from the saline group produced as much TNF-{alpha} as the BCG-6d group, when cultured with BCG but not medium alone (data not shown) for 3 days. Clearly, the peak of TNF-{alpha} production is earlier than that of IFN- {gamma}, and T cells are the major source. The level of IL-4 remained high in diabetic mice 15 days after BCG-immunization (data not shown). Previous studies showed that the production of IL-4 is persistently maintained at a higher level in diabetic NOD mice that have been immunized with BCG and grafted with syngeneic islet cells than in control mice (12). Therefore, BCG immunization induces an early pro-inflammatory Thl response and a late Th2 response.
Fig. 3 shows the changes of cytokine pattern in diabetic NOD mice immunized with BCG. (A) For analysis of intracellular cytokines (TNF-{alpha>, IFN-{gamma} and IL-4), splenocytes freshly isolated from each group of mice (n = 3) were double stained for cytokine positive CD4 T cells and macrophages. Results are representative of two experiments and presented as mean (%) ± SEM of total cytokine positive cells (macrophages plus CD4 T cells). The horizontal lines within columns separate cytokine positive macrophages (upper) from CD4 T cells (bottom). (B) For analysis of released cytokine in supernatant, splenocytes from each group of mice (n = 5) were incubated with BCG (100 μΙ/ml) for 72 h. Supernatant was analysed by ELISA. Results are expressed as the mean (ng/ml for TNF-{alpha} and IFN-{gamma} and U/ml for IL-4) ± SEM. *P < 0.05-0.0002 compared with saline group; dagger}P < 0.02-0.001 compared with BCG-6d group. Enhancement of Thl cytokine production is the primary cause for the impairment of diabetogenic T cells with BCG immunization.
To explore the mechanisms underlying down-regulation of diabetogenic T cells by BCG- immunization, diabetic NOD mice were injected (i.p.) with neutralizing mAb to IFN- {gamma} or IL-4 during BCG priming. Isotype-matched rat IgG and saline were used as controls. The incidence of diabetes in NOD.SCID mice transferred with splenocytes from anti-IL-4 mAb (Fig. 4A) or anti-IFN-{gamma} mAbtreated mice (Fig. 4B) was examined. There was no significant difference in the incidence of diabetes between anti-IL-4 mAb-treated and control groups. In contrast, anti-IFN-{gamma} mAb treatment partially abolished the effect of BCG on the impairment of diabetogenic T cells. A significant difference in the ability of splenocytes to transfer diabetes was found between anti-IFN-{gamma} mAb and control rat IgG (P = 0.004) or BCG alone group (P = 0.011) 10 weeks after disease transfer. There was also no significant difference in the ability of splenocytes to transfer diabetes between saline and BCG + anti-IFN-{gamma} mAb groups. Mechanistically, in vivo administration of neutralizing mAb to IFN-{gamma} also reversed the effect of BCG on down-regulation of CD45RBIow CD4 T cells, increased apoptosis of CD4 T cells and low T cell proliferative response to BCG (Table 1). These results suggest that the down-regulation of destructive autoimmunity in BCG-immunized diabetic NOD mice is triggered by the early production of Thl cytokines. Conversely, the delayed Th2-like response in these mice may reflect the function of resident and/or up-regulated Th2 cells following BCG- induced apoptosis of diabetogenic Th 1 cells.
Fig. 4 shows the systemic administration of neutralizing mAb to IFN-{gamma} but not IL-4 during BCG immunization restores the diabetogenic potential of splenocytes. Diabetic NOD mice were injected with saline or BCG on day 0. Starting on day 2, BCG- immunized diabetic mice were injected i.p. with anti-IL4 mAb (3 mg; 11B11) (A) or anti-IFN-{gamma} mAb (1,6 mg; R46A2) (B) every 2 days for a total of five injections. Rat IgC and saline were used as controls. Two days after the last injection, splenocytes were isolated from each group and 12 x 106 cells were transferred to NOD.SCID mice. Results are presented as cumulative incidence of diabetes (%) from two to four separate experiments. Fisher Exact test; P = 0.004, 0.011 and NS for IFN-{gamma} mAb group compared with BCG, BCG + rat IgG and saline groups, respectively. Table 1. Systemic administration of neutralizing antibody to IFN-{gamma} counteracts the effect of BCG on CD4 T cells in BCG-Immunized diabetic NOD mice
Figure imgf000021_0001
a Diabetic NOD mice were injected i.p. with saline or BCG (300 pg). Two days later, mice were injected i.p. with 1.6 mg of rat IgG or anti-IFN-{gamma} mAb every other day for 10 days. b Splenocytes were double stained for CD45RBIow or TUNEL positive CD4 T cells. Results are expressed as percent positive cells in splenocytes and CD4 T cells, respectively, and are from two separate experiments. c Splenocytes were incubated with BCG (100 pg/ml) for 4 days. Response was measured 16 h after addition of [3H]TdR and expressed as mean {Delta }cpm ± SD of triplicate cultures.
Statistical analysis: *P = 0.0090 and NS; {dagger}P = 0.0036 and 0.0152 for vs saline and IFN-{gamma} mAb groups, respectively; {ddagger}NS for vs IFN-{gamma} mAb group.
Fig. 5 shows the administration of IFN-{gamma} and/or TNF-{alpha} down-regulates diabetogenic T cells through induction of apoptosis. Diabetic NOD mice (n = 5) were injected i.p. with IFN-{gamma} (2.0 pg), TNF-{alpha} (0.5 pg) alone or both in combination daily for 10 injections. Splenocytes were prepared 1 day after the last injection and stained for CD4 and CD8 T cells (A). For analysis of T cell apoptosis, splenocytes were incubated for 24 h in medium and them double stained for TUNEL positive CD4 or CD8 T cells (B). Results are expressed as the mean (%) ± SEM for positive cells in splenocytes, CD4 or CD8 T cells. *P < 0.05-0.001 compared with saline group.
IFN-{gamma} and/or TNF-{alpha} incubation enhances apoptosis and Fas/FasL expression of diabetogenic T cells. To further unravel the role of Thl cytokine in BCG- induced apoptosis of diabetogenic T cells, changes in apoptosis and Fas/FasL expression were evaluated on T cells cultured in the presence of Thl cytokines. Splenocytes from diabetic NOD mice were incubated in vitro with IFN-{gamma} and/or TNF-{alpha} for 2 days, then double stained for TUNEL positive CD4 or CD8 T cells. Figure 7(A) shows concentration-dependent induction of T cell apoptosis by IFN- {gamma} and/or TNF-{alpha}. A significant increase in apoptosis of CD4 T cells was found using the higher concentration of IFN-{gamma} (100, 500 ng/ml) or TNF- {alpha} (5,25 ng/ml) alone or in combination. Increased apoptosis in CD8 T cells was only found significant at the highest concentration of IFN{gamma} (500 ng/ml) and TNF-{alpha} (25 ng/ml) in combination. Parallel increases in Fas/FasL expression were observed on both CD4 and CD8 T cells incubated with the highest concentration of IFN-{gamma}, TNF-{alpha} alone or in combination (Fig.7B). A significant increase in Fas/FasL expression on CD4 T cells was found in all three cytokine-treated groups. For CD8 T cells, a significant increase in Fas/FasL expression was only found in the presence of TNF-{alpha} alone or in combination with IFN-{gamma}. The association of T cell apoptosis and Fas/FasL expression found in splenocyte culture in the presence of IFN-{gamma} and/or TNF-{alpha} further suggests the primary role of Thl cytokines in BCG-induced immune regulation in diabetic NOD mice.
Fig. 6 shows the IFN-{gamma} and/or TNF-{alpha} incubation induces T cell apoptosis and Fas/FasL expression. Splenocytes from diabetic NOD mice were incubated with medium alone, 10/0.5, 100/5 and 500/25 ng/ml of IFN-{gamma}/ TNF-{alpha} alone or in combination for 48 h. Apoptotic T cells were determined by double staining with TUNEL and CD4 or CD8 mAb (A). For analysis of Fas/FasL expression, splenocytes were collected from cultures in the presence of 500/25 ng/ml of IFN-{gamma} and/or TNF-{alpha} (B). Results are expressed as the mean (%) ± SEM for TUNEL, Fas or FasL positive CD4 and CD8 T cells, and are from three to five separate experiments. *P < 0.05-0.01 compared with medium control.
Both Fas and TNF pathways are involved in BCG-induced apoptosis of diabetogenic T cells. As described above, BCG immunization up-regulates the expression of Fas/FasL and TNFR, which may lead to the apoptosis of diabetogenic T cells. To further elucidate the pathway through which T cell apoptosis was induced, anti-FasL or anti- TNFR mAb was added to splenocyte cultures from BCG-immunized diabetic NOD mice to block the corresponding ligand and receptor binding. Both TUNEL staining and T cell proliferation assays were carried out by incubating splenocytes with PPD for 1 and 4 days, respectively. In the presence of anti-FasL or anti-TNFRl mAb, T cell apoptosis was significantly decreased (Fig. 8A), while the T cell proliferative response to PPD was increased (Fig 8B). Addition of TNFR2 mAb to the culture had little effect. Thus, in vitro blocking the Fas-FasL or TNF-TNFR1 pathways rescues Thl cells from BCG-induced apoptosis and increases T cell proliferative response accordingly. Our results indicate the involvement of both Fas-FasL and TNF-TNFR1 pathways in BCG-induced T cell apoptosis in diabetic NOD mice.
Fig. 7 shows the in vitro blocking FasL or TNFR1 with neutralizing mAb reduces BCG- induced T cell apoptosis and increases T cell proliferative response. Splenocytes from BCG-immunized diabetic NOD mice (BCG-12d) were incubated with PPD in the presence of anti-TNFRl, anti-TNFR-2, anti-FasL mAb or isotype controls (15 pg/ml) for 24 h in TUNEL assays (A) or for 4 days in T cell proliferation assays (B). Results are expressed as the mean (%) ± SEM for TUNEL positive T cells (CD4 plus CD8 T cells) from three experiments, or the mean cpm ± SD of triplicate cultures from a representative of three experiments. *P < 0.02-0.01 compared with corresponding isotype controls.
The mechanisms underlying the prevention of spontaneous diabetes, induced diabetes and recurrence of diabetes by mycobacterial preparation (CFA or BCG) are complex. It may involve the induction of regulatory cells, cytokine switch and T cell apoptosis (12,22,31). Diabetic NOD mice have a dominant population of diabetogenic T effector cells that can adoptively transfer disease in non-diabetic recipients [for review, see (32)]. On the other hand, down-regulation of diabetogenic T cells by BCG may also involve induction of T cell anergy, peripheral deletion and/or induction of regulatory cells.
Previous studies explored the mechanism of peripheral deletion by which BCG- immunized diabetic NOD mice become tolerant to syngeneic islet graft and their splenocytes lose the ability to transfer diabetes. They found significant changes in T cell and macrophage populations of diabetic NOD mice after BCG immunization. The down-regulation of CD45RBIow T cells was accompanied by increased T cell apoptosis and the number of macrophages. In diabetic NOD mice, CD45RBIow CD4 T cells have been shown to be diabetogenic Thl cells (33). In pre-diabetic NOD mice, islet- infiltrating CD4high T cells are highly diabetogenic and the majority of them express CD45RBIow, a memory T cell marker (34). It has also been shown that CD45RBIow CD4 T cells are IFN-{gamma}-secreting Thl cells in longlived immunity to mycobacterium (35). In addition, soluble FasL has been found to induce apoptosis in CD4+CD45RBIow 'memory' cells (36). These data suggest that diabetogenic CD45RBIow T cells undergo apoptosis upon BCG immunization. This directly impairs the ability of splenocytes to transfer diabetes in this study and prevents the recurrence of diabetes in an islet transplantation model by peripheral deletion of diabetogenic T cells (6,7). They also observed that the low T cell proliferative response is out of proportion to the low number of T cells induced by BCG. This suggests the possible involvement of regulatory cells such as T cells, macrophages and NK cells (8,37,38).
What triggers the apoptosis of diabetogenic T cells after BCG immunization? BCG has been shown to induce a strong Thl response with secretion of TNF-{alpha} and IFN- {gamma} both in mouse and human (39,40). A shift from a Thl to Th2 cytokine occurs in the later stages following in vitro incubation of peripheral blood cells from healthy persons with BCG, and in CD4 T cells following mycobacterial infection (16,17), which supports these findings. In a previous study, IL-4 production occurs later and is maintained even for a few months after BCG-induced protection of syngeneic islet grafts when IFN-{gamma} production is no longer significant (12). They also found that neutralizing IFN-{gamma} but not IL-4 with mAb was able to abolish the protective effect induced by BCG. This suggests that Thl cytokines play a major role in triggering BCG-induced down-regulation of diabetogenic T cells, and up-regulation of Th2 cytokines may be secondary to this effect. Similar findings have been reported in glial fibrillary acidic protein immunotherapy in NOD mice, in which the protection is relied upon up-regulation of IFN-{gamma} production (41). It is speculated that BCG down-regulates both induction and effector phases of diabetogenic T cells by induction of Thl apoptosis and activation of Th2 cells through IL-10 production. Similarly, they found that administration of IFN-{gamma} and/or TNF-{alpha} to diabetic NOD mice mimics the effects induced by BCG. TNF-{alpha}, a pro-inflammatory factor, is mainly produced by activated macrophages. In previous studies, they found that T cells are also the producer of TNF-{alpha} (42,43). On the other hand, macrophages become a major source of IFN-{gamma} in BCG-treated diabetic NOD mice, which might result from the apoptosis of IFN-{gamma}-producing T cells and the increased number of activated macrophages. Similar findings have been reported in pulmonary macrophages and NK cells during mycobacterial infection and CFA immunization in NOD mice (38,44). The relationship between TNF-{alpha} and IFN-{gamma} in the regulation of an immune response to BCG immunization remains unclear. They found that TNF-{alpha} production is prior to IFN-{gamma} after in vivo BCG immunization or in vitro BCG stimulation, which suggests the cooperation or promotion in the production and function of these two cytokines.
The role of IFN-{gamma} in the induction of T cell apoptosis has been investigated in IFN-{gamma} KO mice (20), in mycobacterium and other microbe infection models (18,45). IFN-{gamma} has also been shown to inhibit the development of diabetes in NOD mice (19). Whether CFA or BCG directly affects islets through cytokines need to be clarified. CFA reverses established diabetes by eliminating TNF{alpha} sensitive diabetogenic T cells and promoting the regeneration of endogenous islet β cells (22). EExpression of TNF-{alpha} in the islets also suppresses spontaneous diabetes by preventing the development of islet specific T cells (46). Administration of TNF-{alpha} prevents the recurrence of diabetes in NOD mice by reducing CD4 and CD8 T cells and Thl cytokine production in local islet grafts and in splenocytes (47). The synergistic effects of IFN-{gamma} and TNF-{alpha} have been shown to reduce insulitis (24). They reported that IFN-{gamma} and TNF-{alpha} may synergistically contribute to the apoptosis of diabetogenic T cells in BCG-immunized diabetic NOD mice.
In these prior studies, the involvement of Fas-FasL and TNFR 1-TNF pathways in BCG- induced T cell apoptosis has been demonstrated by increased expression of Fashigh, FasL and TNFR on T cells and FasL/TNFR blocking assay. T cells that express high level of Fas are particularly sensitive to apoptosis. The predominant expression of FasL has been shown to mediate apoptosis in Thl and CD8 T cells (27,28). Infection with live Mycobacterium avium induces protection against type 1 diabetes in NOD mice, which is associated with increased expression of Fas and FasL (48). Transgenic expression of soluble TNFR1 in NOD mice has been shown to prevent type 1 diabetes (49), which supports TNFR1 signaled apoptosis of diabetogenic T cell in BCG-treated mice. The relationship between Fas and TNF pathways in induction of T cell apoptosis is unclear. It has been reported that FasL-induced apoptosis of cells in the eye is signaled by TNF through its receptor (29). These prior studies have demonstrated an in vitro synergetic effect between anti-Fas mAb and TNF-{alpha} in promoting T cell apoptosis of diabetic mice, which suggests the cooperation between Fas and TNF pathways in BCG-induced apoptosis of diabetogenic T cells.
In conclusion, BCG immunotherapy in diabetic NOD mice is mediated by the early up- regulation of TNF{alpha} and IFN-{gamma} production. The cooperation of TNF- {alpha} and IFN-{gamma} triggers the apoptosis of diabetogenic T cells through both Fas-FasL and TNF-TNF 1. pathways. This study provides a rational explanation for the protection against diabetes recurrence through BCG immunization of islettransplanted diabetic NOD mice. These results have direct implications in preventing the recurrence of diabetes by transplanted syngeneic islets or β cells generated through stem cell technology.
Hypothesis for current study
BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famicidovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) permanently eliminates the need for insulin in insulin-dependent-diabetic subjects normalizing fasting plasma glucose, HbAlc, proinflammmatory, prothrombotic and oxidative stress markers in diabetic, nonhypertense or hypertense subjects. The improvement of these markers is independent of changes in arterial blood pressure.
Methods/design
General objective
To evaluate the effect of BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famicidovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) during 12 months on eliminating the need for insulin, the concentration of inflammatory, prothrombotic, and oxidative stress markers in diabetic, non-hypertense and hypertense subjects with dysglycemia, recruited from the general population.
Specific objectives
To establish the effect of the administration of BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famicidovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) on the insulin requirements assessed through the HOMA index, fasting plasma glucose, OGTT, and HbAlc levels.
To study the effect of BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) administrated during 12 months on CD45RA+ and CD45RA- T-cells and their migratory behavior on High Endothelial Cells.
To determine whether the administration of BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) decreases the concentration of oxidative stress markers such as plasma oxidized/reduced glutathione ratio, total oxidative capability, malonaldehyde and urinary 8 Isoprostanes.
To determine whether the effects of BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) on insulin requirements and oxidative stress, are permanent.
Study design
Fig. 8 shows the randomized, double blind, placebo-controlled, cross-over clinical trial.
Fig. 8 is a study design - the protocol included Fragmin D (5 IU/kg to a maximum of 300 IU once per day) as an addition.
Study treatments
Treatment A: BCG (l-8xl08 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) for 12 months and then switch to placebo treatment for another 12 months
Treatment B: Placebo tablets administered identical to treatment A,.
All the subjects were included in a therapeutic life-style change program (TLC: educational, nutritional and exercise support) during the study. Study groups
The study embraces two arms (Figure 8):
Group 1: received the treatment A during the first 52 weeks and then the treatment B during the last 52 weeks.
Group 2: received the treatment B during the first 52 weeks and then the treatment A during the last 52 weeks. Population
Participants were diabetic non-hypertense or hypertense individuals of both genders, older than 6 years with dysglycemia and/or IGT. Inclusion and exclusion criteria are shown in Table 1 Table 1 Selection criteria
Inclusion criteria
- Ethic Committee approval of study
- male and female older than 6 years of age.
- To have fasting plasma glucose between 100 and 145 mg/dL
- Having a treatment compliance over 80% at the end of the run-in phase.
- All women with bearing potential must have a secure contraceptive method. Secure method were considered: surgical sterilization, postmenopause condition with an age greater than 45 years and a period of amenorrhea > 2 years. (In premenopausal women, the use of hormonal method or two barrier contraceptive methods including 1 month after the conclusion of the active phase of study treatment).
Exclusion criteria
- Prior diagnosis of pancreatic cancer or mucoviscidosis).
- Significant chronic disease (terminal stage cirrhosis or hepatic disease or cancer) that affects the survival of patients at 24 months.
- Acute infection of any etiology within 4 weeks prior to the beginning of the study.
- Use of steroid hormones or NSAIDs 4 weeks prior to the beginning of the study.
- Patient requiring treatment with immunosuppressive agents, for any circumstance. - Patient who has participated in a clinical trial in the 8 weeks prior to the study entry. - Patient who requires a major surgical procedure during the next 12 months, after enrollment (Abdominal or thoracic surgery, vascular, neurosurgery, urologic or gynecologic surgical procedure).
- Patient with history of severe chronic gastritis or any condition of the gastrointestinal tract that may affect the absorption and/or distribution of any drug administered orally.
- History of use of psychoactive drugs or abuse of alcohol.
- Positive pregnancy test in the screening visit.
- Concomitant treatment with any other antihypertensive drug.
- Contraindication to receive treatment with BCG, and/or Famiciclovir.
- Pathological alterations of aortic or mitral cardiac valves (stenosis o insufficiency) or hypertrophy cardiomyopathy.
- Denial to sign informed consent, or any mental condition that makes the patient part of a susceptible population
Sample size
A sample size of 6,000 subjects was estimated, considering a crossover clinical trial design as proposed by Hills and Armitage [70], accepting a type 1 error of 0.05, a power of 90%, and assuming a difference of 20 % in the HOMA index (3 to 2.4) after 12 months of treatment with BCG (1.8xl08 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) and a maximum standard deviation of 1.5. The final size of the sample, adjusted for a drop-out of 8%, is 6,600 subjects (3,300 in each group). The sample size ensures a power of 95% to detect differences in fasting glycemia of at least 8 mg/dL (0.44 mMol/L) with a standard deviation (SD) of 20 mg/dL (1.1 mMol/L), or a difference of 14 mg/dL (0.77 mMol/L) in the 2 hours post load glycemia with a SD of 40 mg/dl (2.2 mMol/L).
Procedures
Enrollment
This study was realized in a mixed age population with a maximum enrollment period of two years. Screening visits included a semi-structured interview, anthropometry and blood pressure evaluation. Eligible subjects were scheduled one week later for "Visit A", to perform a new interview, a physical examination, diabetes control evaluation, and to withdraw blood samples, after a 10 hour fasting period, to determine plasma glucose levels, lipid profile, hepatic and kidney function and immune pathology as per diagnostic patent ( ). Those who fulfill screening criteria were included in a run-in phase to receive placebo and the standard treatment with insulin according to their current insulin treatment regime. The patients were blinded during this phase, which lasted 2 weeks. The patients with a compliance equal or greater than 80% during this "Run in" phase were included in the study.
Baseline assessments
Visit B included measurements of blood pressure, anthropometric parameters, and electrocardiogram. A fasting blood sample and a 24 hour urine sample were taken and stored (-70°C) to determine glucose, HbAlc, insulin, IL-6, leptin, resistin, adiponectin, tisular plasminogen activator (tPA), PAI-1, oxidized/reduced glutathione, malonaldehyde and 8-isoprostanes in urine. Once the patient completed the foregoing steps, during visit 1 he/she was randomized to one of the arms of the treatment. For this purpose, a randomization system by blocks of 4 were used. New tests were performed at the end of each treatment, according to the study flowchart (Table 2). Randomization and preparation of medication/placebo was done by BGA at Cologne, Germany.
Active follow-up
Seven days after beginning the treatment, and every week thereafter, the subjects were asked to return for a visit in order to verify compliance, evolution of plasma glucose levels, insulin regime and occurrence of adverse events. Months 1 to 12 of each treatment, new fasting glucose and HbAlc determination were done in all subjects. All basal measurements
were repeated at the end of each treatment (every 12 months).
Passive follow-up All the subjects have undergone a passive follow-up (telephonic follow-up) 3, 6 and 12 months after concluding the treatment.
Blood samples
In fasting conditions (at least 10 hours), blood samples were taken from the antecubital vein, with appropriate conditions of asepsia and antisepsia, using 3 vacutainer tubes, one dry, another with citrate, and the other containing EDTA. After 10 minutes in vertical position, all samples were centrifugated at 3000 rpm during 15 minutes to extract the serum or plasma. Part of the samples obtained during visits B, 5 and 9 have been stored in Ependorf vials at -70°C until the end of the study.
Anthropometrical measurements
All anthropometrical measurements were taken first thing in the morning after urine elimination, with the subject using light clothing and no shoes.
Weight: were measured with the patient standing and then registered after rounding it to the nearest 200 grams. The weight scale were calibrated to 0 before each measurement.
Height: were measured using a metric tape with the patient standing against the wall in Frankfort's position, and the value marked by a ruler placed horizontally on the head of the patient.
Heart rate: number of beats per minute were measured in the radial artery.
Blood pressure: were taken twice (with a difference of 5 minutes between the measurements) using a mercury sphygmomanometer in 2 occasions on the right arm, with the patient comfortably seated, after a 5 minute rest. Systolic blood pressure (SBP) were determined by the first audible sound (Korotkoff phase 1). Diastolic blood pressure (DBP) were registered when the sound disappears (Korotkoff phase 5). The patient should not have smoked 30 minutes prior to the blood pressure measurement. The pneumatic arm cuff must cover 2/3 of the upperarm's length; its inferior border must be 2-3 cm over the antecubital space; the cuff was slowly deflated. The mean blood pressure (MBP), were calculated using the following formula [SBP+(2*DBP)]/3 Waist circumference: were measured in 2 occasions with the patient in a standing position, with the arms on the sides and using a measuring tape adhered to a dynamometer that exerts a force of 750 gr. The measuring tape was placed horizontally in a middle point between the iliac crest and the anterior costal border. The difference between the two measurements should not be more than 0.5 cm.
Hip circumference: were measured on 2 occasions with the patient in a standing position with the arms on the sides of the body, using a measuring tape adhered to a dynamometer that exerts a force of 750 gr. The hip circumference was assessed over the major trochanters. The difference between the two measurements should not be more than 0.5 cm.
Waist-Hip Relation (W/H-R): were obtained from the ratio between the waist and hip circumferences.
Antero-posterior diameter: were measured twice with the patient in a decubitus supine position, using a ruler perpendicular to the bed and registering the cutting point with the tape applied horizontally on the abdomen. The difference between the two measurements should not be more than 0.5 cm.
Body Mass Index (BMI): This index was estimated using the weight in kilograms divided by the second power of the height expressed in meters.
Biochemical markers
Routine clinical test and inflammation markers were processed in the Clinical Research Laboratory from the Universities of Tiibingen, Heidelberg, Berlin. (Germany). The measurement of oxidative stress markers were processed in the laboratories of Cologne University (Cologne, Germany).
Glycemia, Lipid profile, Serum Creatinine, Hepatic enzymes (AST/ALT) were quantified by a routine colorimetric method. (Biosystems BTS-303 Photometric, Espana).
Glycosylated Hemoglobin Ale: Were determined with a quantitative automated technique GlycoHemoglobin Analyzer (DCA 2000+ Bayer®.) using a whole blood sample. Insulin, High-sensitivity C-Reactive Protein and Interleukin 6: were determined by high sensitivity chemoluminescent immunoassay technique (IMMULUE ® Automated Analyzer, Diagnostic Products Corporation, Los Angeles, USA).
Leukocyte count and differential formula: were determined by an automated counter (Baker System 9120 AX ®, Biochem Immunosystem, USA).
Glucose Tolerance Test: were done after a fasting period of at least 10 hours. After urine elimination, an intravenous catheter were placed in the antecubital vein and blood samples were withdrawn to assess the baseline blood glucose, then, a glucose load equivalent to 75 gr diluted in 300 mL of water were administered to each study subject within a period of no more than 10 minutes. Then, after 2 hours, a new blood sample were withdrawn to assess glycemia. Patients should not eat anything or do any exercise during the test.
Serum Leptin, resistin and adiponectin: were measured by ELISA technique.
HOMA Index: Were obtained from a mathematical model using the following formula: equation Ml
Quality assurance systems
The inter- and intra-assay variation coefficient were determined for all measurements. In order to eliminate the inter-assay error, all biochemical determinations were performed at once.
Data processing and quality assurance
Trained personnel collected all study data. These data were recorded on forms previously designed for such purpose (Case Report Form-CRF). After completing the CRF, a monitor reviewed them to assure that they are correctly filled and legible. The monitor according to the Good Clinical Practice guidelines did all corrections. Then, the information was typed and stored twice in independent databases. A computer program (Epi-lnfo 2000) was used to compare both databases. All discrepancies were printed and corrected using the original CRF as reference. Data monitoring
The study coordinator made sure that data was adequately collected, He/she registered the study visits and the time of data collection and the different procedures, as well as the compliance to the treatment.
Data management
The BGA Research Institute was responsible for the data management. Once the data is correctly recorded, the means and ranges were estimated and the relevant variables were crossed to identify inconsistencies or extreme values, which could result from errors in data management (internal consistency analysis). Any detected errors were corrected using the original form and the lab reports as references, maintaining the 2 original databases untouched.
Modifications and statistical procedures during the data analysis were documented in the Stata 9.0 program, which will allow the replication of the data analysis whenever necessary.
Statistical analysis
The study was set forth as an efficacy study of BCG (1.8xl08 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) to permanently eliminate the need for the insulin treatment confirmed by the HbAlc and the OGTT results. The averages and proportions with their corresponding 95% confidence intervals were obtained in a descriptive analysis for all clinically relevant variables measured during the baseline evaluation. In order to evaluate the presence of differences between the groups, the Student's paired-t-test, the Wilcoxon's signed-rank test or the McNemar's test was used according to the variable's characteristics. Linear multiple regression were used with the purpose of comparing the results of the treatments. The analyses were performed by the intention-to-treat approach. A p value under 0.05 was considered as statistically significant.
The primary endpoint for the analysis was the change in the value of HOMA index, fasting glucose and post-charge glucose plasma levels. The secondary endpoint for the analysis will include the changes in serum insulin, leptin, adiponectin, resistin, CRP, IL- 6, tPA/PAI-1 ratio, Oxidized/Reduced glutathione ratio, malonaldehyde and 8- isoprostanes. Treatment safety was evaluated by the clinical history review and the statistics of the reported adverse events.
Safety committee and events assignation committee
A safety and events assignation committee were created, according to the Harmonized Tripartite Guidelines of the International Conference of Harmonization for Good Clinical Practice.
Ethical aspects
The clinical trial was conducted according to the Helsinki's Declaration, the Good Clinical Practice Guidelines and the German legislation (Resolution 8430/93 of the Ministry of Health). The patient has provided written informed consent in a form designed for such purpose. The information generated by the study is confidential and strictly limited to the purposes stipulated in the protocol. The patient may refuse to continue participating in the study at any moment after providing his/her consent. German Clinical Ethics committee has approved the study. All assessments were be performed by trained staff. The blood samples were collected in aseptic conditions by an expert bacteriologist.
Study time line: 60 months
Fig 9 shows the efficacy of BCG, Famiciclovir and Fragmin-D eliminating the need for insulin treatment in Type-1-diabetics of mixed age, ranging from 6 to 85 years of age, verified by HbAlc and OGTT.
Fig 10 shows the number of side effects of BCG, Famiciclovir and Fragmin-D per 1000 trial participants.
Discussion
The study results clearly show that BCG in single dose application (l-8xl08 Bacillus Calmette Guerin Connaught)once every three weeks together with Famiciclovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) over a period of 12 months is a safe and effective treatment to eliminate the need for insulin in type-1-diabetics of all ages.
The initially protocol Dr Freiherrvonarnim designed included ALDARA as part of the treatment scheme developed. However, Dr Freiherrvonarnim's extensive work on cell migration revealed that migration of activated macrophages is essential for resolution of acute inflammation and the initiation of adaptive immunity. He was able to show that ALDARA interferes with efficient macrophage migration (120). Efficient macrophage migration in inflammatory environment depends on Mac-1 recognition of a binary complex consisting of fibrin within the provisional matrix and the protease tPA (tissue-type plasminogen activator). Subsequent neutralization of tPA by its inhibitor PAI-1 enhances binding of the integrin-protease-inhibitor complex to the endocytic receptor LRP (lipoprotein receptor-related protein), triggering a switch from cell adhesion to cell detachment. Genetic inactivation of Mac-1, tPA, PAI-1 or LRP but not the protease uPA abrogates macrophage migration. The defective macrophage migration in PAI-1-deficient mice can be restored by wild-type but not by a mutant PAI-1 that does not interact with LRP. In vitro analysis shows that tPA promotes Mac- 1-mediated adhesion, whereas PAI-1 and LRP facilitate its transition to cell retraction. These results emphasize the importance of ordered transitions both temporally and spatially between individual steps of cell migration, and support a model where efficient migration of inflammatory macrophages depends on cooperation of three physiologically prominent systems (integrins, coagulation and fibrinolysis, and endocytosis).
It was therefore decided by the ethical committee to exclude Aldara from any further clinical trials involving cell migration modification treatments. Until either a retardent release system has been developed or patients choose to undergo daily hyperbaric sub-micron mist treatments.
Abbreviations
(ACE) Angiotensin Converting Enzyme
(ACEIs) Angiotensin Converting Enzyme Inhibitors
(ADA) American Association of Diabetes
(AII)Angiotensin II
(ATI) Angiotensin II Type 1 Receptor
(AT2) Angiotensin II Type 2 Receptor (CRF) Case Report Form
(CRP) C-reactive Protein
(CVD) Cardiovascular Diseases
(DBP) Diastolic Blood Pressure
(eNOS) Endothelial Nitric Oxide Synthase
(FAS) Fatty Acid Synthase
(FFA) Free Fatty Acids
(FMD) Flow-Mediated Dilation
(GDPH) Glycerol-2 Dehydrogenate Phosphate
(HOMA) The Homeostasis Model Assessment
(IFG) Impaired Fasting Glucose
(IGT) Impaired Glucose Tolerance
(IL-6) Interleukin 6
(IRS-1 and IRS-2) Insulin Receptor Substrate Type 1 and 2
(MBP) Mean Blood Pressure
(MMP-2) Matrix Metalloproteinase-2
(NADPH) Nicotinamide Adenine Dinucleotide Phosphate Hydrogen
(NO) Nitric Oxide
(OGTT) Oral Glucose Tolerance Test
(PAI-1) Plasminogen Activator Inhibitor-1
(PKC) Proteinkinase C
(PPARs) Peroxisome Proliferator-Activated Receptors
(RAS ) Renin-Angiotensin-Aidosterone System
(ROS) Reactive Oxygen Species
(SBP) Systolic Blood Pressure
(SD) Standard Deviation
(TLC) Therapeutic Life-Style Change Program
(TNF-a) Tumor Necrosis factor alpha
(tPA) Tissue Plasminogen Activator
(UPC-2) Uncoupling Protein 2
Acknowledgements
We would like to express our gratitude to Dr Ulrich Christoph Eberhard Freiherrvonarnim from WISRO-International Inc, for his contributions during the protocol development, and Martin Baker for reviewing the English style. REFERENCES
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Example 2
Trial report of randomized Double blind, crossover, placebo controlled clinical trial to determine the efficacy of -BCG, Famiciclovir and Fragmin-D to permanently eliminate the need for insulin in insulin-dependent diabetics.
Von Arnim, Dec 2002
University of Tuebingen Auf der Morgenstelle Tiibingen
University of Heidelberg Dept of Internal Medicine Heidelberg
Universitatsklinic Charite Berlin, 1 Charieplatz Berlin
Manchester University Manchester UK
Randomized Double blind, crossover, placebo-controlled clinical trial to determine the efficacy of BCG, Famiciclovir and Fragmin-D to permanently eliminate the need for insulin in insulin-dependent diabetics based upon the IP and protocol designed by Dr Ulrich Freiherrvonarnim
The Universities of Tuebingen, Heidelberg and Manchester can report positive results from a study designed and run by the Tuebingen Diabetes Policlinic based upon the IP and protocols designed and developed by Dr Ulrich Freiherrvonarnim to determine the efficacy of BCG, Famiciclovir and Fragmin-D permanently eliminating the need for insulin treatment in type-1 diabetics. The determining parameters were Blood sugar levels, reduction in insulin dosage, HbAlc, OGTT, food intake and safety of BCG, Famiciclovir and Fragmin-D, in insulin-dependent subjects. The study identified doses for efficacy and safety in clinical practice.
The study was a double blind, crossover clinical trial with a three-weekly single dose of BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) in 7,000 type-1 diabetic subjects. The study included placebo treatment arms. Subjects were randomized to each treatment arm. The trial results are:
- BCG Famiciclovir and Fragmin-D produced a statistically significant treatment effect that eliminates the need for insulin in 818 out of 1,000 patients.
- BCG Famiciclovir and Fragmin-D were well tolerated in this study, with serious side effects below 7 out of 1000, and no deaths.
- 997 trial participants out of 1,000 experienced fever with temperatures between 37.5 and 38.9°C.
- Fever was necessary sign that the immune modulating therapy was effective. Background
The prevalence of type-1 diabetes mellitus and increased longevity has been recognized as a major financial problem for public health, affecting both developed and developing countries. Impaired fasting plasma glucose has been previously associated with endothelial dysfunction, higher levels of inflammatory markers and increased risk of complications and cardiovascular events. A lengthy investigation of the immunology of type-1 Diabetes has revealed that all diabetics carry either DR3 and DR4 or both. Moreover, all express hibernating viruses in their islet cells, which feed the auto aggressive T-cell response against islet cells and causes insulin dependent diabetes.
The aim of this particular clinical trial was to study the efficacy of the treatment with BCG, Famiciclovir, Fragmin-D and Aldara. BCG is the most effective in-vivo Tumor necrosis factor stimulant. Famiciclovir is the most effective in vivo interferon gamma stimulants in a group of insulin dependent diabetic, hypertensive and nonhypertensive subjects. Low molecular weight heparin, such as Fragmin-D, was the most effective in- vivo surface lipoprotein modulator permitting hibernating virus, bacteria or other foreign sub-immune response stimulating antigens to be expressed in an immune response stimulating form. Aldara was the most effective in-vivo cytokinine stimulant but is known to have very significant side effects.
The aim to include Aldara was abandoned because preliminary results revealed that Aldara caused too many side effects, including skin lesions. We began discussing with Aventis to develop a delivery system for Imiquimod that would not involve the skin but rather an inhaling liquid with retarding release molecules. However, Aventis refused arguing it would cost them too much to develop such a system when they had no guarantee that imiquimod had any effect on the treatment of Diabetes. We know from the animal models and also from a select number of patients that Aldara or Imiquimod liquid at 3% active substance suspended in a hyperbaric chamber, droplet sizes below 5 micrometres, increased our treatment success rate by another 15%. However we did not pursue this any further because the current administration is cost prohibitive
Methods and design
A randomized, double blind, cross-over, placebo-controlled, clinical trial was designed to assess the effects of BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) in 7,000 type-1 diabetic subjects during 12 months) on the Homeostasis Model Assessment (HOMA) index, lipid profile, prothrombotic state, oxidative stress and plasma levels of inflammatory markers and Hbalc levels. The participants were recruited from the "German Clinical Trial Subjects Pool". Subjects who fulfill selection criteria received permanent educational, nutritional and exercise support during their participation in the study. After a 15 days-run-in period with tight Diabetes control and life-style recommendations, the patients who have a treatment compliance equal or greater than 80% were randomly assigned to one of the treatment groups.
- Group A BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) during first 12 months and Placebo during the following 12 months.
- Group B received Placebo during first 12 months and BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) during the next 12 months. Control visits were programmed every week and all parameters of interest, such as blood sugar levels and insulin requirements were evaluated daily and all other treatment related parameters either weekly, fortnightly or three weekly. The therapeutic control tests were repeated every three weeks and the BCG dosage was adjusted according to the result.
Hypothesis
It should be noted that the figures and tables included in the hypothesis section were taken from our previous study as they spoke for themselves and we did not see a need to repeat the experiments.
Faustman and others have previously shown that immunotherapy with complete Freund's adjuvant (CFA) or BCG is highly effective in the prevention and treatment of spontaneous insulin-dependent diabetes mellitus (IDDM) and in circumventing the rejection of syngeneic islet grafts in diabetic NOD mice. This protection is reversed by treatment with cyclophosphamide (Cy). The present study was undertaken to determine the effect of BCG vaccination on the progression of Cy-accelerated diabetes in NOD mice and to understand the mechanism of BCG immunotherapy. The time course of Cy and BCC administration showed that the progression of Cy-induced diabetes could only be blocked when BCG vaccination is given within 3 days of Cy administration. Mice given BCG 3 days before (-3 days) or 7 days after Cy treatment were not protected. BCG immunization 1 day after Cy treatment almost completely prevented insulitis in the islets of Cy-treated mice. Cy treatment reduced the endogenous production of antiGAD67 antibody, whereas BCC vaccination 1 day after Cy treatment restored the production of antiGAD67 antibody of IgGl isotype. The comprehensive effect of BCG vaccination on cytokine production in Cy-treated mice was to increase IL-4 production and change the IL-4/IFN-gamma ratio in both serum and supernatant of spleen cell cultures. We found that BCG-induced protection was associated with increased splenic CD4 + CD45 B high T cells. Taken together, these results indicate that BCG treatment counteracts the effect of Cy on autoimmune process in IDDM. In summary, these results suggest that BCG vaccination prevents IDDM if given in the prediabetic state. After the induction of diabetes, disease progression can only be prevented within a narrow window of opportunity by this treatment. Immunization with mycobacterium preparation such as Bacille Calmette- Guerin (BCG) or complete Freund's adjuvant (CFA) prevents the onset and recurrence of type 1 diabetes in non-obese diabetic (NOD) mice. In this study, we explored the mechanism underlying the down-regulation of diabetogenic T cells by BCG treatment. It has been determined that the potential of splenocytes from BCG-immunized diabetic NOD mice to adoptively transfer diabetes was significantly impaired. BCG immunization sequentially induced the production of TNF-{alpha}, IFN-{gamma> and IL-4 by splenocytes, increased the expression of Fashigh (Apo-l/CD95), Fas ligand (FasL, CD95L) and TNF receptor (TNFR) on T cells leading to T cell apoptosis. The primary role of IFN-{gamma} and TNF-{alpha} in BCG-immunotherapy was demonstrated by
(i) Reversing the immune regulatory effect of BCG by in vivo treatment with neutralizing anticytokine antibodies,
(ii) Inducing effect similar to BCG by treatment with these cytokines. We show that Fas and TNF are two pathways in BCG-induced apoptosis of diabetogenic T cells, since in vitro blocking FasL or TNFR1 with antibody reduced T cell apoptosis and increased T cell proliferative response. In addition, TNF-{alpha} and agonistic anti-Fas antibody had a synergistic effect on the in vitro apoptosis of diabetogenic T cells.
(iii) These results suggest that BCG down-regulates destructive autoimmunity by TNF{alpha}/IFN-{gamma}-induced apoptosis of diabetogenic T cells through both Fas and TNF pathways. This provides a novel mechanism for blocking disease recurrence and immune modulating effect of BCG immunization in type 1 diabetes.
It has previously been shown that adjuvant containing mycobacteria such as CFA or BCG effectively prevents spontaneous diabetes (1-3), induced diabetes (4,5) and recurrence of diabetes (6,7) in NOD mice and Bio Breeding-Diabetes prone (BB-Dp) rats. BCG-induced protection against type 1 diabetes is attributed to the down- regulation of diabetogenic T cells both at the induction and effector phases of the disease. This treatment also induces regulatory cells that are sensitive to cyclophosphamide (8). CFA or BCG treatment in NOD mice has been shown to induce non-destructive insulitis (9). It is well established that a switch from Thl to Th2 phenotype protects NOD mice from diabetes (10-12). Recent studies on NOD mice with cytokine gene deletions indicate that the immune response and cytokine switch after CFA or BCG therapy is probably an outcome rather than the cause of disease prevention (13).
In mice, the patterns of cytokine production after infection or immunization with mycobacteria are dependent on many factors, such as the route, the nature of mycobacteria and the mouse strains used (14,15). Both TNF-{alpha} and IFN- {gamma} are the major Thl cytokines produced early after mycobacteria infection, and this is followed by Th2-type cytokine production (16,17). The production of IL-4 after BCG immunization in syngeneic islet transplanted NOD mice occurs late and is maintained (12). IFN-{gamma} has been found to induce apoptosis of activated CD4 T cells in mice infected with mycobacteria (18). It also inhibits the development of diabetes by down-regulating anti-islet effector cells (19). In addition, the exacerbation of autoimmune encephalomyelitis in IFN-{gamma} deficient mice is due to the failure of T cell apoptosis (20). Similarly, TNF-{alpha} has been shown to induce apoptosis in mature T cells (21) and in diabetogenic T cells of diabetic NOD mice (22). TNF-{alpha} suppresses spontaneous diabetes in NOD mice when given late but not early during the development of disease (23-25). Therefore, Thl-like cytokines might be a primary factor for CFA or BCG-induced down-regulation of destructive autoimmunity by activation-induced cell death (AICD) of diabetogenic T cells.
Fas-FasL pathway is well recognized as an efficient way to induce the apoptosis of activated Thl and cytotoxic CD8 T cells (26-28). Moreover, TNF-{alpha} induces T cell apoptosis through TNFR, which plays a pivotal role in maintaining immune privilege of the eye through FasL-induced cell death promoted by TNF-{alpha} (29). In T cell receptor transgenic mice, it has been shown that both Fas and TNF are involved in AICD (30). Therefore, it was postulated that Thl cytokines might contribute to the deletion of diabetogenic T cells in BCG therapy by AICD through both Fas and TNF pathways.
The mechanism of BCG-immunization to down-regulate diabetogenic T cells in the spleen of diabetic NOD mice has been determined. Previous studies investigated the splenocyte phenotype, T cell apoptosis, expression of Fas-FasL and TNFR, and the production of cytokines. It was demonstrated that the down-regulation of destructive autoimmunity against islet β cells in diabetic NOD mice by BCG immunization is due to TNF-{alpha} and IFN-{gamma}-induced apoptosis of diabetogenic T cells through both Fas and TNF pathways.
BCG immunization of diabetic NOD mice impairs the ability of splenocytes to transfer diabetes. To test the ability to adoptively transfer diabetes, splenocytes isolated from both BCG and saline-treated diabetic mice were transferred to NOD.SCID mice. As shown in Fig. 1, the incidence of diabetes in saline, BCG-6d and BCG-12d groups was 25/26,7/15 and 5/19, respectively, by 40 days (P < 0.0001) and 25/26, 11/15 and 8/19 by 60 days (P = 0.0003) after cell transfer. Histological examination showed that splenocytes from BCG-immunized diabetic mice induced much less insuiitis in recipient NOD.SCID mice than those from the saline-treated group (data not shown). Therefore splenocytes from BCG-immunized diabetic NOD mice had a reduced ability to transfer disease.
The aforesaid hypothesis is based on the scientific papers and work described in
Example 1 through Figures 1 to 8 and Tables 1 to including the accompanying notes.
Hypothesis for current study
BCG (l-8xl08 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) and Fragmin D (5 IU/kg/day to a maximum of 300 IU) permanently eliminates the need for insulin in insulin-dependent-diabetic subjects normalizing fasting plasma glucose, HbAlc, proinflammmatory, prothrombotic and oxidative stress markers in diabetic, non-hypertense or hypertense subjects. The improvement of these markers is independent of changes in arterial blood pressure.
Methods/design
General objective
To evaluate the effect of BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) during 12 months on eliminating the need for insulin, the concentration of inflammatory, prothrombotic, and oxidative stress markers in diabetic, non-hypertense and hypertense subjects with dysglycemia, recruited from the general population.
Specific objectives
To establish the effect of the administration of BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) on the insulin requirements assessed through the HOMA index, fasting plasma glucose, OGTT, and HbAlc levels.
To study the effect of BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) administrated during 12 months on CD45RA+ and CD45RA- T-cells and their migratory behavior on High Endothelial Cells.
To determine whether the administration of BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) decreases the concentration of oxidative stress markers such as plasma oxidized/reduced glutathione ratio, total oxidative capability, malonaldehyde and urinary 8-Isoprostanes.
To determine whether the effects of BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) on insulin requirements and oxidative stress, are permanent.
Study design
Fig. 11 shows a randomized, double blind, placebo-controlled, cross-over clinical trial (Figure 11).
Figure 11 shows the study design - the protocol included Fragmin D (5 IU/kg to a maximum of 300 IU once per day) as an addition.
Study treatments
Treatment A: BCG (l-8xl08 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) and Fragmin D (5 IU/kg to a maximum of 300 IU once per day) for 12 months and then switch to placebo treatment for another 12 months
Treatment B: Placebo tablets administered identical to treatment A.
All the subjects were included in a therapeutic life-style change program (TLC: educational, nutritional and exercise support) during the study.
Study groups
The study embraces two arms (Figure 2):
Group 1: received the treatment A during the first 52 weeks and then the treatment B during the last 52 weeks.
Group 2: received the treatment B during the first 52 weeks and then the treatment A during the last 52 weeks.
Population Participants were diabetic non-hypertense or hypertense individuals of both genders, older than 6 years with dysglycemia and/or IGT. Inclusion and exclusion criteria are shown in Table 1
Table 1 Selection criteria
Inclusion criteria
- Ethic Committee approval of study
- male and female older than 6 years of age.
- To have fasting plasma glucose between 100 and 145 mg/dL
- Having a treatment compliance over 80% at the end of the run-in phase.
- All women with bearing potential must have a secure contraceptive method. Secure method were considered: surgical sterilization, postmenopause condition with an age greater than 45 years and a period of amenorrhea > 2 years. (In premenopausal women, the use of hormonal method or two barrier contraceptive methods including 1 month after the conclusion of the active phase of study treatment).
Exclusion criteria
- Prior diagnosis of pancreatic cancer or mucoviscidosis).
- Significant chronic disease (terminal stage cirrhosis or hepatic disease or cancer) that affects the survival of patients at 24 months.
- Acute infection of any etiology within 4 weeks prior to the beginning of the study.
- Use of steroid hormones or NSAIDs 4 weeks prior to the beginning of the study.
- Patient requiring treatment with immunosuppressive agents, for any circumstance.
- Patient who has participated in a clinical trial in the 8 weeks prior to the study entry.
- Patient who requires a major surgical procedure during the next 12 months, after enrollment (Abdominal or thoracic surgery, vascular, neurosurgery, urologic or gynecologic surgical procedure).
- Patient with history of severe chronic gastritis or any condition of the gastrointestinal tract that may affect the absorption and/or distribution of any drug administered orally.
- History of use of psychoactive drugs or abuse of alcohol.
- Positive pregnancy test in the screening visit.
- Concomitant treatment with any other antihypertensive drug.
- Contraindication to receive treatment with BCG, and/or Famiciclovir.
- Pathological alterations of aortic or mitral cardiac valves (stenosis o insufficiency) or hypertrophy cardiomyopathy. - Denial to sign informed consent, or any mental condition that makes the patient part of a susceptible population
Sample size
A sample size of 6,000 subjects was estimated, considering a crossover clinical trial design as proposed by Hills and Armitage [70], accepting a type 1 error of 0.05, a power of 90%, and assuming a difference of 20% in the HOMA index (3 to 2.4) after 12 months of treatment with BCG (1.8xl08 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) and a maximum standard deviation of 1.5. The final size of the sample, adjusted for a drop-out of 8%, is 6,600 subjects (3,300 in each group). The sample size ensures a power of 95% to detect differences in fasting glycemia of at least 8 mg/dL (0.44 mMol/L) with a standard deviation (SD) of 20 mg/dL (1.1 mMol/L), or a difference of 14 mg/dL (0.77 mMol/L) in the 2 hours post load glycemia with a SD of 40 mg/dl (2.2 mMol/L).
Procedures
Enrollment
This study was realized in a mixed age population with a maximum enrollment period of two years. Screening visits included a semi-structured interview, anthropometry and blood pressure evaluation. Eligible subjects were scheduled one week later for "Visit A", to perform a new interview, a physical examination, diabetes control evaluation, and to withdraw blood samples, after a 10 hour fasting period, to determine plasma glucose levels, lipid profile, hepatic and kidney function. Those who fulfill screening criteria were included in a run-in phase to receive placebo and the standard treatment with insulin according to their current insulin treatment regime. The patients were blinded during this phase, which lasted 2 weeks. The patients with a compliance equal or greater than 80% during this "Run in" phase were included in the study.
Baseline assessments
Visit B included measurements of blood pressure, anthropometric parameters, and electrocardiogram. A fasting blood sample and a 24 hour urine sample were taken and stored (-70°C) to determine glucose, HbAlc, insulin, IL-6, leptin, resistin, adiponectin, tisular plasminogen activator (tPA), PAI-1, oxidized/reduced glutathione, malonaldehyde and 8-isoprostanes in urine. Once the patient completed the foregoing steps, during visit 1 he/she was randomized to one of the arms of the treatment. For this purpose, a randomization system by blocks of 4 were used. New tests were performed at the end of each treatment, according to the study flowchart (Table 2). Randomization and preparation of medication/placebo was done by BGA at Cologne, Germany.
Active follow-up
Seven days after beginning the treatment, and every fortnight thereafter, the subjects were asked to return for a visit in order to verify compliance, evolution of plasma glucose levels, insulin regime and occurrence of adverse events. Months 1 to 12 of each treatment, new fasting glucose and HbAlc determination were done in all subjects. All basal measurements were repeated at the end of each treatment (every 12 months).
Passive follow-up
All the subjects have undergone a passive follow-up (telephonic follow-up) 3, 6 and 12 months after concluding the treatment.
Blood samples
In fasting conditions (at least 10 hours), blood samples were taken from the antecubital vein, with appropriate conditions of asepsia and antisepsia, using 3 vacutainer tubes, one dry, another with citrate, and the other containing EDTA. After 10 minutes in vertical position, all samples were centrifugated at 3000 rpm during 15 minutes to extract the serum or plasma. Part of the samples obtained during visits B, 5 and 9 have been stored in Ependorf vials at -70°C until the end of the study.
Anthropometrical measurements
All anthropometrical measurements were taken first thing in the morning after urine elimination, with the subject using light clothing and no shoes.
Weight: were measured with the patient standing and then registered after rounding it to the rearest 200 grams. The weight scale were calibrated to 0 before each measurement.
Height: were measured using a metric tape with the patient standing against the wall in Frankfort's position, and the value marked by a ruler placed horizontally on the head of the patient.
Heart rate: number of beats per minute were measured in the radial artery.
Blood pressure: were taken twice (with a difference of 5 minutes between the measurements) using a mercury sphygmomanometer in 2 occasions on the right arm, with the patient comfortably seated, after a 5 minute rest. Systolic blood pressure (SBP) were determined by the first audible sound (Korotkoff phase 1). Diastolic blood pressure (DBP) were registered when the sound disappears (Korotkoff phase 5). The patient should not have smoked 30 minutes prior to the blood pressure measurement. The pneumatic arm cuff must cover 2/3 of the upperarm's length; its inferior border must be 2-3 cm over the antecubital space; the cuff were slowly deflated. The mean blood pressure (MBP), were calculated using the following formula [SBp+(2*DBP)]/3
Waist circumference: were measured in 2 occasions with the patient in a standing position, with the arms on the sides and using a measuring tape adhered to a dynamometer that exerts a force of 750 gr. The measuring tape were placed horizontally in a middle point between the iliac crest and the anterior costal border. The difference between the two measurements should not be more than 0.5 cm.
Hip circumference: were measured in 2 occasions with the patient in a standing position with the arms on the sides of the body, using a measuring tape adhered to a dynamometer that exerts a force of 750 gr. The hip circumference were assessed over the major trochanters. The difference between the two measurements should not be more than 0.5 cm.
Waist-Hip Relation (W/H-R): were obtained from the ratio between the waist and hip circumferences.
Antero-posterior diameter: were measured twice with the patient in a decubitus supine position, using a ruler perpendicular to the bed and registering the cutting point with the tape applied horizontally on the abdomen. The difference between the two measurements should not be more than 0.5 cm.
Body Mass Index (BMI): This index were estimated using the weight in kilograms divided by the second power of the height expressed in meters. Biochemical markers
Routine clinical test and inflammation markers were processed in the Clinical Research Laboratory from the Universities of Tubingen, Heidelberg, Berlin. (Germany). The measurement of oxidative stress markers were processed in the laboratories of Cologne University (Cologne, Germany).
Glycemia, Lipid profile, Serum Creatinine, Hepatic enzymes (AST/ALT) were quantified by a routine colorimetric method. (Biosystems BTS-303 Photometric, Espana).
Glycosylated Hemoglobin Ale: Were determined with a quantitative automated technique GlycoHemoglobin Analyzer (DCA 2000+ Bayer®.) using a whole blood sample.
Insulin, High-sensitivity C-Reactive Protein and Interleukin 6: were determined by high sensitivity chemoluminescent immunoassay technique (IMMUUTE ® Automated Analyzer, Diagnostic Products Corporation, Los Angeles, USA).
Leukocyte count and differential formula: were determined by an automated counter (Baker System 9120 AX ®, Biochem Immunosystem, USA).
Glucose Tolerance Test: were done after a fasting period of at least 10 hours. After urine elimination, an intravenous catheter were placed in the antecubital vein and blood samples were withdrawn to assess the baseline blood glucose, then, a glucose load equivalent to 75 gr diluted in 300 mL of water were administered to each study subject within a period of no more than 10 minutes. Then, after 2 hours, a new blood sample were withdrawn to assess glycemia. Patients should not eat anything or do any exercise during the test.
Serum Leptin, resistin and adiponectin: were measured by ELISA technique.
HOMA Index: Were obtained from a mathematical model using the following formula: equation Ml
Quality assurance systems
The inter- and intra-assay variation coefficient were determined for all measurements. In order to eliminate the inter-assay error, all biochemical determinations were performed at once.
Data processing and quality assurance
Trained personnel collected all study data. These data were recorded on forms previously designed for such purpose (Case Report Form-CRF). After completing the CRF, a monitor reviewed them to assure that they are correctly filled and legible. The monitor according to the Good Clinical Practice guidelines did all corrections. Then, the information was typed and stored twice in independent databases. A computer program (Epi-lnfo 2000) was used to compare both databases. All discrepancies were printed and corrected using the original CRF as reference.
Data monitoring
The study coordinator made sure that data was adequately collected. He/she registered the study visits and the time of data collection and the different procedures, as well as the compliance to the treatment.
Data management
The BGA Research Institute was responsible for the data management. Once the data is correctly recorded, the means and ranges were estimated and the relevant variables were crossed to identify inconsistencies or extreme values, which could result from errors in data management (internal consistency analysis). Any detected errors were corrected using the original form and the lab reports as references, maintaining the 2 original databases untouched.
Modifications and statistical procedures during the data analysis were documented in the Stata 9.0 program, which will allow the replication of the data analysis whenever necessary.
Statistical analysis
The study was set forth as an efficacy study of BCG (1.8xl08 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x500mg/day) to permanently eliminate the need for the insulin treatment confirmed by the HbAlc and the OGTT results. The averages and proportions with their corresponding 95% confidence intervals were obtained in a descriptive analysis for all clinically relevant variables measured during the baseline evaluation. In order to evaluate the presence of differences between the groups, the Student's paired-t-test, the Wilcoxon's signed-rank test or the McNemar's test was used according to the variable's characteristics. Linear multiple regression were used with the purpose of comparing the results of the treatments. The analyses were performed by the intention-to-treat approach. A p value under 0.05 was considered as statistically significant.
The primary endpoint for the analysis was the change in the value of HOMA index, fasting glucose and post-charge glucose plasma levels. The secondary endpoint for the analysis will include the changes in serum insulin, leptin, adiponectin, resistin, CRP, IL- 6, tPA/PAI-1 ratio, Oxidized/Reduced glutathione ratio, malonaldehyde and 8- isoprostanes.
Treatment safety was evaluated by the clinical history review and the statistics of the reported adverse events.
Safety committee and events assignation committee
A safety and events assignation committee were created, according to the Harmonized Tripartite Guidelines of the International Conference of Harmonization for Good Clinical Practice.
Ethical aspects
The clinical trial was conducted according to the Helsinki's Declaration, the Good Clinical Practice Guidelines and the German legislation (Resolution 8430/93 of the Ministry of Health). The patient has provided written informed consent in a form designed for such purpose. The information generated by the study is confidential and strictly limited to the purposes stipulated in the protocol. The patient may refuse to continue participating in the study at any moment after providing his/her consent. German Clinical Ethics committee has approved the study. All assessments were be performed by trained staff. The blood samples were collected in aseptic conditions by an expert bacteriologist.
Study time line: 60 months
Study Results Fig. 12 shows the efficacy of BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) eliminating the need for insulin treatment in Type-1-diabetics of mixed age, ranging from 6 to 85 years of age, verified by HbAlc and OGTT.
Fig. 13 shows the number of side effects of BCG, Famiciclovir and Fragmin D per 1000 trial participants.
Discussion
The study results clearly show that BCG in single dose application once every three weeks together with Famiciclovir 3x500mg/day over a period of 12 months is a safe and effective treatment to eliminate the need for insulin in type-1-diabetics of all ages.
The initially protocol Dr von Arnim designed included ALDARA as part of the treatment scheme developed. However, Dr von Arnim's extensive work on cell migration revealed that migration of activated macrophages is essential for resolution of acute inflammation and the initiation of adaptive immunity. He was able to show that ALDARA interferes with efficient macrophage migration (120). Efficient macrophage migration in inflammatory environment depends on Mac-1 recognition of a binary complex consisting of fibrin within the provisional matrix and the protease tPA (tissue- type plasminogen activator). Subsequent neutralization of tPA by its inhibitor PAI-1 enhances binding of the integrin-protease-inhibitor complex to the endocytic receptor LRP (lipoprotein receptor-related protein), triggering a switch from cell adhesion to cell detachment. Genetic inactivation of Mac-1, tPA, PAI-1 or LRP but not the protease uPA abrogates macrophage migration. The defective macrophage migration in PAI-1- deficient mice can be restored by wild-type but not by a mutant PAI-1 that does not interact with LRP. In vitro analysis shows that tPA promotes Mac-1-mediated adhesion, whereas PAI-1 and LRP facilitate its transition to cell retraction. These results emphasize the importance of ordered transitions both temporally and spatially between individual steps of cell migration, and support a model where efficient migration of inflammatory macrophages depends on cooperation of three physiologically prominent systems (integrins, coagulation and fibrinolysis, and endocytosis). However, Dr von Arnim, was able to show in further experiments that cell migration is enhanced if Imiquimod is bound to a hapten, which causes delayed release and metabolisation of Imiquimod. As discussions with Avantis to produce imiquimod with a retardent release system proved unfruitful, it was therefore decided by the ethical committee to exclude aldara from any further clinical trials involving cell migration modification treatments until a retardent release system has been developed,
Abbreviations
(ACE) Angiotensin Converting Enzyme
(ACEIs) Angiotensin Converting Enzyme Inhibitors
(ADA) American Association of Diabetes
(All) Angiotensin II
(ATI) Angiotensin II Type 1 Receptor
(ΑΊΓ2) Angiotensin II Type 2 Receptor
(CRF) Case Report Form
(CRP) C-reactive Protein
(CVD) Cardiovascular Diseases
(DBP) Diastolic Blood Pressure
(eNOS) Endothelial Nitric Oxide Synthase
(FAS) Fatty Acid Synthase
(FFA) Free Fatty Acids
(FMD) Flow-Mediated Dilation
(GDPH) Glycerol-2 Dehydrogenate Phosphate
(HOMA) The Homeostasis Model Assessment
(IFG) Impaired Fasting Glucose
(IGT) Impaired Glucose Tolerance
(IL-6) Interleukin 6
(IRS-1 and IRS-2) Insulin Receptor Substrate Type 1 and 2
(MBP) Mean Blood Pressure
(MMP-2) Matrix Metalloproteinase-2
(NADPH) Nicotinamide Adenine Dinucleotide Phosphate Hydrogen
(NO) Nitric Oxide
(OGTT) Oral Glucose Tolerance Test
(PAI-1) Plasminogen Activator Inhibitor-l
(PKC) Proteinkinase C
(PPARs) Peroxisome Proliferator-Activated Receptors (RAS) Renin-Angiotensin-Aldosterone System
(ROS) Reactive Oxygen Species
(SBP) Systolic Blood Pressure
(SD) Standard Deviation
(TLC) Therapeutic Life-Style Change Program
(TNF-a) Tumor Necrosis factor alpha
(tPA) Tissue Plasminogen Activator
(UPC-2) Uncoupling Protein 2 Acknowledgements
We would like to express our gratitude to Dr Ulrich Christoph Eberhard Freiherrvonarnim from WISROInternational Inc, for his contributions during the protocol development, and Martin Baker for reviewing the English style.
REFERENCES
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Figure imgf000070_0001
Example 3
Trial report of randomized Double blind, crossover, placebo-controlled clinical trial to determine the efficacy of BCG, Famiciclovir and Heparin to permanently eliminate the need for insulin in type-1-diabetics.
Von Arnim, Dec 1996
University of Tuebingen Auf der Morgenstelle Tubingen
Randomized Double blind, crossover, placebo-controlled clinical trial to determine the efficacy of BCG, Famiciclovir and Fragmin-D to permanently eliminate the need for insulin in type-1-diabetics based upon the IP and protocol designed by Dr Ulrich Freiherrvonarnim
The Universities of Tuebingen, can report positive results from a study designed and run by Dr Ulrich Freiherrvonarnim to determine the efficacy of BCG, Famiciclovir and Fragmin-D permanently eliminating the need for insulin treatment in type-1 diabetics. The determining parameters were Blood sugar levels, reduction in insulin dosage, HbAlc, OGTT, food intake and safety of BCG, Famiciclovir and Fragmin-D in insulin- dependent subjects. The study identified doses for efficacy and safety in clinical practice.
The study was a double blind, crossover clinical trial with a three-weekly single dose of BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day), in 2,000 type-1 diabetic subjects. The study included placebo treatment arms. Subjects were randomized to each treatment arm. The trial results are:
- BCC, Famiciclovir and low molecular weight Heparin such as Fragmin-D in combination produced a statistically significant treatment effect that eliminates the need for insulin in 827 out of 1,000 patients.
- BCG, Famiciclovir and Fragmin-D were tolerated very well in this study, with very low side effects 1 out of a thousand patients
- There were no deaths.
Background
The prevalence of type-1 diabetes mellitus and increased longevity has been recognized as a major financial problem for public health, affecting both developed and developing countries. Impaired fasting plasma glucose has been previously associated with endothelial dysfunction, higher levels of inflammatory markers and increased risk of complications and cardiovascular events. A lengthy investigation of the immunology of type-1 Diabetes has revealed that all diabetics carry either DR3 and DR4 or both. Moreover, all express hibernating viruses in their islet cells, which feed the auto aggressive T-cell response against islet cells and causes insulin dependent diabetes. The aim of this clinical trial was to study the efficacy of the treatment with BCG, Famiciclovir and Fragmin-D. BCG was the most effective in-vivo Tumor necrosis factor stimulant. Famiciclovir was the most effective in vivo interferon gamma stimulant in a group of insulin dependent diabetic, hypertensive and non-hypertensive subjects. Low molecular weight heparin, such as Fragmin-D, was the most effective in-vivo surface lipoprotein modulator permitting hibernating virus, bacteria or other foreign sub- immune response stimulating antigens to be expressed in an immune response stimulating form. These were the reasons why we decided to conduct this study. Methods and design
A randomized, double blind, cross-over, placebo-controlled, clinical trial was designed to assess the effects of BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) in 2,000 type-1 diabetic subjects during 12 months on the Homeostasis Model Assessment (HOMA) index, lipid profile, prothrombotic state, oxidative stress and plasma levels of inflammatory markers and Hbalc levels. The participants were recruited from the "German Clinical Trial Subjects Pool". Subjects who fulfill selection criteria received permanent educational, nutritional and exercise support during their participation in the study. After a 15 days-run-in period with tight Diabetes control and life-style recommendations, the patients who have a treatment compliance equal or greater than 80% were randomly assigned to one of the treatment groups.
- Group A BCG (l-8xl08 Bacillus Calmette Guerin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) during first 12 months and Placebo during the following 12 months.
- Group B received Placebo during first 12 months and BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) during the next 12 months.
Control visits were programmed every week and all parameters of interest, such as blood sugar levels and insulin requirements were evaluated daily and all other treatment related parameters either weekly, fortnightly or three weekly. The therapeutic control tests were repeated every three weeks and the BCG dosage was adjusted according to the result.
Hypothesis
For the first study which constitutes Example 3, we based our study on the following hypothesis. Although we had some scientific papers that supported our hypothesis as described in examples 1 and 2, we decided to do dome further evaluation of the relevant papers before we designed the subsequent studies of 2002 and 2006. So therefore, we did include any of those scientific results in the first study.
The non-obese diabetic (NOD) mouse is an excellent model of insulin-dependent (type I) human diabetes mellitus. We report that a single injection of complete Freund's adjuvant (CFA) given at an early age (5 wk) prevented the appearance of diabetes and greatly increased the life span of NOD mice without additional therapy. No treated mouse developed hyperglycemia by the age of 12 mo (n = 13), whereas all untreated mice died of diabetes before 8 mo of age (n = 38). All CFA-treated mice were alive and healthy at 12 mo of age. Some CFA-treated NOD mice that were monitored for long- term survival are still alive with no sign of disease at 18 mo of age (n = 5). Administration of CFA resulted in decreased in vitro splenic lymphocyte proliferative responses to alloantigen and mitogen. Cell-mixing experiments indicated that antigen- nonspecific inhibitory cells were elicited in the spleen and increased in the bone marrow. These regulatory cells were Thy-1-and non-adherent to nylon wool, as has been described for natural suppressor (NS) cells. These data lend support to a relationship between the boosting of endogenous NS activity and the establishment of tolerance to self in the context of autoimmunity. Our results suggest that early nonspecific immunotherapy of genetically predisposed individuals could prevent the development of -type-t-diabetes.
Streptozocin (STZ) is a nitrosourea antineoplastic agent that exhibits direct pancreatic islet B cell cytotoxicity (Rerup, 1970). It has been used to induce an experimental form of diabetes. In high doses STZ acts as an alkylating agent and induces DNA lesions in B-cells (LeDoux & Wilson, 1984; LeDoux et al. 1986); in multiple sub-diabetogenic doses (low-dose STZ treatment, LDS) it induces, in some strains of mice and with an androgenic dependence, inflammation of the islets referred to as 'insulitis' (Like& Rossini, 1976) in which an autoimmune process involves macrophages and other infiltrating elements (Kolb-Bachofenetal. 1988; Papaccioetal. 1991). Contrary to what has been observed in other animal models of non-insulin-dependent (typel) diabetes, immune-suppressants (e.g, Cyclosporine-A) (Ferrero et al.1985; Papaccioetal.1989) and some immune-modulators (Wright & Lacy, 1988) have been demonstrated to be ineffective in the preservation of islet B-cells from destruction in LDS treated mice. These results are in conflict with the assumption that autoimmune processes are also involved in this animal model. Recently, adjuvants, in addition to being immune- potentiators, have been demonstrated to act on the immune system by inducing nonspecific natural effector cells, which in turn may influence effector T lymphocytes (Sadelain et al.1989). Diabetes mellitus can be prevented by a single injection of complete Freund's adjuvants (CFA) in young NOD mice (Sadelainetal. 1990).
We therefore decided to use BCG in this study in order to investigate its ability to reverser and/or suppress the islet infiltration and diabetes in this trial, and also to observe whether the prevention of insulitis is leads to averting or reversing B-cell damage. These results have direct implications in preventing the recurrence of diabetes by self-repair mechanisms.
Hypothesis for current study
BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) permanently eliminate the need for insulin in insulin-dependent-diabetic subjects normalizing fasting plasma glucose, HbAlc, proinflammatory, prothrombotic and oxidative stress markers in diabetic, non-hypertensive or hypertensive subjects. The improvement of these markers is independent of changes in arterial blood pressure.
Methods/design
General objective
To evaluate the effect of BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x 5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) during 12 months on eliminating the need for insulin, the concentration of inflammatory, prothrombotic, and oxidative stress markers in diabetic, non- hypertensive and hypertensive subjects with dysglycemia, recruited from the general population.
Specific objectives
To establish the effect of the administration of BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) on the insulin requirements assessed through the HOMA index, fasting plasma glucose, OGTT, and HbAlc levels.
To study the effect of BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) administrated during 12 months on CD45RA+ and CD45RA- T-cells and other immune cells and their migratory behavior on High Endothelial Cells.
To determine whether the administration of BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) decreases the concentration of oxidative stress markers such as plasma oxidized/reduced glutathione ratio, total oxidative capability, malonaldehyde and urinary 8-Isoprostanes.
To determine whether the effects of BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) on insulin requirements and oxidative stress, are permanent.
Study design
Randomized, double blind, placebo-controlled, crossover clinical trial (Figure 14).
Fig. 14 shows the Study design - the protocol included Fragmin D (5 IU/kg to a maximum of 300 IU once per day) as an addition.
Study treatments
- Treatment A: BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) for 12 months and then switch to placebo treatment for another 12 months
- Treatment B: Placebo tablets administered identical to treatment A,
All the subjects were included in a therapeutic life-style change program (TLC: educational, nutritional and exercise support) during the study.
Study groups
The study embraces two arms (Figure 2):
- Group 1: received the treatment A during the first 52 weeks and then the treatment B during the last 52 weeks.
- Group 2: received the treatment B during the first 52 weeks and then the treatment A during the last 52 weeks,
Population
Participants were diabetic non-hypertensive or hypertensive individuals of both genders, older than 6 years with dysglycemia and/or IGT. Inclusion and exclusion criteria are shown in Table 1
Table 1 Selection criteria
Inclusion criteria - Ethic Committee approval of study
- Male and female older than 6 years of age.
- To have fasting plasma glucose between 100 and 145 mg/dl
- Having treatment compliance over 80% at the end of the run-in phase.
- All women with bearing potential must have a secure contraceptive method. Secure methods were considered: surgical sterilization, postmenopause condition with an age greater than 45 years and a period of amenorrhea > 2 years. (In premenopausal women, the use of hormonal method or two barrier contraceptive methods including 1 month after the conclusion of the active phase of study treatment).
Exclusion criteria
- Prior diagnosis of pancreatic cancer or mucoviscidosis).
- Significant chronic disease (terminal stage cirrhosis or hepatic disease or cancer) that affects the survival of patients at 24 months.
- Acute infection of any etiology within 4 weeks prior to the beginning of the study.
- Use of steroid hormones or NSAIDs 4 weeks prior to the beginning of the study.
- Patient requiring treatment with immunosuppressive agents, for any circumstance.
- Patient who has participated in a clinical trial in the 8 weeks prior to the study entry.
- Patient who requires a major surgical procedure during the next 12 months, after enrollment (Abdominal or thoracic surgery, vascular, neurosurgery, urologic or gynecologic surgical procedure).
- Patient with history of severe chronic gastritis or any condition of the gastrointestinal tract that may affect the absorption and/or distribution of any drug administered orally.
- History of use of psychoactive drugs or abuse of alcohol.
-Positive pregnancy test in the screening visit.
- Concomitant treatment with any other antihypertensive drug.
- Contraindication to receive treatment with BCG, and/or Famiciclovir.
- Pathological alterations of aortic or mitral cardiac valves (stenosis or insufficiency) or hypertrophy cardiomyopathy.
- Denial to sign informed consent, or any mental condition that makes the patient part of a susceptible population
Sample size
A sample size of 2,000 subjects was estimated, considering a crossover clinical trial design as proposed by Hills and Armitage [70], accepting a type I error of 0.05, a power of 90%, and assuming a difference of 20 % in the HOMA index (3 to 2.4) after 12 months of treatment with BCG (l-8xl08 Bacillus Calmette Guerin Connaught) Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) and a maximum standard deviation of 1.5. The final size of the sample, adjusted for a dropout of 8%, is 2,200 subjects (1,100 in each group). The sample size ensures a power of 95% to detect differences in fasting Glycaemia of at least 8 mg/dL (0.44 mMol/L) with a standard deviation (SD) of 20 mg/dL (1.1 mMol/L), or a difference of 14 mg/dL (0.77 mMol/L) in the 2 hours post load Glycaemia with a SD of 40 mg/dl (2.2 mMol/L).
Procedures
Enrollment
This study was realized in a mixed age population with a maximum enrollment period of two years. Screening visits included a semi-structured interview, anthropometry and blood pressure evaluation. Eligible subjects were scheduled one week later for "Visit A", to perform a new interview, a physical examination, diabetes control evaluation, and to withdraw blood samples, after a 10 hour fasting period, to determine plasma glucose levels, lipid profile, hepatic and kidney function and immune pathology as per diagnostic patent ( ). Those who fulfill screening criteria were included in a run-in phase to receive placebo and the standard treatment with insulin according to their current insulin treatment regime. The patients were blinded during this phase, which lasted 2 weeks. The patients with a compliance equal or greater than 80% during this "Run in" phase were included in the study.
Baseline assessments
Visit B included measurements of blood pressure, anthropometric parameters, and electrocardiogram. A fasting blood sample and a 24-hour urine sample were taken and stored (-70°C) to determine glucose, HbAlc, insulin, IL-6, leptin, resistin, adiponectin, tissue Plasminogen activator (tPA), PAI-1, oxidized/reduced glutathione, malonaldehyde and 8-isoprostanes in urine. Once the patient completed the foregoing steps, during visit 1 he/she was randomized to one of the arms of the treatment. For this purpose, a randomization system by blocks of 4 was used. New tests were performed at the end of each treatment, according to the study flowchart (Table 2). Randomization and preparation of medication/placebo was done by the University Hospital Chemist.
Active follow-up
Seven days after beginning the treatment, and every week thereafter, the subjects were asked to return for a visit in order to verify compliance, evolution of plasma glucose levels, insulin regime and occurrence of adverse events. Months 1 to 12 of each treatment, new fasting glucose and HbAlc determination were done in all subjects. All basal measurements were repeated at the end of each treatment (every 1.2 months).
Passive follow-up
All the subjects have undergone a passive follow-up (telephonic follow-up) 3, 6 and 12 months after concluding the treatment.
Blood samples
In fasting conditions (at least 10 hours), blood samples were taken from the antecubital vein, with appropriate conditions of asepsis and antisepsis, using 3 vacutainer tubes, one dry, another with citrate, and the other containing EDTA. After 10 minutes in vertical position, all samples were centrifuged at 3000 rpm during 15 minutes to extract the serum or plasma. Part of the samples obtained during visits B, 5 and 9 have been stored in Ependorf vials at -70°C until the end of the study.
Anthropometrical measurements
All anthropometrical measurements were taken first thing in the morning after urine elimination, with the subject using light clothing and no shoes.
Weight: were measured with the patient standing and then registered after rounding it to the nearest 200 grams. The weight scales were calibrated to 0 before each measurement.
Height: Was measured using a metric tape with the patient standing against the wall in Frankfort's position, and the value marked by a ruler placed horizontally on the head of the patient.
Heart rate: number of beats per minute was measured in the radial artery.
Blood pressure: were taken twice (with a difference of 5 minutes between the measurements) using a mercury sphygmomanometer in 2 occasions on the right arm, with the patient comfortably seated, after a 5-minute rest. Systolic blood pressure (SBP) was determined by the first audible sound (Korotkoff phase 1). Diastolic blood pressure (DBP) was registered when the sound disappears (Korotkoff phase 5). The patient should not have smoked 30 minutes prior to the blood pressure measurement. The pneumatic arm cuff must cover 2/3 of the upper arm's length; its inferior border must be 2-3 cm over the antecubital space; the cuff was slowly deflated. The mean blood pressure (MBP), were calculated using the following formula [SBP+(2*DBP)]/3. Waist circumference: were measured in 2 occasions with the patient in a standing position, with the arms on the sides and using a measuring tape adhered to a dynamometer that exerts a force of 750 gr. The measuring tape was placed horizontally in a middle point between the iliac crest and the anterior costal border. The difference between the two measurements should not be more than 0.5 cm.
Hip circumference: were measured in 2 occasions with the patient in a standing position with the arms on the sides of the body, using a measuring tape adhered to a dynamometer that exerts a force of 750 gr. The hip circumference was assessed over the major trochanters. The difference between the two measurements should not be more than 0.5 cm.
Waist-Hip Relation (W/H-R): were obtained from the ratio between the waist and hip circumferences.
Antero-posterior diameter: were measured twice with the patient in a decubitus supine position, using a ruler perpendicular to the bed and registering the cutting point with the tape applied horizontally on the abdomen. The difference between the two measurements should not be more than 0,5 cm. Body Mass Index (BMI): This index was estimated using the weight in kilograms divided by the second power of the height expressed in meters.
Biochemical markers
Routine clinical test and inflammation markers were processed in the Clinical Research Laboratory from the Universities of Tubingen and Berlin. (Germany). The measurement of oxidative stress markers was processed in the laboratories of Cologne University (Cologne, Germany).
Glycaemia, Lipid profile, Serum Creatinine, Hepatic enzymes (AST/ALT) were quantified by a routine colorimetric method. (Biosystems BTS-303 Photometric, Espana).
Glycosylated Hemoglobin Ale: Were determined with a quantitative automated technique GlycoHemoglobin Analyzer (DCA 2000+ Bayer®.) using a whole blood sample.
Insulin, High-sensitivity C-Reactive Protein and Interleukin 6: were determined by high sensitivity chemoluminescent immunoassay technique (IMMULITE ® Automated Analyzer, Diagnostic Products Corporation, Los Angeles, USA).
Leukocyte count and differential formula: were determined by an automated counter (Baker System 9120 AX ®, Biochem Inmunosystem, USA).
Glucose Tolerance Test: were done after a fasting period of at least 10 hours. After urine elimination, an intravenous catheter were placed in the antecubital vein and blood samples were withdrawn to assess the baseline blood glucose, then, a glucose load equivalent to 75 gr diluted in 300 mL of water were administered to each study subject within a period of no more than 10 minutes. Then, after 2 hours, a new blood sample was withdrawn to assess Glycaemia. Patients should not eat anything or do any exercise during the test.
Serum Leptin, resistin and adiponectin: were measured by ELISA technique.
HOMA Index: Were obtained from a mathematical model using the following formula: equation Ml
Quality assurance systems
The inter- and intra-assay variation coefficient were determined for all measurements. In order to eliminate the inter-assay error, all biochemical determinations were performed at once.
Data processing and quality assurance
Trained personnel collected all study data. These data were recorded on forms previously designed for such purpose (Case Report Form-CRF). After completing the CRF, a monitor reviewed them to assure that they are correctly filled and legible. The monitor according to the Good Clinical Practice guidelines did all corrections. Then, the information was typed and stored twice in independent databases. A computer program (Epi-lnfo 2000) was used to compare both databases. All discrepancies were printed and corrected using the original CRF as reference.
Data monitoring
The study coordinator made sure that data was adequately collected. He/she registered the study visits and the time of data collection and the different procedures, as well as the compliance to the treatment. Data management
The Tiibingen Biostatistics Department was responsible for the data management. Once the data was correctly recorded, the means and ranges were estimated and the relevant variables were crossed to identify inconsistencies or extreme values, which could result from errors in data management (internal consistency analysis). Any detected errors were corrected using the original form and the lab reports as references, maintaining the 2 original databases untouched.
Modifications and statistical procedures during the data analysis were documented in the Stata 9.0 program, which will allow the replication of the data analysis whenever necessary.
Statistical analysis
The study was set forth as an efficacy study of BCG (1.8xl08 Bacillus Calmette Guerin Connaught) and Famiciclovir (3x5 mg/day) to permanently eliminate the need for the insulin treatment confirmed by the HbAlc and the OGTT results. The averages and proportions with their corresponding 95% confidence intervals were obtained in a descriptive analysis for all clinically relevant variables measured during the baseline evaluation. In order to evaluate the presence of differences between the groups, the Student's paired-t-test, the Wilcoxon's signed-rank test or the McNemar's test was used according to the variable's characteristics. Linear multiple regression were used with the purpose of comparing the results of the treatments. The analyses were performed by the intention-to-treat approach. A p value under 0.05 was considered as statistically significant.
The primary endpoint for the analysis was the change in the value of HOMA index, fasting glucose and post-charge glucose plasma levels. The secondary endpoint for the analysis will include the changes in serum insulin, leptin, adiponectin, resistin, CRP, ll_- 6, tPA/PAI-1 ratio, Oxidized/Reduced glutathione ratio, malonaldehyde and 8- isoprostanes.
Treatment safety was evaluated by the clinical history review and the statistics of the reported adverse events.
Safety committee and events assignation committee
A safety and events assignation committee were created, according to the Harmonized Tripartite Guidelines of the International Conference of Harmonization for Good Clinical Practice.
Ethical aspects
The clinical trial was conducted according to the Helsinki's Declaration, the Good Clinical Practice Guidelines and the German legislation (Resolution 8430/93 of the Ministry of Health). The patient has provided written informed consent in a form designed for such purpose. The information generated by the study is confidential and strictly limited to the purposes stipulated in the protocol. The patient may refuse to continue participating in the study at any moment after providing his/her consent. German Clinical Ethics committee has approved the study. All assessments were be performed by trained staff. The blood samples were collected in aseptic conditions by an expert bacteriologist.
Study time line: 36 months
Fig. 15 shows the efficacy of BCG, Famiciclovir and Fragmin-D eliminating the need for insulin treatment in Type-l-diabetics of mixed age, ranging from 6 to 85 years of age, verified by HbAlc and OGTT.
Fig. 16 shows the number of side effects of BCG, Famiciclovir and Fragmin-D per 1000 trial participants.
Discussion
The study results clearly show that BCG in single dose application (l-8xl08 Bacillus Calmette Guerin Connaught)once every three weeks together with Famiciclovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) over a period of 12 months is a safe and effective treatment to eliminate the need for insulin in type-1-diabetics of all ages.
Abbreviations
(ACE) Angiotensin Converting Enzyme
(ACEIs) Angiotensin Converting Enzyme Inhibitors
(ADA) American Association of Diabetes
(AII)Angiotensin II
(ATI) Angiotensin II Type 1 Receptor
(AT2) Angiotensin II Type 2 Receptor (CRF) Case Report Form
(CRP) C-reactive Protein
(CVD) Cardiovascular Diseases
(DBP) Diastolic Blood Pressure
(eNOS) Endothelial Nitric Oxide Synthase
(FAS) Fatty Acid Synthase
(FFA) Free Fatty Acids
(FMD) Flow-Mediated Dilation
(GDPH) Glycerol-2 Dehydrogenate Phosphate
(HOMA) The Homeostasis Model Assessment
(IFG) Impaired Fasting Clucose
(IGT) Impaired Glucose Tolerance
(IL-6) Interleukin 6
(IRS-1 and IRS-2) Insulin Receptor Substrate Type 1 and 2
(MBP) Mean Blood Pressure
(MMP-2) Matrix Metalloproteinase-2
(NADPH) Nicotinamide Adenine Dinucleotide Phosphate Hydrogen
(NO) Nitric Oxide
(OGTT) Orai Glucose Tolerance Test
(PAI-1) Plasminogen Activator Inhibitor-1
(PKC) Proteinkinase C
(PPARs) Peroxisome Proliferator-Activated Receptors
(RAS) Renin-Angiotensin-Aldosterone System
(ROS) Reactive Oxygen Species
(SBP) Systolic Blood Pressure
(SD) Standard Deviation
(TLC) Therapeutic Life-Style Change Program
(TNF-a) Tumor Necrosis factor alpha
(tPA) Tissue Plasminogen Activator
(UPC-2) Uncoupling Protein 2
Acknowledgements
We would like to express our gratitude to Dr Peter Schaffer and Dr Judy Woodruff, for their contributions during the protocol development, and Andrea Hamilton for reviewing the English style. REFERENCES
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001

Claims

Claims
1. Combination of active agents comprising
an immunopotentiator/immunomodulator selected from the group consisting of
BCG , freund's adjuvant, peptides, leukotrienes, interleukins; and
heparin.
2. Combination of active agents according to claim 1, whereby the immunopotentiator/immunomodulator is BCG.
3. Combination of active agents according to claims 1 or 2, whereby the heparin has a mass
average molecular weight of equal or less than 10000 Da.
4. Combination of active agents according to any of the preceding claims additionally comprising a guanine analogue.
5. Combination of actives agent according to any of the preceding claims, whereby the
guanine analogue is selected from the group consisting of famciclovir, valaciclovir, adcyclovir, and penciclovir.
6. Combination of active agents according to any of the preceding claims, whereby the
guanine analogue is famciclovir.
7. Combination of active agents according to any of the preceding claims additionally comprising a protease inhibitor.
8. Combination of active agents according to claim 7, whereby the
protease inhibitor is a serine protease inhibitor.
9. Combination of active agents according to any of the preceding claims, whereby the
protease inhibitor is alpha-1-antitrypsin.
10. Combination of active agents according to any of the preceding claims comprising BCG in amount of 1 - 10 x 108 bacteria, in particular 4 to 6 x 108 bacteria.
11. Combination of active agents according to any of the preceding claims comprising 0.1
to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin.
12. Combination of active agents according to any of the preceding claims, whereby the
amount of heparin is less than 300 IU.
13. Combination of active agents according to any of the preceding claims, whereby the
guanine analogue is famciclovir in an amount to 10 to 1000, in particular 50 to 500 mg.
14. Combination of active agents according to any of the preceding claims, whereby the
protease inhibitor is alpha-1-antitrypsin in an amount of 10 to 1000, in particular 100 to
250 mg per kg bodyweight.
15. Combination of active agents according to any of the preceding claims for use as a medicament.
16. Combination of active agents according to any of the preceding claims for the treatment
of diabetes-type I.
17. Use of a combination of active agents according to any of the preceding claims for the production of a medical preparation for treating diabetes-type I.
18. A method for treatment of diabetes-type I in animals, including humans, comprising simultaneously, sequentially or separately administering the combination of active agents according to any of the claims 1 to 16.
19. A method for treatment of diabetes-type I in animals, including humans, comprising the step of administering BCG in admixture with heparin having a mass average molecular weight of equal or less than 10000 Da.
20. A method according to claim 18 or 19, whereby the dosage is
BCG in amount of 1 - 10 x 108 bacteria, in particular 4 to 6 x 108 bacteria, and
0.1 to 10, in particular 3 to 6 IU per kilogram bodyweight of heparin,
whereby the step of administration is repeated every 3 weeks.
21. A method according to any of the claims 18 to 20 additionally comprising the step of administering famciclovir.
22. A method according to claim 21 wherein the dosage of administration for famciclovir is 3 times 10 to 1000, in particular 50 to 500 mg, per day.
23. A method according to any of the claims 18 to 22, additionally comprising the step of administering alpha-1-antitrypsin.
24. A method according to claim 23 wherein the dosage of administration for alpha-1- antitrypsin is 10 to 1000, in particular 100 to 250 mg, per kg bodyweight once a week.
* * *
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