JP2014521492A - Injection filler (filler) {INJECTABLEFILLER} - Google Patents

Abstract

The system and method are published for the following purposes.
• Crosslink heteropolysaccharides to form a single cross-linked material to form a biocompatible cross-linked polymer with an interpenetrating polymer network (IPN) • Additional cross-links in addition to the single cross-linked material To form a multiple cross-linked material. Among them, multiple cross-linked materials have one or more IPN regions that resist biodegradation in the human body than single cross-linked materials, and one or more single cross-linked extensions extend from the IPN. The combination of IPN and expansion also provides biodegradation resistance, soft touch and ease of introduction into the body.
[Selection] Figure 1

Description

    This application claims priority based on provisional application No. 61/558669 filed on November 11, 2011 and utility application No. 13301785 filed on November 22, 2011, and was described in that application. All the descriptions are incorporated.

  The present invention relates to biocompatible viscoelastic polymer gel slurries, methods for their preparation and formulations containing them.

  As the age of a person, wrinkles and creases in the face develop according to the loss of facial fat and the decrease in skin elasticity. For many years, doctors have tried various methods and materials to combat the loss of facial volume of facial soft tissue. One of the most common methods is autologous fat transfer. Using this surgical technique, the person's own fat is collected from other parts of the body, such as the abdomen, and then processed to restore the volume so that it has a more youthful appearance by reducing wrinkles and folds. Prepared for injection into the requested facial skin and soft tissue areas. Autologous fat transfer provides good and desirable results, but is a surgical procedure that is expensive for the sick, painful, time consuming, has a long recovery time and is associated with complications associated with any surgical procedure.

  An object of the present invention is to provide a conventional apparatus for solving such problems.

  The following are some detailed aspects.

I. Continuous cross-linking II. HA molecular weight manipulation III. Free radical scavengers: vitamins, enzymes and the like
IV. Anti-hyaluronidase and anti-elastase
Continuous cross-linking
In one aspect, each system and method is disclosed for soft tissue cosmetic enhancement using cross-linked HA optimized for:

1. Easy product delivery 2. Local organization compliance Greater cohesion to control the movement of implant material
4). Biodegradation profile
Use of cross-links by forming unique cross-linked HAs and regions of cross-linked HA (IPN) of cross-linked HA by their further cross-linking. The IPN structure provides these utilities for this cross-linking HA only for this cosmetic enhancement application. The IPN core (imagined as tapioca balls) is more resistant to biodegradation in the human body than a single cross-linked material that is normalized for the same level of cross-linking. In addition, the various physical properties that continuously change from core to radiation make the polymer rugged and conform to local tissue at the same time for better tissue and device biocompatibility, making the hand feel more natural.

  The HA cross-linking method described above has been optimized for cosmetic enhancement in certain cases where the drug substance needs to be controlled to tailor the local tissue response to the polymer. The pharmaceutical ingredient is compounded with the other phases to form a cross-linked HA polymer.

  Implementations of the above aspects will include one or more of the following. The system is biocompatible and ensures that regulated medicines are released at strategic timing simultaneously with major organizational events. For example, a rapid drug release profile without delay may be suitable for controlled release of anesthetics such as lidocaine so as to relieve severe pain experienced by patients associated with surgery. The system is also a media release profile and media delay for corticosteroids or steroids, such as dexamethasone or triamcinolone, to coincide with a physiological inflammatory foreign body reaction. The system is customized to have an uncontrolled healing and media to delay the release profile and longer delay before initiating the release of antiproliferative agents such as paclitaxel, sirolimus, 5-fluorouracil, etc. Stop poor control healing and excessive remodeling.

1. Molecular weight manipulation
Another aspect of the present invention includes a method for optimizing biodegradation profiles and controlling the movement of implant materials through various types of molecular weight manipulations. The system optimizes the biodegradation profile and controls the movement of the implant material. The system optimizes the biodegradation profile from hypervolume to isovolumetric and isovolumetric, and various types of molecular weights such as Mn, Mw, Mz etc. and their polydispersity index (PDI) Can be formulated around.

2. Free radical scavenger vitamins and enzymes
HA in the body is biodegraded by two main mechanisms: oxidation and hydrolysis. These mechanisms are enzymatic hydrolysis by three enzymes, namely hyaluronidase (hyase), BD-glucuronidase, and β-N-acetyl-hexosaminidase, in mammalian cells. Oxidation by radicals (sometimes also called reactive oxygen species (ROS)). These are an odd number of electrons (unpaired atoms) or a group of atoms, which can be formed by the interaction of oxygen with specific molecules.

ROS is produced as a normal product of cellular metabolism. In particular, one of the main factors leading to oxidative damage is hydrogen peroxide (H ₂ O ₂ ) converted from superoxide leaking from mitochondria. Catalase and superoxide dismutase convert these compounds to oxygen and water, which are benign molecules, and convert the benign molecules to hydrogen peroxide by converting the damage effect of hydrogen peroxide and superoxide into these compounds. And improve the superoxide's obstacle effect. However, this conversion is not 100% efficient and residual peroxide is retained in the cell. ROS is produced as a product of normal cell function, but excessive amounts can cause deleterious effects. Memory power decreases with age, as is apparent in human degenerative diseases such as Alzheimer's disease with accumulating oxidative damage. Since oxidative damage is a member of aging, current studies have shown that ROS accumulation can reduce the fitness of an organism. In particular, the accumulation of oxidative damage can lead to cognitive impairment, as demonstrated in studies where older mice were given mitochondrial metabolites and cognitive tests. The results suggest that mice can behave better after receiving metabolites, suggesting that metabolites have reduced oxidative damage and improved mitochondrial function. The accumulation of oxidative damage can subsequently affect mitochondrial effects and further increase the rate of ROS production. The accumulation of oxidative damage and its effect on aging depends / depends on the specific tissue type in which the damage occurred. Results from further experiments suggest that oxidative damage is involved in aging-related declines in brain function. Older gerbils were found to have higher levels of oxidized protein compared to gerbils. Treatment of gerbils and gerbils aged with spin trapping compounds resulted in decreased levels of oxidized protein in older gerbils, but no effect on gerbils. Moreover, older gerbils have improved their ability to cognitive tasks during treatment, but when they stopped treatment they lost their functional ability.

Furthermore, once these highly reactive radicals are formed, the chain reaction can be initiated.
Damage caused when reacting with important cellular components such as DNA and cell membranes leads to their main danger. If this happens, the cells may work poorly or die. The body has an antioxidant defense system to prevent free radical damage. Free radicals and antioxidants react quickly and easily.

  The decomposition reaction of HA with oxygen-derived free radicals was the result of studies using HA in synovial fluid. HA indicates that it was easily decomposed by free radicals of superoxide. This reaction is most preferred in the case of secondary free radicals. Neutrophils (polymorphonuclear leukocytes) produce oxygen-derived free radical species that can ingest HA molecules. These WBCs (leukocytes) are far more exclusive destroyers of HA, with the mechanism of oxygen-derived free radicals. Thus, one aspect of the present invention suppresses the effects of free radicals before the free radicals decompose HA using free radical scavengers such as antioxidant vitamins.

  Antioxidants are closely related to the prevention of cell damage as a common pathway leading to cancer, aging and various diseases. Antioxidants are molecules that can interact safely with free radicals and terminate chain reactions before important molecules are damaged. Despite the presence of several enzyme systems that trap free radicals in the body, the principle that the antioxidants of micronutrients (vitamins) are vitamin E, beta carotene, and HA is vitamin C is an exception. Is done. In addition, selenium, a trace metal required for one proper function of the body's antioxidant enzyme system, is sometimes included in this category. Because the body itself cannot produce micronutrients, it must be supplied to the diet.

  The following are example antioxidant vitamins, their roles and recommended daily doses.

  Vitamin E: d-alpha tocopherol is a fat-soluble vitamin present in nuts, seeds, vegetable oils, whole fish oil, whole grains (especially wheat germ), fortified grains and apricots. Recommended daily intake (RDA) is 15 IU for men and 12 IU for women.

  Vitamin C: Except when harmful to the longevity of HA, vitamin C is ascorbic acid, a water-soluble vitamin present in citrus and its juices, peppers, cabbage, spinach, broccoli, kale, muskmelon, kiwi and strawberries It is. RDA is 60 milligrams per day. Ingestion above 2000 mg may have some side effects depending on the person.

Vitamin A: Beta carotene is a precursor of vitamin A (retinol) and is present in liver, egg yolk, milk, butter, spinach, carrot, pumpkin, broccoli, yam, tomato, cantaloupe, peach, and cereal. Beta carotene is not particularly required because it is converted to vitamin A in the body. Instead, RDA is expressed as retinol equivalent (RE) to clarify the relationship. (Note: Vitamin A has no antioxidant properties and can be quite toxic if taken excessively)
Glutathione: (GSH) is a peptide having a gamma peptide bond between the amine group of cysteine and the carboxyl group of the glutamic acid side chain that are linked by a peptide bond perpendicular to glycine. This is an antioxidant that prevents damage to important cellular components caused by reactive oxygen species such as free radicals and peroxides. Thiol groups are reducing agents that are present in animal cells at a concentration of about 5 mM. Glutathione functions as an electron donor to reduce the disulfide bond formed in the cytoplasmic protein to cysteine. In this process, glutathione is converted to its oxidized form, glutathione disulfide (GSSG), also called L (-)-Glutathione (L (-)-glutathione).

  Once oxidized, glutathione may be reduced again by glutathione reductase using NADPH as an electron donor. The ratio of reduced glutathione to oxidized glutathione in the cell is often used as a measure of cytotoxicity.

  Uric acid: This is the most important plasma antioxidant in humans and a carbon, nitrogen, oxygen, hydrogen heterocyclic compound of the formula C5H4N4O3. Uric acid forms a salt with ions known as urate and monosodium urate, such as acidic ammonium urate. Uric acid is a product of metabolic degradation of purine nucleotides. High concentrations of uric acid in the blood can lead to a type of arthritis known as gout. Chemicals are associated with other medical illnesses including diabetes and the formation of kidney stones of ammonium acid urate.

  Another aspect of the present invention is the use of antioxidant enzymes to protect the lifetime of HA. These enzymes can reduce free radicals and protect against ROS. They are: α-1-microglobulin, superoxide dismutase, catalase, lactoperoxidase, glutathione peroxidase, peroxiredoxin.

3. Anti-hyaluronidase and anti-elastase
With respect to the field of cosmetic enhancement to rejuvenate aging skin using cross-linked HA, one aspect of the present invention is the depolymerization of HA with hyaluronidase inhibitors (anti-HA), particularly with hyaluronidase. And maintain the longevity of the HA. The maintenance of HA's longevity is important because it is directly related to wrinkling and signs of aging.

  HA is an important molecule for everything living on this planet. In this regard, it is a multifunctional degrading high molecular weight polysaccharide found throughout the animal kingdom, particularly in the extracellular matrix (ECM) of soft connective tissue. HA is involved in many biological processes, and its levels are thought to rise significantly during embryogenesis, cell migration, wound healing, malignant transformation, and tissue turnover. The enzyme that degrades HA, hyaluronidase (HAases) is expressed in both prokaryotes and eukaryotes. These enzymes are known to be involved in physiological and pathological processes from fertilization to aging. Hyaluronidase-mediated degradation of HA increases connective tissue permeability, decreases fluid viscosity, and is involved in bacterial pathogenesis, toxin / venom spread, acrosome reaction / egg fertilization, cancer progression . In addition, these enzymes can facilitate direct contact between the pathogen and the host cell surface. Moreover, the depolymerization of HA adversely affects the role of ECM and weakens the activity as a host of growth factors, cytokines and various enzymes involved in signal transduction. Therefore, inhibition of HA degradation may be important in reducing disease progression and the spread of toxins / venoms and bacterial pathogenesis. Hyaluronidase inhibitors are powerful and universal regulators involved in maintaining the balance of HA assimilation and catabolism. Hyaluronidase inhibitors function as contraceptives and antitumor agents, and may have antibacterial, antitoxin and antitoxin activities. In addition, these molecules can be used as pharmacological tools to study the physiological and pathophysiological roles of HA and hyaluronidase.

  The mechanism of hyaluronidase in HA degradation generally follows the following five steps.

The following table shows examples of hyaluronidase inhibitors.

1 illustrates an exemplary system for producing multiple cross-linked HA in series. Figure 3 shows another exemplary system for producing multiple cross-linked HA in series. An exemplary diagram of the resulting multi-crosslinked HA is shown.

  First, the preparation of hyaluronic acid is consulted, and the addition of additional chemicals that enhance the use of hyaluronic acid for skin or subcutaneous use is consulted.

A simple treatment can be used to isolate the hyaluronic acid from the medium since the hyaluronic acid of the recombinant Bacillus cells is expressed directly in the medium. First, Bacillus cells and cell debris are physically removed from the medium. The medium may be diluted first to reduce the viscosity of the medium as needed. Many methods for removing cells from the culture medium, such as centrifugation or microfiltration, are well known to those skilled in the art. If necessary, the remaining supernatant may be filtered, such as by ultrafiltration, to concentrate and remove small molecule contaminants from hyaluronic acid. Following removal of cells and cell debris, simple precipitation of hyaluronic acid from the medium is performed by known mechanisms. A salt, alcohol, or a combination of the salt and alcohol can be used to precipitate hyaluronic acid from the filtrate. Once reduced to a precipitate, hyaluronic acid can be easily isolated from solution by physical means. Hyaluronic acid can be dried or concentrated from the filtrate solution using evaporation techniques well known to those skilled in the art, such as freeze drying or spray drying.
Molecular weight The content of hyaluronic acid can be determined according to a modified carbazole method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334). Moreover, the number average molecular weight of hyaluronic acid is described in, for example, Ueno et al., 1988, Chem. Pharm. Bull. 36, 4971-4975; Wyatt, 1993, Anal. Chim. Acta 272: 1-40; Wyatt Technologies, 1999, “Light Scattering Universities”, “Methods such as“ DAWN EOS Manual ”,“ Wayt Technologies ”, and“ DAWN EOS Manual ”. Can be determined.
In one embodiment, the hyaluronic acid or salt thereof of one embodiment has a molecular weight of about 10,000 to about 10,000,000 Da. In a more preferred embodiment, it has a molecular weight of about 25,000 to about 5,000,000 Da. In the most preferred embodiment, the hyaluronic acid has a molecular weight of about 50,000 to about 3,000,000 Da.

  In other embodiments, the hyaluronic acid or salt thereof has a molecular weight within the range of 300,000 to 3,000,000. Preferred is in the range of 400,000 to 2,500,000, more preferred is in the range of 500,000 to 2,000,000, and most preferred is in the range of 600,000 to 1,800,000. Within range.

  In yet another embodiment, hyaluronic acid or a salt thereof has a low number average molecular weight in the range of 10,000 to 800,000 Da. Preferred is in the range of 20,000 to 600,000 Da, more preferred is in the range of 30,000 to 500,000 Da, and even more preferred is in the range of 40,000 to 400,000 million Da. And most preferred is in the range of 50,000 to 300,000 Da.

Illustration
Example 1 Preparation of DVS cross-linked fine particles in emulsion
This example illustrates the preparation of DVS crosslinked fine particles. Sodium hyaluronate (HA, 580 kDa 1.90 g) was stirred vigorously at room temperature for 3 hours until a homogeneous solution was obtained and dissolved in aqueous NaOH (sodium hydroxide) (0.2 M, 37.5 mL) I was allowed to. Sodium chloride (0.29 g) was added and mixed in a short time. Mineral oil (10.0 g) and ABIL® EM 90 surfactant (Cetyl PEG / PPG-10 / 1 Dimethicone, 1.0 g) were mixed by stirring.

  Divinyl sulfone (DVS, 320 microliters) was added to the aqueous alkaline HA solution and mixed for 1 minute to obtain a uniform distribution in the liquid phase. The aqueous phase was then added to the oil phase within 2 minutes by mechanical stirring at low speed. An emulsion formed immediately and stirring was continued for 30 minutes at room temperature. The emulsion was left overnight at room temperature. The emulsion was neutralized to pH 7.0 by adding aqueous HCl (4 M, about 2.0 mL) and stirred for about 40 minutes.

Example 2 Preparation of DVS cross-linked microparticles in a neutralized emulsion using a pH indicator
This example illustrates the preparation of DVS-crosslinked fine particles by a neutralization method using a pH indicator. Sodium hyaluronate (HA, 580 kDa 1.88 g) was stirred vigorously at room temperature for 2 hours until a homogeneous solution was obtained and dissolved in aqueous NaOH (sodium hydroxide) (0.2 M, 37.5 mL) I was allowed to. Bromothymol blue pH indicator (corresponding to a pH range of 6.6-6.8) was added (15 drops, blue in solution). Sodium chloride (0.25 g) was added and mixed briefly.

  Mineral oil (10.0 g) and ABIL® EM90 surfactant (cetyl PEG / PPG-10 / 1 dimethicone, 1.0 g) were mixed with stirring.

  Divinyl sulfone (DVS, 320 microliters) was added to the aqueous alkaline HA solution and mixed very vigorously in 30-60 seconds to obtain a uniform distribution in the liquid phase. The aqueous phase was then mechanically stirred at 400 RPM and added to the oil phase within 30 seconds. An emulsion formed immediately and stirring was continued for 30 minutes at room temperature. Neutralization was performed by adding aqueous HCI (4 M, 1.6 ml) and the emulsion was left at room temperature with magnetic stirring for 4 hours. The color of the pH indicator contained in the gel particles changed to green. The pH of the emulsion was measured with a 3-4 pH stick. The emulsion was left in the refrigerator overnight. The pH indicator contained in the gel particles turned yellow.

Example 3 Phase separation of emulsion and swelling / isolation of fine particles
This example illustrates the cutting of W / O emulsion (water-in-oil emulsion) and the phase separation and dialysis. Cross-linked HA microparticles were separated from the W / O emulsion by organic solvent extraction. The W / O emulsion (5 g) was mixed vigorously with a mixture of n-butanol / chloroform (1/1 v% 4.5 mL) by swirling in a test tube at room temperature. Extra MQ-water (20 ml) was added to obtain phase separation. The test tube was centrifuged to obtain three phases: the lower phase of the organic phase, the middle phase of the gel particles, and the upper phase of the clear aqueous solution. The upper and lower phases were discarded and the middle phase of the gel particles was transferred into a dialysis tube (MWCO 12-14,000, diameter 29 mm, volume / length 6.4 ml / cm). Samples were dialyzed overnight at room temperature with MilliQ® water. The dialysate was changed two more times and left overnight. The resulting gel was thick and viscous and swollen to a volume of about 50 m, equivalent to 0.004 g HA / cm ³ .

Example 4 Preparation of DVS crosslinks in emulsion and separation of microparticles
This example illustrates the preparation of DVS cross-linked HA microparticles. Sodium hyaluronate (HA, 580 kDa, 1.89 g) was dissolved in aqueous NaOH (0.2 M 37.5 mL). Sodium chloride (0.25 g) was added and the solution was magnetically stirred at room temperature for 1 hour until a homogeneous solution was obtained. TEGOSOFT® M (10.0 g) oil and ABIL® EM 90 surfactant (cetyl PEG / PPG-10 / 1 dimethicone, 1.0 g) were mixed with stirring.

  Divinyl sulfone (DVS, 320 microliters) was added to the aqueous alkaline HA solution and mixed for 1 minute to obtain a uniform distribution in the liquid phase. The aqueous phase was then added to the oil phase within 2 minutes by mechanical stirring (300 RPM). An emulsion formed immediately and stirring was continued for 30 minutes at room temperature.

  The emulsion was neutralized by adding an equivalent amount of HCI (4 M, 1.8 mL) and stirring for about 40 minutes. The emulsion was broken by adding n-butanol / chloroform mixture (1/1 v% 90 mL) and extra MilliQ®-water (100 mL), followed by magnetic stirring. The upper phase was separated into a volume of about 175 ml. The organic phase was mixed with mQ water (30 ml) as a final wash. The combined water / gel phase (205 mL) was transferred to a dialysis tube (MWCO 12-14,000, 29 mm diameter, volume / length 6.4 mL / cm) and overnight against MilliQ® water at room temperature. Dialyzed. The conductivity decreased to 0.67 micro-Sievert / cm after subsequent changes in water (3 times) and overnight dialysis (2 nights). The microparticles were evaluated by microscopy (DIC 200x). Refer to Figure 1. The cross section of one microparticle was shown and labeled as “21,587.92 nm”.

Example 5 Phase separation of emulsion and isolation of fine particles
This example illustrates the failure of a W / O emulsion (water-in-oil emulsion) and the isolation of gel particles. Gel particles were separated from the W / O emulsion by organic solvent extraction. An example of an organic solvent used for this extraction is a butanol / chloroform mixture in a volume ratio (v%) of 75:20 to 20.80, respectively. The weight ratio (w%) between the W / O emulsion and the organic solvent is about 1: 1.
Small scale separation: The W / O emulsion (5 g) was weighed in a centrifuge tube (50 mL). A butanol / chloroform mixture was prepared (1: 1 v%), from which 4.5 mL (5 g equivalent) of the mixture was added to the test tube. The test tube was carefully mixed to ensure that the entire emulsion was dissolved. The test tube was mixed by swirl mixing and left at room temperature for phase separation. Phase separation with an upper phase of the aqueous phase, a middle phase of the emulsion phase, and a lower phase of the organic phase was often observed. The addition of more aqueous and organic phases improves the separation. The aqueous phase was separated by decanting the supernatant and further purified or characterized.

Example 6 Preparation of W / O emulsion (water-in-oil emulsion)
In this example, a composition in which HA fine particles are formed is illustrated.

  A cold temperature procedure that incorporates the cold aqueous phase B into the hot oil phase can be used, reducing manufacturing time. Non-limiting examples of formulation are as follows:

Phase A:
2.0% ABIL® EM 90 (cetyl PEG / PPG-10 / 1 dimethicone)
20.0% mineral oil (or TEGOSOFT (registered trademark) M)
Phase B:
0.5% sodium chloride
3.8% hyaluronic acid
0.2 M NaOH (aqueous solution) up to 100%
Phase C:
About 0.6% divinyl sulfone
Preparation:
1. Mix Phase A at room temperature.

  2. Phase B: Hyaluronic acid (Hyacare®) is solubilized in aqueous NaOH with stirring, NaCl is added and stirred.

  3. Add DVS to Phase B and stir for 1 minute.

  4. Stir while slowly adding Phase B to Phase A.

  5. Homogenize in a short time, or leave as it is to stir and react.

  6. Agitation and swelling.

  7. Continue stirring at a temperature below 30 ° C.

  8. Neutralize.

Example 7 Preparation and separation of DVS cross-linked fine particles
Sodium hyaluronate (HA, 580 kDa, 1.88 g) was dissolved in aqueous NaOH (0.2 M, 37.5 mL). Sodium chloride (0.25 g) was added and the solution was magnetically stirred at room temperature for 1 hour until a homogeneous solution was obtained. The TEGOSOFT® M (10.0 g) oil and ABIL® EM 90 (cetyl PEG / PPG-10 / 1 dimethicone, 1.0 g) surfactant were mixed with stirring. Divinyl sulfone (DVS, 320 microliter) was added to the aqueous alkaline HA solution and mixed for 1 minute to obtain a uniform distribution in the liquid phase. The aqueous phase was then added to the oil phase within 2 minutes by mechanical stirring (300 RMP). An emulsion formed immediately and stirring was continued for 30 minutes at room temperature.

  The emulsion was neutralized by adding an equivalent amount of HCI (4 M, 1.8 mL) and stirring for about 40 minutes. The emulsion was transferred to a separatory funnel and broken by attaching n-butanol / chloroform mixture (1: 1 v%, 90 mL) and extra MilliQ® water (100 mL) and then shaking vigorously. . The upper phase was separated into a volume of about 175 ml. The organic phase was mixed and washed with milliQ ™ water (100 ml). The bound water / gel phase was transferred to a dialysis tube (MWCO 12-14,000, diameter 29 mm, volume / length 6.4 mL / cm) and dialyzed overnight against MilliQ® water at room temperature. . The conductivity decreased to 10 micro-Sievert / cm after subsequent changes in water (3 times) and overnight dialysis (2 nights).

Example 8 Cleaning method for fine particle purification
This example illustrates the final isolation and purification of microparticles.

  Previously isolated 100 mL particles were resuspended in Na2HPO4 / NaH2PO4 buffer (0.15 M, 400 mL) and stirred slowly for 0.5 h. The suspension was left at 5 ° C. for 2 hours to remove the solid oil. The solution was then filtered through a mesh and further washed with 2 × 50 mL of buffer. The particles could be drip dried before characterization.

Example 9: Investigation of rheological properties of fine particles
This example illustrates conducting a rheological study on particles. The particle sample is analyzed on an Anton Paar rheometer (Anton Paar Limited, Graz Austria, Physica MCR 301, software: Rheoplus) using a 50 mm 2 ° cone plate shape. First, the linear range of the material viscoelastic model G ′ (storage elastic modulus) and G ″ (loss elastic modulus) is determined by an amplitude sweep with a variable strain γ. Next, the harmonic sweep is performed, and the viscoelastic model Based on the value and the values of G ′ and G ″, the tangent δ can be calculated as a strong / weak gel behavior value.

Example 10: Investigation of syringe capability experiment in texture analysis
This example illustrates conducting a study of the force applied to inject at a constant rate as a function of sample homogeneity. The particle sample is transferred to a syringe with a 27G × 1/2 ″ or 30G × 1/2 ″ needle and a texture analyzer (Stable, Microsystems Stable Micro Systems, Surrey, UK, TA. XT Plus, SoftWare: It is placed in the sample tool at Texture Component 32). The experiment is performed at an injection speed of 12.5 mm / min at a constant distance.

Example 11 Preparation of DVS Cross-linked HA Hydrogel
This example illustrates the preparation of a DVS cross-linked HA hydrogel with simultaneous swelling and pH adjustment.

  Sodium hyaluronate (HA, 770 kDa, 1 g) was dissolved in 0.2 M NaOH to give a 4% (weight / volume) solution that was stirred at room temperature for about 1 hour at about 20 ° C. Three replicates were prepared. A sufficient amount of divinyl sulfone (DVS) was then added to the HA solution to obtain a weight ratio of 10: 1, 7: 1, 5: 1 HA / DVS, respectively. These mixtures were stirred at room temperature for 5 minutes and then allowed to stand at room temperature for 1 hour. The gel was then swelled in 160 mL of phosphate buffer (pH 4.5 or 6.5) as shown in Table 1 in 24 hours.

The pH of the gel was stabilized during the swelling process. After swelling, excess buffer was removed by filtration and the hydrogel was easily homogenized using an IKA® ULTRA-TURRAX® T25 homogenizer (Ika Labortechnik, DE). Gel volume and pH were measured. (See Table 2)

The pH of the hydrogel was in the range of 7.1 to 7.6 (Table 2). This confirmed that the swelling step can be used to adjust the pH in this process. All hydrogels occupied a volume of 70 mL corresponding to about 1.4% of HA concentration (weight / volume (w / v)). These were transparent, homogeneous and sticky. Softness increased with decreasing degree of crosslinking (see Table 2).

Example 12 Preparation of a homogeneous DVS cross-linked HA hydrogel
This example illustrates the preparation of a very homogeneous DVS cross-linked HA hydrogel.

Sodium hyaluronate (770 kDa, 2 g) was dissolved in 0.2 M NaOH at room temperature in about 1 hour to give an 8% (body weight / volume) solution. DVS was added so that the HA / DVS weight ratio was 7: 1. After stirring at room temperature for 5 minutes, one of the samples was heat treated at 50 ° C. for 2 hours without stirring and left overnight at room temperature. The resulting cross-linked gel was swollen in 200 ml phosphate buffer (pH 5.5) at 37 ° C. for 42 or 55 hours and then washed twice with 100 ml of water finally discarded. The volume, pH, and pressure required to inject through a 27G * 1/2 injection needle were also measured. (See Table 3)
The cross-linked HA hydrogel prepared according to this example showed a higher swelling ratio and increased softness compared to the non-heat treated control hydrogel (Table 3). The pressure applied during injection through the 27G * 1/2 needle is more stable than that of the latter sample, indicating that the crosslinked HA hydrogel is more homogeneous.

  Features of DVS-HA hydrogel

EXAMPLE 13 Biological stability of DVS cross-linked HA hydrogel
This example illustrates the in vitro biological stability of a DVS cross-linked HA hydrogel using enzymatic degradation.

Bovine testicular hyaluronidase (HAase) solution (100 U / mL) was prepared with 30 mM citric acid, 150 mM Na ₂ HPO _4, 150 mM NaCl (pH 6.3). A sample of DVS-HA cross-linked hydrogel (approximately 1 mL) was placed in a safety lock glass vial, lyophilized, and weighed ((W ₀ ; formula 1). Enzyme solution (4 mL, 400 U) was used for each sample. Added and the vials were incubated by shaking gently (100-200 rpm) at 37 ° C. At regular intervals, the supernatant was removed and the sample was washed thoroughly with distilled water to remove residual salts. It was then lyophilized and finally weighed (Wt, formula 1).

  Biodegradation is expressed as the ratio of sample weight loss to initial weight (Formula 1). Weight loss was calculated from the weight loss for each sample before and after the enzymatic degradation test. Each biodegradation experiment was repeated three times. A DVS-HA hydrogel prepared as described in Example 2 ('with heat treatment') was compared to a non-heat treated DVS-HA hydrogel ('without heat of treatment'). Both types of gels degraded faster during the first 4 hours, but gradually slowed down to completion in the next 24 hours. Importantly, there was a significant variation in the weight loss of the unheated sample compared to the hydrogel prepared using the heat treatment process as described in Example 2. This clearly shows that a very homogeneous DVS cross-linked hydrogel can be obtained with the method described in Example 2.

Example 14 Preparation of Cosmetic W / O Emulsion (Water-in-oil Emulsion) In this form and in the following examples, DVS cross-linked HA hydrogels are creams and serums that increase skin wetness and elasticity when extended to the skin. The immediate anti-aging effect and the film formation effect which were mix | blended in are obtained.

  A typical formulation of a W / O emulsion contains 2% DVS cross-linked HA. Each phase (A to E) was adjusted separately by mixing each determined component (see Table 4). Phase B was then added to Phase A and stirred with a stirrer propelled by mechanical force below 40 ° C. Phase C, and Phase D and Phase E were added and stirred. Formulations with HA hydrogel concentrations of 4%, 6%, and 8%, respectively, were also made in Phase D to give a range of W / O formulations.

Another exemplary formulation of a W / O emulsion containing 2% DVS cross-linked HA is shown in Table 5. Each phase (A to F) in Table 5 was adjusted separately by mixing the determined components. Phase B was mixed with Phase A and the resulting oil phase was heated at 75 ° C. Phase C was also heated to 75 ° C. The oil phase was added to phase C at 75 ° C. and stirred with a mechanical stirrer propelled by mechanical force. The emulsion was then cooled to below 40 ° C. Phase D, and Phase E and Phase F were added and stirred. Formulations with HA hydrogel concentrations of 4%, 6% and 8%, respectively, were also made in Phase E to give a range of W / O formulations.

Example 15 Preparation of Silicone Serum As shown in Table 6, a typical formulation of silicone serum containing 2% DVS cross-linked HA was prepared. All ingredients were mixed simultaneously with high speed stirring below 40 ° C. (see Table 6). Formulations with HA hydrogel concentrations of 4%, 6%, and 8%, respectively, were also made in Phase E to give a range of serum.

Example 16 pH equilibrium during swelling; kinetic study
Kinetic studies have shown that neutral pH DVS cross-linked HA hydrogels are obtained after swelling with phosphate buffer (pH 7.0) in 8-14 hours, depending on the degree of cross-linking. A set of DVS cross-linked HA hydrogels, prepared as described above, was prepared using 4% to 8% HA solution and various amounts of DVS crosslinker and is shown in Table 7.

Periodically (every 2 hours), the hydrogel was removed during the heat treatment, the supernatant liquid was gently transferred, and the pH was measured (see FIG. 2). Fresh swelling buffer was used after each measurement. As a result, the pH of all hydrogels was between 11-12 for 2 hours after swelling and then gradually decreased to 7.2-7.5.

  The slower the hydrogel cross-linking, in other words, the higher the HA / DVS ratio, the faster it decreased. The pH drop for the 6% HA solution and for the two different ratios of HA and DVS is shown in FIG. Among them, when the HA / DVS ratio is 10: 1, it is labeled with a triangle, and when the HA / DVS ratio is 15: 1, it is labeled with a square. In these two cases, the pH was neutralized within 8 hours. In contrast, neutral pH could be achieved 14 hours after swelling of hydrogels with higher HA concentration (eg 8%) or higher degree of crosslinking (eg HA / DVS ratio of 2.5). These observations indicate that HA molecules with low cross-linked hydrogels exhibit greater freedom and flexibility, resulting in sufficient hydration and thereby faster pH equilibration.

Example 17 Attributes of viscoelastic model of hydrogel based on DVS cross-linked HA
Rheological measurements were performed on a Physica MCR rheometer (Anton Paar, Ostfildern, Germany) using plate plate geometry at a certain temperature. The viscoelastic behavior of the sample was investigated in a dynamic amplitude shear vibration test where the material was subjected to sinusoidal shear strain. First, strain / amplitude sweep experiments were performed to evaluate the region of deformation where linear viscoelasticity was effective. The typical range of distortion was 0.01-200% and the frequency was set to 1 Hz. Next, in the region of linear viscoelasticity, the values of shear storage (elasticity) modulus (or elastic modulus G ′) and shear loss elastic modulus (or viscosity modulus G ″) are constant shear strain (10%) and 0.1. Recorded from frequency sweep experiments as frequencies between -10 Hz, shape, NF, and gap were PP25, 2, 1 mm, respectively.

  G ′ provides information about the elasticity or energy stored in the material being deformed, while G ″ describes the energy consumed as a viscous feature or heat. Provides information about its ability to sustain and return to its initial configuration after strain stress or deformation.In all experiments, each material was measured at least three times.

  For a hydrogel with a higher degree of cross-linking (ie lower HA / DVS ratio: 10/1), G ′ is an order of magnitude higher than G ″. This indicates that this sample behaves as a strong gel material. Simply put, the overall rheological response is due to the contribution of physical and chemical cross-links and topological interactions between macromolecules, which are inherent in their ability to relieve stress. This results in a reduction in mobility, so that the material behaves as a three-dimensional network in which the fundamental mode of load stress adaptation (regulation) is performed by deformation of the network. (Ie higher HA / DVS ratio: 15: 1) In fact, the number of permanent covalent crosslinks and the number of entanglements The higher the Kere, elastic response of the hydrogel is also high.

Example 18 Crosslinked HA / DVS Hydrogel with Preservative DVS Crosslinked HA Hydrogel uses 1.5 g sodium HA in 0.2 M NaOH to obtain a 6% (w / v) solution. Prepared. The HA / DVS weight ratio was 10: 1. The hydrogel was prepared in 3 iterations until the swelling step of the procedure described in Example 2. Then, it processed as follows. After incubating in an oven at 50 ° C. for 2 hours, the hydrogel is Na2HPO4 / NaH2PO4 buffer (1 L, containing preservative (2-phenoxyethanol / 3 [(2-ethylhexyl) oxy] 1,2-propandiol)). 50 mM, pH 7.0).

The concentration of the preservative is 10 mL / mL and targets a final concentration of 1% (v / v) in the swollen hydrogel. It is expected that the preservative will diffuse into the hydrogel during incubation, while at the same time preventing microbial contamination in the buffer.
The container was covered with parafilm and placed in an oven at 37 ° C. After 1 hour, the swelling bath was removed and the hydrogel was swollen with fresh phosphate buffer containing 10 mL / mL preservative in 6-7 hours. This process was repeated until the swelling time was 12 hours and swollen in fresh phosphate buffer containing 10 mL / mL preservative for 6-7 hours. This process was repeated until the swelling time was 12 hours, after which the pH was measured. Swelling was continued for another 2.5 hours to reach a neutral pH.

  The amount of preservative incorporated into the hydrogel was determined by a UV-spectrophotometer (Thermo Electron, Nicolet, Evolution 900, equipment nr. 246-90). A solution of A1% (v / v) of preservative in phosphate buffer was first analyzed to select the wavelength. About 5 mL of hydrogel was collected using a pipette. Samples are usually taken at the center of the swollen round hydrogel and at the “side” of the north, east, south and west of the round gel.

  The sample was then transferred to a cuvette and the absorbance was read at 292 nm. Each sample was read three times and the absorbance was zeroed against a blank DVS cross-linked HA hydrogel without preservatives.

  The results showed that the amount of preservative incorporated into the DVS-HA hydrogel ranged between 0.91% and 1.02% (see Table 10). There was very good reproducibility during replication. Importantly, no significant difference between samples from the same hydrogel is observed, indicating uniform diffusion of the preservative in the hydrogel.

Example 19-Biodegradable polymer options
The decomposition time can be adjusted based on the polymer mixture in Table 1 below. Examples 1 and 2 below are examples of drug or drug matrix incorporation into biodegradable polymers to control drug release.

Table 1: Biodegradation time and components Polymer degradation time (mos)
50:50 DL-PLG1-2
65:35 DL-PLG 3-4
75:25 DL-PLG4-5
85:15 DL-PLG 5-6
DL-PLA 12-16
L-PLA> 24
PGA 6-12
PCL> 24
Various types of biodegradable polymers can be used to control degradation time and / or degradation by-products. The following are some biodegradable polymers.

  PGA, PLA and their copolymers are the most commonly used biodegradable polymer materials due to their attributes that can be adjusted by changing the polymer composition within the basic PLA / PGA theme It is.

o Polyglycolic acid (PGA) is prone to hydrolysis o Polylactic acid (PLA) is a biodegradation due to the crystalline regions that form when in a mixture that limits the degree of hydrolysis These results in timing are present in a mixture of D and / or L enantiomers.

・ Polydioxanone (PDS) Polydioxanone (PDS)
Poly (ε-caprolactone) poly (ε-caprolactone)
Poly (DL-lactide-co-ε-caprolactone) Poly (DL-lactide-co-ε-caprolactone)
Surfactant options:
The microcapsule particle size is directly controlled by the interfacial chemistry of the organic and aqueous phases. Surfactants are often used to mediate surface chemistry between oily substances and aqueous environments. A surfactant is a detergent that is in an aqueous solution. Surfactants are macromolecules with both polar and nonpolar ends. Polar ends of molecules and polar molecules stick to water. The nonpolar ends of the molecule attract NAPL (non-aqueous phase liquid) composites.

  Examples of surfactants used for solubilization are as follows.

1. Sioponic 25-9: linear alcohol with a solubilization of 2.75 g / g 2. Tergitol: ethylene oxide / propylene oxide with solubilization of 1.21 g / g Tergitol XL-80N: primary alcohol, ethylene oxide / propylene oxide alkoxylate with a solubilization of 1.022 g / g
4). 4. Tergitol N-10: trimethyl nonal ethoxylate with a solubilization of 0.964 g / g Rexophos 25/97: Phosphorylated nonylphenol ethoxylate with a solubilization of 0.951 g / g
Example 20: Biodegradable microparticles containing anti-inflammatory and corticosteroids
a. 30 days delay b. 120-day release control Organic phase:
Make 20% DLPLG polymer with methylene chloride. DLPLA polymer contains 65% DL and 35% PLG. Weigh 0.02 g of triamcinolone into a glass vial. Add 20% DLPLG polymer solution to a vial containing triamcinolone. Dispense 2 mL ・ Use orbital mixer to completely dissolve the drug Aqueous phase:
Make 100 mL of 0.1 molar SDS (sodium dodecyl sulfate) in DI water. Dispense 8 mL of 0.1 molar SDS solution into drug / polymer solution. Solvent evaporation:
Place a glass vial containing the reaction mixture under the impeller mixer. Launch the mixer to 1200 RPM. Start slowly as long as the speed required to produce the desired particle size is known. Gradually increase the speed of the impeller so that it can produce the diameter size. Once the speed to produce the desired particle size is reached, start heating the container with continuous mixing in an 80 ° C water bath. When all the methylene chloride in the organic phase has been boiled off, the heating is stopped. (The time in this case is 45 minutes)
Continue mixing and slowly cool the reaction to room temperature Cooling and mixing rates result in mutual agglomeration between particles SDS can be washed by continuously exchanging DI water and mixture solution.

Collect particles by filtration Dry particles in a vacuum oven at 80 ° C. Fluid bed encapsulation Make 3% and 5% PL: PLG 50:50 polymer composition in methylene chloride Dry Place the drug containing the particles into the fluidized bed-Deposit a uniform layer of polymer onto the drug containing the particles using a 5% polymer solution. Adjust spray rate and air flow to get an optimized particle bed.

  • Use a 3% polymer solution to end the process of ensuring that there are no pinholes to unwanted early release, with the ultimate cause being no pinholes causing undesired early release of the drug.

Example 21 Biodegradable microparticles containing an antiproliferative drug
a. 60 days delay b. 365 days release control Organic phase:
Make 20% DLPLG polymer with methylene chloride. DLPLA polymer contains 100% PLA. Weigh 0.02 g of sirolimus into a glass vial. Dispense 2 mL of 20% DLPLG polymer solution into a vial containing triamcinolone.・ Use an orbital mixer to completely dissolve the drug Aqueous phase:
Make 100 mL of 0.1 molar SDS (sodium dodecyl sulfate) in DI water. Dispense 8 mL of 0.1 molar SDS solution into drug / polymer solution. Solvent evaporation:
Place a glass vial containing the reaction mixture under the impeller mixer.

  • Bring up the mixer to 1200 RPM.

  As long as the speed required to produce the desired particle size is known, start slowly and gradually increase the speed of the impeller to produce the desired particle size.

  When the speed to produce the desired particle size is reached, begin heating the container in a 80 ° C. water bath with continuous mixing.

• When all the methylene chloride in the organic phase has been boiled off, stop heating. (The time in this case is 45 minutes)
Continue mixing and slowly cool the reaction to room temperature.

  • The rate of cooling and mixing results in mutual aggregation between the particles.

  The SDS can be cleaned by continuously exchanging the DI water and the mixture solution.

Collect particles by filtration Dry particles in a vacuum oven at 80 ° C. Fluid bed encapsulation Make 3% and 5% PL: PLG 65:35 polymer composition in methylene chloride Dry Place the drug containing the particles into the fluidized bed-Deposit a uniform layer of polymer onto the drug containing the particles using a 5% polymer solution. Adjust spray rate and air flow to get an optimized particle bed.

  • Use a 3% polymer solution to end the process of ensuring that there are no pinholes to unwanted early release, with the ultimate cause being no pinholes causing undesired early release of the drug.

Example 22. Skin filler composition containing anesthetic, corticosteroid and antiproliferative agent
a. Biodegradable microcapsules containing corticosteroids with 30 day delay and 120 day release control b. 60-day delayed, 365-day release controlled, biodegradable microcapsule composition mixture (dry) with anti-proliferative drug
・ Hyaluronic acid, cross-linking 60% -95%
・ Anti-inflammatory including microparticles 5% -20%
・ Antiproliferative agent containing fine particles 5% -20%
・ Anesthetic (lidocaine hydrochloride) 0.1% -5%
Reconstitute in phosphate buffered saline at a concentration of 0.024 g / mL.

Example 23: Encapsulation of antiproliferative agent, biodegradable acrylic acid copolymer
Eggshell-forming phase Dissolve the following to constitute the organic phase.

・ 0.25 g of biodegradable acrylic acid copolymer
・ 0.75g of sirolimus
・ 2 mL of methylene chloride
・ 0.1 mL of ethanol
Aqueous phase 75 mL of 0.5% polyvinyl alcohol solution maintained at room temperature
Disperse the two phases using a mechanical mixer at an appropriate speed to obtain 1200 rpm or the desired particle size Add the appropriate amount of amine (in this case triethylamine)
Continue mixing in a reaction bath in an 80 ° C. water bath for 2 hours Add 0.1 mL of Jeffamine (T-403) to harden the capsule surface Continue mixing and cool the reaction slowly to room temperature Cooling and mixing This rate results in mutual aggregation between the particles. Polyvinyl alcohol can be washed by continuously exchanging the mixture solution with fresh DI water.

Collect the particles by filtration Dry the particles at 80 ° C. in a vacuum oven Fluid bed encapsulation Make 3% and 5% PL: PLG 65:35 polymer composition in methylene chloride.

  Place drug containing dry particles in fluidized bed.

  Deposit a uniform layer of polymer on the drug containing particles using a 5% polymer solution. Adjust spray rate and air flow to get an optimized particle bed.

  • Use a 3% polymer solution to end the process of ensuring that there are no pinholes to unwanted early release, with the ultimate cause being no pinholes causing undesired early release of the drug.

  In addition to biocompatibility, another important feature of gel slurries according to one embodiment that determines the usefulness of gel slurries in various medical fields is a complex combination of their rheological attributes. These attributes include viscosity and its shear modulus dependence, the ratio between elastic and viscous properties in dynamic mode, relaxation behavior and several factors described in detail below. In general, the rheology of an embodiment product can be controlled over a very wide range basically by two methods. In the first method, the rheological properties of each of the two phases forming the viscoelastic gel slurry are controlled to give the desired rheology of the final product. The second method of controlling the rheology of the gel slurry consists of selecting the appropriate ratio of the two phases. However, because these parameters, ie the rheology of the two phases and their ratio, determine some other important properties of the product of an embodiment, the best way to control the rheology is Depending on the case, it should be chosen on the fly.

  Gels suitable for use in products according to one embodiment can represent a variety of different types of rheological bodies that vary from hard and fragile gels to very soft and deformable gels such as fluids. Usually, in the case of gels formed without a cross-linking reaction, such as conventional gelatin, the hardness and elasticity increase with increasing polymer density. The rheological properties of the crosslinked gel are usually a function of several parameters such as the crosslinking density in the gel, the polymer concentration, the composition of the solvent in which the crosslinked polymer is swollen, and the like. Gels with different rheological properties based on hyaluronic acid and hylan are described in the aforementioned US Pat. Nos. 4,605,691, 4,582,865, and 4,713,448. According to these patents, the rheological properties of the gel can be controlled primarily by changing the polymer concentration in the initial reaction mixture and the ratio of polymer to vinyl sulfone crosslinker. These two parameters determine the ratio of equilibrium swelling of the resulting gel and thus also determine the polymer concentration in the final product and its rheological properties.

  A substantial amount of the solvent can be removed from the gel that has previously reached an equilibrium swelling state by mechanical compression of the gel. Compression is obtained by applying pressure to the gel in a closed container with a screen that allows solvent to pass but not gel. The pressure is conveniently applied directly to the gel with any suitable device or through a gas layer or air. In order to compress the gel, there is a method in which centrifugal force is applied to the gel in the container having the above-described semipermeable membrane. The compressibility of the polymer gel slurry depends on many factors, such as gel chemistry, gel particle size, polymer concentration, and the presence of free solvent in the gel slurry. In general, a gel slurry is followed by a much slower removal of solvent from the gel particles when subjected to pressure such that the removal of free solvent in the slurry proceeds rapidly. The kinetics of solvent removal from the gel slurry depends on parameters such as pressure, temperature, equipment configuration, gel particle size, and the initial polymer concentration of the gel. Typically, increasing pressure, temperature, and filtration surface area and decreasing gel particle size, initial polymer concentration, lead to increased solvent removal rates.

              Partial removal of the solvent from the gel slurry makes the slurry more tacky and substantially changes the rheological properties of the slurry. The magnitude of the change depends on the degree of compression, defined below as the ratio of the initial volume of the slurry to the volume of the compressed material.

  The achievable degree of compression, ie the compression of the gel slurry, varies from gel to gel. For example, a hylan gel slurry in saline is easy to reach a compression ratio of 20 or higher.

  Reconstitution of the compressed gel with the same solvent as the initial polymer concentration produces a gel identical to the original. This is evidenced by measuring rheological properties and the kinetics of solvent removal from the gel by centrifugation.

  It should be understood that the polymer concentration in the gel phase of a viscoelastic mixture according to one embodiment can vary over a wide range depending on the desired properties of the mixture, which in turn is determined by the end use of the mixture. However, in general, the polymer concentration in the gel phase can be 0.01 to 20%, preferably 0.05 to 20%. In the case of a hylan and hyaluronic acid pure or mixed gel, the polymer concentration in the gel is preferably in the range of 0.1 to 10%. More preferably, it is 0.15 to 5% when the swelling solvent is a physiological saline solution (0.15 M aqueous sodium chloride).

  As noted above, the selection of soluble polymer for the second phase of the viscoelastic gel slurry according to one embodiment is governed by a number of considerations that are determined by the end use of the product. The polymer concentration in the soluble polymer phase may vary over a wide range depending on the preferred properties of the final mixture and the properties of the gel phase. If the rheological properties of the viscoelastic gel slurry are of greatest concern, the soluble polymer concentration can be selected accordingly with due consideration of the polymer chemistry and its molecular weight. In general, the polymer concentration in the soluble phase is between 0.01% and 70%. Moreover, Preferably it is 0.02%-40%. When hylan or hyaluronic acid is used as a soluble polymer, its concentration is in the range of 0.01 to 10%, preferably in the range of 0.02 to 5%. When other glycosaminoglycans such as chondroitin sulfate or dermatan sulfate are used as soluble polymers, they have a much lower molecular weight, so the concentration may be significantly higher.

  The two phases forming the viscoelastic gel slurry according to one embodiment may be mixed in any conventional manner, such as a stirrer or a mixer. In order to achieve a uniform distribution of the gel phase in the polymer solution, the mixing should continue for a sufficient time. As mentioned above, the gel phase may already be a slurry obtained by collapsing the gel with any conventional method of pushing the gel with a mesh or plate with openings or stirring at high speed with a suitable stirrer. There is. Alternatively, a viscoelastic mixed gel slurry can be prepared by mixing a large gel with a polymer solution and then disintegrating with a mixture with the formation of a viscoelastic slurry by any conventional method described above. In the former case where a mixed gel slurry is prepared according to one embodiment, the gel slurry phase is made of an equilibrium swollen gel where there is no free solvent between the gel particles or some free solvent. In the latter case, this free solvent dilutes the concentration of the polymer solution used as the second phase. The third type of gel slurry used as the gel phase in the mixture is a compressed gel whose properties are described above. When a compressed gel is mixed with a polymer solution, the thermodynamics of each component and their mixture may cause solvent from the solution phase to enter the gel phase and further swell the gel phase to equilibrium.

  The composition of the viscoelastic mixed gel slurry according to one embodiment can vary within wide limits. In the mixture, the polymer solution accounts for 0.1 to 99.5%, preferably 0.5 to 99%, more preferably 1 to 95%, and the rest is the gel phase. The selection of an appropriate composition for the mixture depends on the properties of the two components and the composition, and is governed by the desired properties of the slurry and its end use.

  In addition to the two main components of the polymer gel slurry and polymer solution, the viscoelastic gel mixture according to one embodiment is in addition to the two main components, depending on the end use of the product, microcrystalline cellulose, metal powder, Many other ingredients may be included such as inorganic salts, insoluble inorganic salts, dyes, surfactants, agents such as oils, viscosity modifiers, stabilizers, various physiologically active substances including fillers.

  A viscoelastic gel slurry according to one embodiment essentially represents a continuous polymer solution matrix in which regular or irregularly shaped individual viscoelastic gel particles are uniformly distributed and act as a fluid flow as a fluid. In other words, these viscoelastic gel slurries exhibit a specific viscosity, elasticity, and plasticity. By changing the composition parameters of the slurry, ie the polymer concentration in the gel phase and the solution phase and the ratio between the two phases, the viscosity in steady flow, elasticity in dynamic mode, relaxation properties, viscous behavior and elastic behavior The rheological properties of the slurry, such as the ratio, can be controlled.

  Another group of properties that are strongly influenced by the composition parameters of the viscoelastic gel slurry according to one embodiment relate to the diffusion of various materials into the slurry and from the slurry to the surrounding environment. The diffusion process is extremely important for some specific applications of viscoelastic gel slurries in the medical field, such as prevention of adhesion formation between tissue and drug delivery, as described in more detail below.

  It is well known that adhesion formation between tissues is one of the most common and highly undesirable complications after all surgeries. The mechanism of adhesion formation involves the formation of a fibrin thrombus that ultimately transforms into scar tissue that joins two different tissues to be separated. Adhesions cause a number of undesirable symptoms such as discomfort and pain, and in certain cases lead to life-threatening situations. Even if there is no guarantee for adhesion formation after re-operation, adhesion formation simply requires another surgery to simply remove the adhesion. One means of removing adhesions is to isolate the affected tissue during surgery with some material that prevents the diffusion of fibrinogen into the space between tissues and eliminate the formation of continuous fibrin clots at that interval It is to be. However, low molecular weight and molecular weight materials in the case of plain gel slurries readily appear between the gel particles, especially when the slurry is mixed with body fluid and the gel particles are separated from each other. On the other hand, when the viscoelastic mixed gel slurry according to one embodiment is injected (embedded) into the body, the polymer solution phase located between the gel particles continues to limit diffusion and prevent adhesion even after dilution with the body fluid. . Furthermore, this effect becomes more remarkable as the polymer concentration in the polymer solution phase increases.

  The same is true when a viscoelastic mixed gel slurry according to one embodiment is used as a drug delivery vehicle. Each or both phases of the slurry can be loaded with a physiologically active agent or any other substance that slowly diffuses from the viscoelastic slurry after it has been injected into the body. The diffusion rate can be conveniently controlled by changing the composition parameter of the slurry.

  The components of the viscoelastic mixed gel slurry according to one embodiment affect the behavior of living cells by slowing the rate of movement of living cells through the medium and preventing their adhesion to various surfaces. The degree of manifestation of these effects is highly dependent on factors such as the composition of the two components of the mixture and their ratio, the nature of the surface and the interaction with the viscoelastic gel slurry, the cell type and the like. However, in any case, this property of the viscoelastic gel slurry can be used to treat medical disorders where the regulation of cell migration and cell adhesion is most important in cases such as cancer growth and metastasis.

  In addition to the above two applications of the biocompatible viscoelastic gel slurry according to one embodiment, other possible applications include the use of materials as a viscous surgical tool in areas such as soft tissue augmentation, ophthalmology and otolaryngology, Wound management, treatment of osteoarthritis in orthopedics, etc. In all of these applications, the following basic properties of the mixed gel slurry are utilized: biocompatibility, controlled viscoelastic and diffusion properties, easy controlled residence time at the injection site, and easy handling of recognized materials. (For example, injection through a small diameter needle). The following method was used to characterize the product obtained by one embodiment. The concentration of hylan or hyaluronic acid in the solution was determined by hexuronic acid analysis test using an automated carbazole method (EA Balazs, et al, Analyt. Biochem. 12, 547-558, 1965). . The concentration of hylan or hyaluronic acid in the gel phase was determined by a modified hexuronic acid assay as described in Example 1 of US Pat. No. 4,582,865.

  Viscosity measurement, vibration and relaxation: The rheological properties can be operated in a controlled shear rate and three modes, computerized rheo, and evaluated with a Borin rheometer system. Rheological properties were evaluated with a Bohlin rheometer system that is a computerized rheometer with controlled shear rate and can operate in three modes: viscosity measurement, vibration, and relaxation. The measurement of shear viscosity at low and high shear rates (velocities) characterizes viscoelastic gel slurries and their pseudoplasticity (ratio of viscosity at different shear rates) which is important for many product applications. The measurement of viscoelastic properties at various frequencies characterized the balance between the properties of elasticity (storage modulus G ′) and viscosity (loss modulus G ″). The relaxation feature is the shear modulus over time. It was evaluated as the change in G and expressed as the ratio of the two elastic modulus values at different relaxation times.

  Next, various HA cross-linking approaches are described. The following reaction mainly focuses on the two most reactive functional groups, the hydroxyl group and the carboxyl group.

1. Bisepoxide, (Bisepoxide)
Ethylene glycol diglycidyl ether (Ethyleneglycol ether)
1,4 butanediol diglycidyl ether (butanediol diglycidyl ether)
This method was originally developed to crosslink agarose. Currently, to crosslink HA, the reaction is in dilute NaOH using biseoxybutane and sodium borohydride. A hydrogel was also obtained from the reaction of ethylene glycol diglycidyl ether and hyaluronic acid in ethanol 0.1 N NaOH at 60 ° C. The resulting gel was high in water content (> 95%) and was investigated for use as a responsive degradable matrix for inflammation (stimulation) and injectable drug delivery.

  Hydrogels prepared from hyaluronic acid and alkaline 1,4-butanediol diglycidyl ether were highly porous. This material is activated with periodoxide and then modified with an 18 amino acid peptide containing a cell adhesion domain called Arg-Gly-Asp (RGD) to enhance cell adhesion with the hydrogel. In an alkaline medium, divinyl sulfone also crosslinks with hyaluronic acid, possibly via reaction with hydroxyl groups.

2. Divinyl sulfone (DVS)
In an alkaline medium, perhaps divinyl sulfone also crosslinks with hyaluronic acid via reaction with hydroxyl groups. .

3. Internal Internal Esterification Autocrosslinking (ACP ™, Fidia) is an internal esterified derivative of hyaluronic acid, both intermolecular and intramolecular bonds between the hydroxyl and carboxyl groups of hyaluronic acid. ACP ™ can be lyophilized to a white powder and hydrated to a clear gel. This new kind of biomaterial is post-operative. . . It is used as a barrier to reduce.

4). Photocrosslinking The methacrylic acid derivative of hyaluronic acid was synthesized by esterification of the hydroxyl group with excess methacrylic anhydride as described above for hyaluronic acid butyrate. This derivative was photocrosslinked to form a stable hydrogel using 1-vinyl-2-pyrrolidone and ethyl eosin in triethanolamine as an initiator under 514 nm argon ion laser irradiation. . The use of in situ photopolymerization of a hyaluronic acid derivative that results in the formation of an adhesive gel that surrounds the damaged tissue can provide separation from the surrounding organs to prevent the formation of adhesions. Preliminary cell capsule testing will be performed successfully using the islets of Langerhans to develop a bioartificial source of insulin.

5. Glutaraldehyde cross-linking Although the chemistry of this process was not confirmed, hyaluronic acid strands extruded from cation-exchanged sodium hyaluronate (1.6 MDa) were cross-linked in aqueous glutaraldehyde solution. The strand surface was then reconstructed with poly-D-lysine and poly-L-lysine attachment. The hyaluronic acid strands that floated on the polypeptide showed good biocompatibility and promoted cell adhesion.

6). Metal cation mediated crosslinking Intergel® (FeHA, LifeCore) is a hydrogel formulation of hyaluronic acid formed by chelation with ferric hydroxide. Similar cross-linking of hyaluronic acid is the basis for formulations using copper, zinc, calcium, barium, and other chelating metals. Reddish FeHA gel is under development to prevent post-surgical adhesions.

7). Carbodiimide cross-linking Incert® is a bioabsorbable sponge (Anika treatment) prepared by cross-linking biscarbodiimide and hyaluronic acid in hydrous isopropanol. This method takes advantage of the undesirable tendency of carbodiimides to react with hyaluronic acid to form N-acylureas. In this application, the formation of two N-acyl urea bonds provides chemical stability and cross-linking without byproducts. Because hydrophobic biscarbodiimide is used, Incert® adheres to tissue without the need to sew and retain its effectiveness even in the presence of blood. Recently, studies on rabbit fecal wear have proved effective in preventing postoperative adhesions.

  A low water content hyaluronic acid hydrogel membrane crosslinks a hyaluronic acid (1.6 MDa) membrane together with a water soluble carbodiimide as a coupling agent in an aqueous mixture containing a water miscible non-solvent of hyaluronic acid. The highest degree of cross-linking that resulted in low hydrous hydrogel acid was obtained in 80% ethanol. This membrane with a moisture content of 60% remains stable for 2 weeks after immersion in the buffer. In the presence of L-lysine methyl ester, the cross-linking of the water-soluble carbodiimide and the hyaluronic acid membrane further extended the biodegradation of the hyaluronic acid membrane.

8). Hydrazide Crosslinking Using the hydrazide chemistry described above, hydrogels have been prepared using bishydrazide, trishydrazide, and polyhydric hydrazide compounds. By adjusting the reaction conditions and molar ratios of the reagents, gels with physicochemical characteristics ranging from soft and injectable gels to mechanically hard and friable gels can be obtained. HA-ADH can be cross-linked using commercially available small molecule homobifunctional cross-linkers.

  More recently, in situ polymerization techniques have been developed by cross-linking HA-ADH with a PEG-dialdehyde polymeric crosslinker under physiological conditions. Biocompatible and biodegradable hyaluronic acid hydrogel membranes with well-defined mechanical strength were obtained after evaporation of the solvent. Macromolecular drugs were slowly released from these hyaluronic acid hydrogel membranes, and these new materials promoted re-epithelialization during wound healing.

1. Cross-linking with residual protein An example of this is Hylan (biological substrate). These are hydrogels or hydrosols formed by crosslinking formaldehyde and residual protein containing hyaluronic acid in a basic solution. 13 soluble hylan is a high molecular weight form (8-23MDa) of hyaluronic acid that exhibits enhanced rheological properties compared to hyaluronic acid. Hylan gel is more elastic and viscous than soluble hylan material and can maintain high biocompatibility of natural hyaluronic acid. Hylan has been studied in numerous medical applications.

2. Multicomponent reactions There are 3-4 component reactions known as (1) Parsellini reaction and (2) Ugi reaction.

  In the Parsellini reaction, an aqueous solution of hyaluronic acid is mixed with hydrous glutaraldehyde (or other water-soluble dialdehyde) and added to a known amount of a highly reactive isocyanate, such as cyclohexyl isocyanide.

  In the four-component eel reaction, diamine is added to the ternary mixture.

  The degree of crosslinking is controlled by the amount of aldehyde and diamine.

3. Surface Modification In one example, it should be performed on the surface of polypropylene (PP), polystyrene (PS) activated with argon gas and ammonia gas plasma to diverge the polymer surface. The emitted surface was then modified with succinic anhydride to obtain surface pendant carboxylic acid functionality. This pendant carboxylic acid functional group was then condensed with HA-ADH in the presence of carbodiimide to obtain a hydrophilic, non-tacky and smooth plastic surface. Metal and glass surfaces can be modified by surface activation followed by covalent chemical attachment of an appropriate hyaluronic acid derivative.

  2. As shown below, there are four different therapeutic modification options for HA.

              1. A: HA can be crosslinked at two locations: (1) the position of the hydroxyl group and (2) the position of the carboxyl group.

              2. B: A drug having a functional group that easily reacts with a hydroxyl group and / or a carboxyl group is bound on the HA molecule, and the HA molecule can act as a carrier for the drug.

              3. C: Individual HA molecules can be grafted or covalently attached to polymer chains with pendant functional groups that are susceptible to reaction with hydroxyl and / or carboxyl groups.

              4). D: HA molecules can be implanted into provided liposomes where their functional groups are susceptible to reaction.

  Options for HA therapeutic modification include cross-linked HA hydrogels, HA drug bioconjugates, HA graft copolymers, HA liposomes.

              HA reaction site

5. Carboxyl group chemical reaction Esterification

  Esterified hyaluronic acid biomaterials are prepared by alkylation of alkyl halides with tetra (n-butyl) ammonium salt of hyaluronic acid in dimethylformamide (DMF) solution. These hyaluronic acid esters can be extruded to produce membranes and fibers, lyophilized to obtain sponges, or processed by spray drying, extraction, and evaporation to produce microspheres. These polymers exhibit excellent mechanical strength when dried, but are not very strong when hydrated. The degree of esterification affects the size of the hydrophobic patch that produces a polymer chain network that is more rigid and stable and less sensitive to enzymatic degradation.

  2. Carbodiimide mediated reaction

3. Chemical modification of the carboxyl function of hyaluronic acid with a carbodiimide compound is generally performed in water at pH 4.75.

6). Hydroxyl group chemical reaction i. Sulfation Hydration of hyaluronic acid with pyridine-sulfur trioxide complex in DMF produces a different degree of sulfation, HyalSx (x = 1-4 per disaccharide). Next, sulfated hyaluronic acid HyalS3.5 was immobilized on plasma treated polyethylene (PE) using a diamine polyethylene glycol derivative and a water soluble carbodiimide. The behavior of the thrombin time test and platelet adhesion showed that preparation of hemocompatible and antithrombotic PE surfaces could be expected with this method. HyalSx was converted to a photolabile azidophenylamino derivative and photofixed on a polyethylene terephthalate (PET) film. Nine surfaces coated with sulfated hyaluronic acid show a marked decrease in cell attachment, contamination, and bacterial growth compared to uncoated surfaces. The coating was stable against degradation by chondroitinase and hyaluronidase.

  Hyaluronic acid butyrate is used in particular as a targeted drug delivery system to tumor cells. Butyric acid induces cell differentiation, and various types of human tumors conjugated to hyaluronic acid through the reaction of butyric anhydride with sym-collidinium salt of low molecular weight hyaluronic acid in DMF-containing dimethylaminopyridine. It is known to inhibit proliferation.

ii. Isourea coupling or cyanogen bromide activation The anthracycline antibiotics adriamycin and daunomycin were conjugated to hyaluronic acid via cyanogen bromide (CNBr) activation. This reaction scheme is generally used to activate oligosaccharide accumulation and generate an affinity matrix via a highly reactive isourea intermediate pair. The therapeutic agent attached to one of the hydroxyl functional groups of glycosaminoglycan via oligosaccharide accumulation or urethane linkages, but there was no spectroscopic verification. Furthermore, the severity of the reaction conditions can jeopardize the integrity and biocompatibility of hyaluronic acid.

iii. Peroxidase oxidation Reactive bisaldehyde functional groups can be generated from proximate secondary alcohols on hyaluronic acid by oxidation of sodium peroxidase.

  It can be generated from a secondary alcohol function adjacent to hyaluronic acid by oxidation with the reactive functional group bisaldehyde sodium peroxidase. This chemical property (phenomenon / action) is a standard method for chemical activation of glycoproteins for affinity immobilization or conversion to fluorescent probes. By conjugating with primary amino acid with peroxidase activated hyaluronic acid, it is possible to obtain a cross-linked, peptide-attached or cell-immobilized material containing a cell adhesion domain. Severe oxidation treatment causes strand scission and potential and immunogenic binding to enter the hyaluronic acid biomaterial.

iv. Reducing End Modification Reductive amination of the reducing end of hyaluronic acid has been used to prepare and insert affinity matrices, fluorophore-labeled materials, hyaluronic acid phospholipids into hyaluronic acid liposomes. For example, low molecular weight hyaluronic acid is covalently bound to phosphatidyl ethanolamine, and this complex is used for protective “sugar decoration” on the surface of low density lipoprotein (LDL) particles. End labeling has not been widely used for hyaluronic acid biomaterials or prodrug applications because there is only one attachment store per glycosaminoglycan.

v. Amide Modification In some formulations, natural hyaluronic acid has many naturally deacylated glucosamine units that can be derivatized. Like the reducing end modification, this provides a very low modification rate. However, modification of the N-acetyl group may be important when a commonly used hydrazine decomposition method is used. Hyaluronic acid's limited hydrazine not only creates a free glucosamine residue on hyaluronic acid, but may also lead to base-induced backbone degradation and reducing end modification.

  In yet other experiments, the material may include the following methods.

1. Experimental method Experiment 001-12: Water-in-oil emulsion cross-linking reaction

1. The reaction is a water-in-oil emulsion reaction. 2. React in 1 hour at room temperature. 3. Collect gel particles with a centrifuge. Wash with acetone

  2. Experiment 001-14

1. First prepare X-Linker mix.

  2. Next, the reaction mixture is prepared.

  3. Add X-Linker mix of “a”-“e” to HA. A reaction takes place.

  4). Mix well with a spatula so that the X-Linker mix goes well into the HA.

  5. Allow each reaction to occur at room temperature with mixing every 30-60 minutes.

  6). The product after 8 hours of reaction is a crosslinked hyaluronic acid gel.

  7). Leave at 52 ° C. for 3 hours with mixing every 30 minutes.

  8). Wash 3X with PBS.

3. Boundary conditions of components in the HA X-linking process Experiment 001-16: X-linker mixed storage period and reaction temperature X-linker mixes must be used within 24 hours of preparation and stored at room temperature.

  1. The reaction temperature of 50 ° C. is too high to hold for 1 hour or more.

Experiment 001-17: Storage period of 1% NaOH A NaOH solution containing X-linker should be used within one hour of preparation.

  3. 1. The NaOH concentration at is too low to obtain a complete reaction product.

1. X-Linker storage period-BDDE
1. Experiment 001-18: Once the mixture containing BDDE is mixed with NaOH, it can be seen that it should be used within 3 hours.

4). X-Linker storage period-DVS “TBD”
5. Experiment 001-19

1. Add A and B together and mix well.

  2. Leave at room temperature for 2 hours, mixing every 30 minutes.

  3. Leave at 50 ° C. for 1 hour with mixing every 30 minutes.

4). The product is very similar to a product called Juvederm.

  6). Experiment 001-21

1. Add together with each of A and B1-B5 and mix well.

  2. Leave at room temperature for 2 hours, mixing every 30 minutes.

  3. Leave at 50 ° C. for 1 hour with mixing every 30 minutes.

  4). The product is very similar to a product called Juvederm.

7). Effect of X-Linking level Experiment 001-22: BDDE (1,4-butanediol diglycidyl ether)

1. Experiment 001-25: DVS (Divinylsulfone)
2.

In one embodiment, the HA can be cross-linked in series to form a system with single phase properties. Biocompatible cross-linked polymers such as IPN cross-link heteropolysaccharides to form a single cross-linked material and perform one or more additional cross-links on the single cross-linked material to form multiple cross-linked materials. The multiple cross-linking material has a core that can last longer in the human body than the single cross-linking material. As a result, a smooth continuous material is formed from a small amount to a highly crosslinked core. A little cross-linked material can allow HA to easily enter the human body with a small gauge syringe, but such a little cross-linked material does not last long in the human body. However, unlike traditional HA skin fillers, highly cross-linked materials do not require long-lasting, regular touch-up of body enhancement in the human body.

  Stable and non-aqueous suspensions of delayed crosslinkers according to preferred embodiments can be controlled by changing any of the following:

1) Cross-linked compound used 2) Particle size of HA in suspension 3) pH of fluid containing HA
4) Concentration of HA suspension (ie load)
5) Solution temperature As an example, when used under similar conditions, the type of molecular weight of the HA compound can be effectively used to control the exact crosslinking time of the aqueous solution. More specifically, a molecular weight HA that is slower than a low molecular weight acid suspension is a cross-linked suspension.

  Regarding the particle size of the suspended hyaluronic acid, the time required for crosslinking the water-soluble polymer solution increases as the particle size increases. Conversely, as the particle size decreases, the time required for crosslinking of the water-soluble polymer solution decreases.

  The pH prior to crosslinking of the water soluble polymer solution can be used to control the crosslinking time. The pH of the water-soluble polymer solution affects the dissolution rate of the stable and non-aqueous suspension of the delayed crosslinker. Specifically, when the suspension contains most of HA particles, the dissolution rate of the crosslinking agent suspension increases as the pH of the water-soluble polymer solution increases. On the other hand, when the suspension contains most of the borax particles, the dissolution rate of the crosslinking agent suspension decreases as the pH of the water-soluble polymer solution decreases. Conversely, when the suspension contains most of the borax particles, the dissolution rate of the crosslinker suspension decreases as the pH of the water-soluble polymer solution decreases, When the suspension contains most of the HA particles, the dissolution rate of the crosslinking agent suspension increases as the pH of the water-soluble polymer solution increases.

  Both the stable and non-aqueous suspension concentration (ie loading) of the delayed HA crosslinker in the water soluble polymer solution and the content of the suspension similarly affect the crosslinking time of the water soluble polymer solution. give. As either the concentration of the suspension of the delayed HA crosslinking agent in the water-soluble polymer solution or the content of the crosslinking agent suspension increases, the crosslinking time of the water-soluble polymer solution decreases. Conversely, as either the concentration of the delayed boron crosslinker suspension in the water soluble polymer solution or the content of the crosslinker suspension decreases, the crosslinking time of the water soluble polymer solution increases. To go.

  The temperature can be used to change the crosslinking time of the water-soluble polymer solution. As the temperature of the water-soluble polymer solution increases, its crosslinking time decreases. Conversely, as the temperature of the water-soluble polymer solution decreases, its crosslinking time increases. Furthermore, the increase or decrease of the crosslinking time of the water-soluble polymer solution depends on the type of clay used to form a stable and non-aqueous suspension of the delayed HA crosslinking agent.

  In addition, materials such as molecular microspheres, polymeric micelles, soluble polymers, hydrogel format materials are used to protect drugs from biochemical degradation and as components of biomedical applications, especially drug delivery devices. As used, it shows great potential. The design and engineering of medical macromolecules (eg, macromolecules for use under physiological conditions) are generally subject to special and stringent requirements. In particular, such polymeric materials must be compatible with the biological environment in which they are used. That is, they exhibit hydrophilic characteristics. They are also suitable biodegradable (ie, they break down into low molecular weight species. High molecular fragments are sequentially metabolized or excreted in the body, leaving no trace). Must prove. Biodegradability is typically achieved by synthesizing or using a polymer having a hydrolytically unstable bond in the backbone. The most common chemical functional groups with this feature are esters, anhydrides, orthoesters, and amides. Chemical hydrolysis of the hydrolytically unstable backbone is a common mechanism for polymer degradation. Biodegradable polymers are either natural or synthetic. Synthetic polymers commonly used in medical applications and biomedical research are polyethylene glycol (pharmacokinetic and immune response modifiers), polyvinyl alcohol (drug carrier), and poly (hydroxypropylmethacrylamide) (drug carrier). including. Natural polymers are also used for biomedical applications. For example, dextran, hydroxyethyl starch, albumin, and partially hydrolyzed proteins prove to be useful in applications from plasma substitutes to radiopharmaceuticals and parenteral nutrition. In general, synthetic polymers have significant advantages over natural materials that can be tailored to provide a wide range of properties and predictable lot-to-lot uniformity.

  In one embodiment, the linker is a dicarboxylic acid with at least 3 atoms between the carbonyls and includes a heteroatom alpha to the carbonyl forming an ester; the linker is a dicarboxylic acid with at least 3 atoms between the carbonyls. If it is an acid and does not contain the heteroatom alpha to the carbonyl forming the ester, the release half-life is just over 100 hours; the linker is a dicarboxylic acid with two atoms between the carbonyl and the tether is nitrogen And the reactive hydrogen, the release half-life of HA is about 0.1 to about 20 hours; the release half-life is such that the complex is PHF-SA-Gly-CPT, PHF- (methyl) SA-Gly- CPT, PHF- (2,2-dimethyl) SA-Gly-CPT, PHF- (2-nonen-2-yl) SA-Gly In 0.05M phosphate buffer, 0.9% saline (pH 7.4) at 37 ° C., not CPT, PHF-SA-Gly-Taxol, or PHF-SA-Gly-Illudin Measured in

  In some embodiments, poly is acetal. In other embodiments, the poly is a ketal. In some embodiments, the acetal is PHF. In some embodiments, Ri is H. In other embodiments, Ri is CH3. In some embodiments, R2 is -CH (Y) -C (O)-, wherein Y is one of the naturally occurring amino acid side chains. In some aspects, R2 is an aryl group. In some embodiments, R2 is a heteroaryl group. In other embodiments, R2 is an aliphatic ring. In some embodiments, when Ri and R2 are attached together with nitrogen, a ring is formed. Other embodiments are well known to those skilled in the art. For example, some embodiments are described in US 2010/036413, the contents of which are incorporated by reference.

  FIG. 1 shows an exemplary system for producing multiple cross-linked HAs in series. In FIG. 1, HA material P-15 and sodium hydroxide P-16 are provided to the gate and measurement unit P-14. The output is provided to mixer P-17. Crosslinker source E9 is provided to chemical reactor I-7 and its output is stored in tank P21. Thereafter, the stored crosslinked HA can be atomized (sprayed?).

  FIG. 2 shows another exemplary system for producing multi-bridge HA in series. In FIG. 2, HA and sodium hydroxide are provided to a chemical reactor that receives multiple crosslinker sources such as PVS1, PVS2, PVS3 sources. The chemical reactor produces multiple cross-linked HA in series, which is then washed in a chamber to remove residues and change the pH to about 7.4. The chamber receives distilled water and PBS (phosphate buffered saline) at a pH of about 7.4. The cleaned output is final assembled and sent to the broadcast equipment.

FIG. 3 shows an exemplary diagram of the resulting multi-crosslinked HA. A mildly cross-linked extension allows the composition to be injected through a small gauge needle, and a second in-line cross-linking center features resistance to absorption by biological processes,
A first portion 300 of a first polymer having a mildly crosslinked extension or arm;
A polymeric second portion 310 having a first serial crosslinking center overlapping the first portion and one or more lightly crosslinked extensions adjacent to the serial crosslinking center;
A third portion 320 of the polymer having a second series cross-linking region overlapping the second portion and one or more lightly cross-linked extensions adjacent to the series cross-linking center.
It is a composition containing this.

  Region 350 may be a multiple cross-link for biodegradation resistance. The polymer is one of collagen, hyaluronic acid, cellulose, protein, carbohydrate, biological extracellular matrix: polymer.

  In other embodiments, the biocompatible cross-linked IPN polymer can be cross-linked with heteropolysaccharides to form a first cross-linked material; It can be carried out by forming a crosslinked material. Results Single phase HA can be used to enhance soft tissue with biocompatible crosslinked polymers.

  In addition to the above-described method of cross-linking both components of the blend to obtain IPN and semi-IPN, semi-IPN comprises both of the components of the blend in the presence of a crosslinker and in the presence of a natural acidic polysaccharide. It can also be obtained by polymerization or its semi-synthetic ester derivatives.

  In the following examples, the proportion of HA composition is varied from 75% to 99% of the total composition and the proportion of crosslinker is varied from 1% to 25% as follows.

As the proportion of HA increases, the material is softer but less resistant to biodegradation. As more cross-linking agent is introduced, the material becomes harder and lasts longer. The multiple series crosslinking process is soft to the touch but has the advantage of long lasting. Changes in mechanical and physical properties that continue to radiate from the IPN and become softer continuously, make the polymer stiffer, and at the same time make it more compliant and better biocompatible, giving a natural feel .

  The multiple cross-linking process is similar to an individual or digital process in which the HA is cross-linked for the first, second, and third before forming an addition of serial cross-links. This individual or digital process is a comparison with a conventional continuous process. In one embodiment, the IPN center is where there is a relative aqueous front.

  It should be noted that for the purpose of HA longevity, hydrolysis is not preferred, so that more hydrophobic cross-linking agents are superior. Sterically crowded crosslinkers are also desirable for the same reasons listed. However, the hydrophobicity in this case reduces the biocompatibility of the HA polymer and is likely to be an illegal and unnecessary foreign body reaction. Cross-linking agents used in all parts of the process also change the properties of longevity, biocompatibility and physical properties. Application requirements determine the ideal polymer composition that provides a balance of their properties.

  Through a series of cross-linking steps, the cross-linked HA (hyaluronic acid) molecular macrostructure is highest on the surface connecting with the basic aqueous medium and lowest cross-linked toward the central core. After the initial cross-linking reaction step, the cross-linked HA chain lost important mobility. Accordingly, the IPN (interpenetrating network) polymer is easily formed by the subsequent sequential crosslinking reaction.

  The rheology of crosslinked HA is characterized as having non-Newtonian fluid behavior. According to the theory of mixing in a two-dimensional cavity flow, the key to effective mixing is to produce repetitive stretching and folding, an operation called a “horseshoe map”. Mixing can be accomplished by mixing viscous Newtonian and non-Newtonian fluids as described on pages 211-252 of Chavan et al., “Mixing of Viscous Newtonian and Non-Newtonian Fluids”. Yes, the contents of this document are incorporated by reference. As another method, Paulo E. et al. It can be mixed as described in “Stretching and mixing of non-Newtonian fluids in time-periodic flows” by Arartia (non-Newtonian fluid expansion and contraction in time-periodic flow). It is incorporated by doing. The mixing process can be scaled as described in Wilkens et al., “How to Scale Up Mixing Processes in Non-Newtonian Fluids”. Are incorporated by reference.

  In the final product, the cross-linking level is non-uniform throughout the HA polymer matrix and the polymer chain is biaxially oriented (biaxial stretching). The orientation is a result of the polar medium in which the HA polymer is present.

  Various implementations for mixing the reactants are described below.

1. Manual mixing:
In this example, mixing is manual. This method is easy to assemble and does not require expensive equipment. The type of crosslinker * and the amount of crosslinker used in various processes ** may be optimized for the desired properties.

a. HA is dissolved in sodium hydroxide (the reactivity of the reaction increases with increasing pH)
b. Cross-linking reaction i. Add a cross-linking agent
ii. Mix the mixture mechanically iii. The number of cross-linking steps can vary depending on the desired properties c. Repeat the “b” step with the same crosslinker and / or another crosslinker type. The amount of cross-linking agent may be the same or different depending on the desired physical properties.

  d. Purification of reaction products: It is important to wash reactive organisms to remove processing aids, unreacted reactants and impurities so that the main product can perform its function without interference .

  Since all chemical components used in this reaction are water soluble and the reaction product is not water soluble, the crosslinked HA polymer can be purified using DI water. Furthermore, water swells the reaction product, creating many folds that easily diffuse impurities away from the polymer. In addition, water can easily diffuse and eliminate impurities from the polymer and swell many pleats of reaction products. Continuous flushing with DI water speeds up the purification process and effectively removes unwanted impurities from the reaction product.

The pH of the water before mixing with the crosslinked HA and after mixing with the crosslinked HA is a good indirect indicator for the cleaning effect throughout the process. The of the water pH before and after shoulder not significant changed.
e. Balance the phosphate buffered saline and average the level where the pH is about 7.4: Drain all the water from the crosslinked HA. Add at least 3 times the volume of cross-linked HA fresh PBS to allow the mixture solution to balance in about 2 hours. . The process is repeated twice more until the pH is around 7.4 ± 0.7.

  2. Mechanical pressure pump: The advantage of this approach is its continuous nature. The advantage of continuous processing is the flexibility in the amount of product that can be produced. Although there is an upper limit, it is different from quantitative batch processing.

  a. HA is dissolved in NaOH, and the concentration of NaOH directly affects the reactive OH terminal of the HA polymer chain.

  b. The cross-linking reaction takes place as soon as the HA in the NaOH solution comes into contact with the cross-linking agent.

  i. The type of cross-linking agent may change.

  ii. The amount of crosslinker may vary.

  c. The rapid and efficient mixing is important for the reproducibility of the final product.

  i. As shown in FIG. 2, mixing in this manner occurs while the reaction mixture moves through various pipe inner diameters.

  ii. The number of times the reaction mixture is transferred to the mixing tube device can be optimized so that the desired physical properties are achieved.

  3. Mechanical peristaltic pump: This approach differs in the mixing components in this method compared to the mechanical pressure pump method. In the mechanical pressure method, the mixing mechanism is a mixing tube device (FIG. 1), and in the peristaltic pump method, the mixing mechanism is in a roller (the pumping mechanism is the same).

4). Biaxially oriented crosslinked HA with molecular macro structure
a. Surfactant In this embodiment, the HA molecule has several hydroxyl ends that may be reactive hydroxyls in an alkaline medium such as easily forming an ether linkage with a vinyl functional termination molecule. The alkaline medium makes many HA modifications a one-step reaction.

1,4-butanediol dimethacrylate,
1,4-butanediol diacrylate,
1, 6-hexanediol diacrylate,
1, 6-hexanediol dimethylacrylate,
Ethylene glycol dimethacrylate
Ethylene glycol diacrylate
Poly (ethylene glycol) * diacrylate
Poly (ethylene glycol) * dimethylacrylate
* Indicates various types of molecular weight polyethylene glycol.

  These hydrophobic crosslinkers are usually not water soluble / water miscible. Surfactants are required to create a favorable environment in which each molecule combines and cause chemical reactions. Because the polar spectrum is mediated by surfactants and media, the crosslinked HA product is highly biaxially oriented. The orientation macrostructure of polar and nonpolar molecules follows the polar orientation of the medium containing the product.

  The opposite molecular macrostructure moves away from the surface of the medium and collects in the central core of the molecule. In this case, a more hydrophobic interpenetrating cross-linked HA network. The molecules maintain their original softness and biocompatibility.

  Furthermore, the molecular macrostructure crosslinked HA is oriented to adjacent molecular species having similar physical properties. This property is unique in that the physical properties are self-adjusting.

b. Use of Pre-Independent Crosslinking HA Other methods used to characterize the product according to one embodiment are described in the following examples that illustrate preferred embodiments of the embodiments, without limitation thereof. Variations and modifications can, of course, be made without departing from the spirit and scope of the invention. For example, HA can be used as a facial filler, skin filler, butt filler, breast filler and other body part filler. The transplanted tissue of the present invention can be instilled with designated drugs and other chemicals or diagnostic agents before or after transplantation. Examples of such agents include, but are not limited to, antibiotics, chemotherapy, other cancer therapeutics, brachytherapy materials for radiation effects, radiopaque for region identification Materials or metal materials, hemostatic agents for bleeding control, growth factor hormones, immune system factors, gene therapy, biochemical indicators or vectors, other types of therapeutic or diagnostic materials that can enhance the patient's therapeutic effect It is done.

  Advantages of one IPN embodiment include one or more of the following. The natural feeling is obtained by viscoelastic harmony of properties between existing tissue and transplanted tissue. This can be done by manipulating the viscous component of the transplanted tissue through the flow characteristics through the ratio of particle size and particle size distribution. The elastic component is inherent in the material tertiary structure (molecular weight and steric hindrance) and crosslink density. Interpenetrating polymer network hydrogels have many desirable properties. These include high moisture content and high tensile strength and are excellent properties of interpenetrating polymer network hydrogels for use in skin filling applications. Other advantages and features are, among other things, longevity without touch-up, degradation of circulating blood volume, liberation compliance and isosmotic pressure.

  Although the present invention has been described with particular reference to breast, buttocks, or body grafts, it will be appreciated by those skilled in the art that the present invention can be applied to other body parts such as the face. Therefore, the present invention can also be applied to replacement of missing or damaged soft tissue / structural tissue / bone, or replacement of cosmetic tissue or aggregate.

  Although the invention has been described with reference to specific embodiments thereof, many variations and modifications and other uses will become apparent to those skilled in the art. Accordingly, it is desired that the invention be limited not by the specific disclosure herein, but only by the appended claims. Other methods used for product characterization according to one embodiment are set forth in the following example that illustrates a preferred embodiment of one embodiment that is not limiting. It will be appreciated that changes and modifications can be effected without departing from the scope and spirit of the invention.

Claims (23)

Cross-linking the heteropolysaccharide to form a single cross-linked material and performing one or more additional cross-links on the single cross-linked material to form a multiple cross-linked material;
Multiple cross-linked materials have one or more IPN regions that resist biodegradation in the human body than single cross-linked materials, and one or more single cross-linked extensions extend from the IPN, and the combination of IPN and extension A method of forming a biocompatible crosslinked polymer having an interpenetrating polymer network (IPN) that provides (characterized by) resistance, soft touch, and ease of introduction into the body.   2. The method of claim 1, comprising injecting a biocompatible crosslinked polymer in a minimally invasive manner.   The method of claim 1, comprising injecting a minimally invasive and biocompatible crosslinked polymer into the skin.   The method of claim 1, comprising injecting the biocompatible crosslinked polymer under the skin using a syringe with minimal invasiveness.   The method of claim 1, comprising injecting the biocompatible crosslinked polymer under the breast or hip or soft tissue using a syringe in a minimally invasive manner.   The method of claim 1, comprising injecting the biocompatible crosslinked polymer beneath the soft tissue using a minimally invasive mechanical pump.   The method according to claim 1, wherein the macromolecule comprises any one of collagen, hyaluronic acid, cellulose, protein, saccharide, and extracellular matrix of biological system.   The method of claim 1, wherein the polymer comprises a thermoplastic that converts the polymer to a thermosetting resin.   The method according to claim 1, comprising using a crosslinking agent, forming a thermosetting polymer, or forming a crosslinked copolymer with another kind of polymer using a polyfunctional monomer. Implanting a composition with a biocompatible viscoelastic gel slurry comprising a two-phase mixture;
The two phases are as follows:
The first phase is a particulate biocompatible gel phase, wherein the gel phase includes chemically cross-linked glycosaminoglycan, and the glycosaminoglycan is selected from at least one other polymer selected from the group consisting of polysaccharides and proteins. Chemically co-cross-links, the gel phase is swollen in a physiologically acceptable aqueous medium and uniformly distributed in the second phase;
The second phase comprises a polymer solution of a hydrophilic biocompatible polymer selected from the group consisting of polysaccharides, polyvinylpyrrolidone, polyethylene oxide in the physiologically acceptable aqueous medium;
The polymer solution in the two-phase mixture is comprised between 0.01% and 99.5%, and the gel phase constitutes the remainder of the body part where enhancement is desired;
The method of claim 1.   The method of claim 1, comprising adding a substance to the composition for biocompatibility.   The method of claim 1, comprising controlling drug release at a predetermined timing according to a physiological event.   2. The method of claim 1, comprising having a biocompatible and sex degradable agent.   2. The method of claim 1 comprising dispensing the drug uniformly throughout the biodegradable polymeric material matrix.     The method of claim 1, wherein the drug is contained in a core shell structure, and the drug release is based on diffusion and solubility.   Polylactide (PLA) and polyglycolide (PGA) tailored to meet the mechanical performance and resorption rate required for applications from non-structural drug delivery polymer applications to biodegradable screw and anchor applications 2) The method of claim 1, comprising providing a polymer having a drug comprising any one of PLA / PGA copolymers.   2. The method of claim 1, comprising releasing the drug into the biological environment at the same rate as the degradation rate of the polymer and the rate of the drug diffusing from the polymer matrix.   The method of claim 1, comprising mixing the drug carrier polymer composition and the filler polymer composition in a predetermined ratio. Anesthetic,
Lidocaine,
A compound that reduces or eliminates an acute inflammatory response,
The method according to claim 1, comprising adding any of the compositions selected from the group consisting of steroids, corticosteroids, dexamethasone, and triamcinolone.   The method of claim 1, comprising providing a sustained release material or an immediate release material. A lightly cross-linked extension allows the composition to be injected through a small gauge needle, and a second in-line cross-linking center features resistance to absorption by biological processes,
A first portion of the first polymer having mild crosslinking;
A first series cross-linking center overlapping the first part and a second part of the polymer having one or more lightly cross-linked extensions adjacent to the series cross-linking center;
A composition comprising: a second series cross-linking center overlying the second part; and a third part of the polymer having one or more lightly cross-linked extensions adjacent to the series cross-linking center.   The composition according to claim 21, wherein the polymer comprises collagen, hyaluronic acid, cellulose, protein, saccharide, and an extracellular matrix of a biological system. The composition according to claim 21, wherein the polymer comprises any of free radical scavengers and / or antioxidants and / or vitamins and / or enzyme inhibitors.