EP2844263A1 - Plant extract for the treatment of an hiv infection as well as products relating thereto - Google Patents

Abstract

The present invention relates to pharmaceutical composition for use in a method of preventing and/or treating an HIV infection, a product suitable for the prevention of HIV transmission, a piece of victuals for the prevention and/or treatment of an HIV infection and a kit comprising at least two HIV medicaments as well as a method of producing a polyphenol-enriched Cistus extract, the extract and its use as a medicament.

Description

Plant extract for the treatment of an HIV infection

as well as products relating thereto

The present invention relates to pharmaceutical compositions for use in a method of preventing and/or treating an HIV infection, a product suitable for the prevention of HIV transmission, a piece of victuals for the prevention and/or treatment of an HIV infection and a kit comprising at least two anti-HIV medicaments as well as a method of producing a polyphenol-enriched Cistus extract, the extract and its use as a medicament.

Acquired immunodeficiency syndrome (AIDS) is a condition in which progressive failure of the immune system results in life-threatening opportunistic infections and cancers to thrive. AIDS is caused by the human immunodeficiency virus (HIV), a lentivirus (a member of the retrovirus family). Infection with HIV occurs by exposure to and transfer of body fluids including blood, semen, vaginal fluid, pre-ejaculate, breast milk and cerebrospinal fluid. Within these body fluids, HIV is present as both free virus particles and within infected cells. The four major routes of transmission are unsafe sex, contaminated needles, breast milk, and transmission from an infected mother to her baby at birth (perinatal transmission). Screening of blood products for HIV has largely eliminated transmission through blood transfusions or infected blood products in the developed world.

There is currently no cure for HIV infection. Since 1996 treatment consists of highly active antiretroviral therapy, or HAART and has been used with many HIV-infected individuals. Current HAART options are combinations (or "cocktails") consisting of at least three drugs belonging to at least two types, or "classes," of antiretroviral agents. Classes of antiretroviral drugs include entry inhibitors, nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs, NtRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors and integrase inhibitors. The majority of approved anti-HIV drugs are NRTIs, NtRTIs, NNRTIs and protease inhibitors. However, HAART has severe side- effects and the timing for initiation of HIV treatment is still subject to debate. The United States Panel on Antiretro viral Guidelines for Adults and Adolescents in 2009 found that "Patients initiating antiretroviral therapy should be willing and able to commit to lifelong treatment and should understand the benefits and risks of therapy and the importance of adherence".

New classes of drugs such as entry inhibitors provide treatment options for patients infected with viruses already resistant to common therapies, although they are not widely available and not typically accessible in resource- limited settings. Anti-retroviral drugs are expensive, and the majority of the world's infected individuals do not have access to medications and treatments for HIV and AIDS.

In summary, there is a constant need for new HIV inhibitors and to improve current treatments by providing a drug or pharmaceutical composition which shows good availability, decreased side effects, simplifies drug regimen, is effective in patients resistant to common anti-HIV drugs and/or is easily accessible.

Surprisingly, it has now been found that an extract from the plant Cistus is a suitable candidate as HIV therapeutic. Particularly it has been shown that the extract inhibits the entry of HIV into a patient's cell. As the entry of the virus is a prerequisite for the cell's or patient's infection, the HIV entry is a keystone in HIV replication. A substance or composition inhibiting HIV entry is an interesting drug for HIV prevention and treatment. Initial experiments (Figure 1) indicating anti-HIV activity of Cistus incanus (Ci) extracts were performed with the commercial decoction of Cistus incanus CYSTUS052® (Dr. Pandalis Urheimische Medizin GmbH und Co. KG, Glandorf, DE). Subsequently, anti- HIV activity was confirmed for Ci extracts prepared from fresh whole plants and parts of plants, including leaves, stems and roots and for Ci extracts prepared with different solvents (water, methanol) (Figure 2). Extracts prepared from a commercial herbal tea consisting of dried whole plants of Cistus incanus also showed anti-HIV activity and are designated here as Ci extracts. Ci extracts inhibited infection of exemplary virus strains that use either CXCR4 or CCR5 as co-receptors for entry, indicating that Ci extracts are active against both X4-tropic and R5-tropic HIV-1 strains (see Figure 3). Pseudotyping experiments confirm the involvement of HIV-1 envelope proteins in antiviral activity of Ci extracts (Figure 3). Assays for classification of the anti-HIV activity show that Ci extracts inhibit virus entry (Figure 4). Pre-incubation of the virus with the Ci extracts decreases virus infectivity, indicating that Ci extracts have virucidal activity (Figure 5). These results show that Ci extracts inhibit HIV infection by preventing entry of the virus into the host cell and strongly suggest interactions of components of Ci extracts with viral envelope proteins (i.e. gpl20, gp41) for virus inhibition. Moreover, Cistus extracts were separated in either polyphenol-free or enriched in polyphenols. Both fractions were tested for anti-HIV activity (Figure 6). The polyphenol-free fraction exhibited a complete loss of inhibitory activity, whereas the enriched Cistus polyphenols retained it completely indicating the relevance of Cistus polyphenols in anti- viral activity. Additionally, enriched Cistus polyphenols showed reduced cellular toxicity towards primary human cells (Figure 7). Furthermore, anti- viral activity was confirmed at DNA and cell level with primary human cells including PBMCs and macrophages that represent the natural HIV target-cells (Figures 8 and 9). Finally, it could be shown that the anti-HIV effect of an extract of Cistus incanus (optionally polyp henol-enriched) is due to an inhibition of HIV entry at the stage of HIV attachment by disturbing the ability of viruses to attach to the target cells (Example 7, Fig. 10). These results also indicate the possibility to modify an extract of Cistus incanus plants by e.g. reduction by depletion of distinct substances (or enrichment of others) which can be used to design an optimized anti-HIV agent as described herein. Accordingly, the present invention relates to a pharmaceutical composition comprising a Cistus extract for use in a method of preventing and/or treating an HIV infection.

Cistus is a genus of flowering plants in the rockrose family Cistaceae, containing about 20 species. They are perennial shrubs found on dry or rocky soils throughout the Mediterranean region, from Morocco and Portugal through to the Middle East, and also on the Canary Islands. The leaves are evergreen, opposite, simple, usually slightly rough- surfaced, 2-8 cm long; in a few species (notably C. ladanifer), the leaves are coated with a highly aromatic resin called labdanum. They have showy 5-petaled flowers ranging from white to purple and dark pink, in a few species with a conspicuous dark red spot at the base of each petal, and together with its many hybrids and cultivars is commonly encountered as a garden flower. Methods of cultivating Cistus are well known in the art.

There are about 20 species in the genus including but not limited to Cistus albanicus, Cistus albidus, Cistus chinamadensis , Cistus clusii, Cistus creticus, Cistus crispus, Cistus heterophyllus, Cistus incanus, Cistus ladanifer - Gum Rockrose, Cistus laurifolius, Cistus libanotis, Cistus monspeliensis - Montpelier Cistus, Cistus munbyi, Cistus ochreatus, Cistus osbeckiaefolius, Cistus parviflorus, Cistus populifolius, Cistus pouzolzii, Cistus psilosepalus (also refered to as Cistus inflatus or Cistus hirsutus), Cistus salviifolius - Salvia Cistus, Cistus sintenisii, Cistus symphytifolius and Cistus varius. The extract is preferably isolated from the species Cistus incanus. Cistus incanus includes two subspecies, Cistus incanus ssp. tauricus as well as C. incanus ssp. undulatus. Of these, the subspecies C. incanus ssp. tauricus and new variety Cistus incanus ssp. PANDALIS (EU 27578) are especially preferably used for extraction.

The extract is isolated from the aerial parts of the plants. Preferably, the aerial shoots of the plants which have regrown in the same year are used. The plant parts are subjected to extraction directly following harvest, i.e. in raw condition. Alternatively, the plant parts can be dried prior to the extraction. Subsequently, the leaves of the plant are minced in a suitable manner, for example by grinding them or cutting them.

Cistus has a long history of use in traditional medicine, e.g. as Ladanum, obtained from Cistus creticus subsp. creticus in the eastern Mediterranean region. In ancient times it was also used for embalming and aphrodisiac purposes. Various classical writers mention its use as an emetic, for weak stomachs and livers, disorders of the spleen and diarrhoea. It has also been used for hair loss, scurvy, catarrh, asthma, stomach ulcers and cancer, as a protection against the plague and as a fumigant. In Spain C. clusii is still used as an antiinflammatory and antirheumatic drug and to improve blood circulation. In Greece an extract from cistus is used for skin inflammation. In Turkey several species, especially C. laurifolius, are still widely used in folk medicine to treat a range of conditions. Infusions of the leaves of various species have been recommended for colds and rheumatism. Additionally, it has been shown that an extract of the rockrose (C. incanus ssp. tauricus) inhibits the propagation of the influenza viruses significantly in vitro. Cistus incanus has been used in traditional medicines for the treatment of various skin diseases and inflammatory conditions for several centuries. Cistus incanus extracts have been attributed with antiinflammatory, antimycotic and antibacterial activites (Kalus et al. 2010). A Cistus incanus extract is commercially available (Dr. Pandalis Urheimische Medizin GmbH und Co. KG, Glandorf, DE). The Cistus incanus plant is rich in polyphenolic compounds (Petereit et al. 1991; Droebner et al. 2007). These compounds are widely spread plant metabolites and are characterized by the presence of more than one phenol group (Bravo 1998). Polyphenolic compounds are known to exhibit a wide range of biological activities.

Due to its long-standing medical use, Cistus can be regarded as safe and as having few side-effects, if at all. Cistus is commercially available, e.g. in fresh form e.g. as a plant (Ruhlemann's Krauter & Duftpflanzen, Horstedt, DE) or seed (Pflanzen-Vielfalt, Weilheim, DE) as well as in processed form e.g. in dried form or as tea, capsule, tablet, creme, salve, brew for gargling, essential oil or spray (e.g. from Naturprodukte Dr. Pandalis GmbH & Co. KG, Glandorf, DE). It is suggested that the beneficial effects are due to the contents and composition of secondary plant substances, particularly of polyphenols, which are present throughout the genus. However, plants and species with higher contents of these compounds are preferred. Polyphenols are widely known for their antioxidant behavior, which refers to their potential to combat free radicals. In the human body, these are especially "reactive oxygen species", which are often caused by environmental influences (UV-rays, chemical noxae), but also by an unbalanced, unhealthy diet. According to latest state-of-the-art knowledge, many diseases can be linked to "reactive oxygen species". Cell damages attributed to radicals can lead to a number of diseases (e.g. immune diseases, mental exhaustion, arthritis) if they are not limited by "antioxidant scavengers". High contents in polyphenols simultaneously indicate a high antioxidant potential.

The fact that Cistus incanus contains a combination of multiple active components is a great advantage for the treatment of HIV-infection, since antiviral measures based on the combined activities of multiple components counteract the formation of resistant viruses. A further advantage of Cistus incanus extracts is the fact that they are very well tolerated (LD₅₀ > 400 μg/ml); extracts are sold as herbal medicine), whereas many current anti-HIV drugs have serious side effects. Another great advantage of polyphenolic compounds like flavonoids is their anti- inflammatory effects (Pan et al. 2010). Acute and chronic inflammations are adverse effects of HIV-infection that underlie multiple serious comorbidities of HIV infection, including various cancers and neurological dysfunctions. Potential application of Ci extracts could be as a kind of food supplement, ingested either during HAART or in the absence of HAART, for example during episodes of HAART interruption or in settings where HAART is not available. The ingestion of Ci extracts during HAART may allow reduction of the required dose of conventional antiviral drugs, reducing the risk of undesired side effects of the conventional drugs and simultaneously protecting from HIV-associated co-morbidities.

In the context of the present invention an extract from Cistus is used. An extract is a product obtained by extracting raw material, often by using a solvent such as ethanol or water. Extraction is a separation process involving the separation of a substance from a mixture of substances (solid or liquid matrix) by using a solvent. It may refer to liquid- liquid extraction or solid phase extraction. The solvent separates the product from the mixture due to higher solubility of the product in the solvent. In pharmacy, extract refers to a drug extracted from a cell (e.g., plant or animal) by a variety of methods (and optionally concentrated) using a solvent such as water, alcohol, or oil. Typically, the extraction may involve disintegration of the matrix, maceration, digestion, expression, absorption, separation of liquid and solid parts and/or distillation.

Extraction is based on different solubilities of the substances of the mixture. Essentially, extraction is carried out (i) by mixing the mixture of substances (solid or liquid matrix) with a solvent, (ii) solving the product in the solvent, (iii) separating the product from the remainder of the matrix and (iv) optionally separating the product from the solvent.

The solvent is to be selected to show selectivity for the product, i.e. the solubility of the product should be higher than that of the remainder of the matrix. Moreover, the solvent should be inert towards the extract, the extraction of the product should be fast, the solvent should be capable to carry a sufficient amount of product and is preferably not flammable, toxic, corrosive and harmful to the environment. It has been shown in the Examples (see Example 1) that water and methanol are suitable solvents. Accordingly, it can be concluded that other relatively small alcohols such as a CI -CIO alcohol, preferably a Cl- C6 alcohol (e.g. methanol, ethanol, propanol, butanol, pentanol or hexanol), especially a CI, C2 or C3 alcohol (e.g. methanol, ethanol, n-propanol or isopropanol) are suitable solvents. Suitable solvents for extracting Cistus suggested in US 2008/0274214 are water, alcohols such as methanol, ethanol or isopropanol, or chlorinated solvents such as dichloromethane, as well as acetone, acetyl acetone, ammonia, or glacial acetic acid. Mixtures of the above solvents may also be used. Preferably, water, methanol or ethanol, or a mixture of water with methanol or ethanol is used. Solvents can be broadly classified into two categories: polar and non-polar. Additionally, solvents with a relative static permittivity greater than 15 can be further divided into protic and aprotic. Protic solvents solvate anions strongly via hydrogen bonding. Aprotic solvents such as acetone or dichloromethane tend to have large dipole moments (separation of partial positive and partial negative charges within the same molecule) and solvate positively charged species via their negative dipole. Water and methanol are polar protic solvents. Accordingly, it can be concluded that other polar protic solvents are also suitable solvents. Examples include but are not limited to formic acid, n-butanol, isopropanol, n- propanol, ethanol, methanol, acetic acid and water.

Extracts may be in form of a liquid or a powder. A tincture is a liquid solution of herbs and a fluid menstruum, usually ethanol. The dried or fresh herbs are combined with solvent, e.g. alcohol or water, and then the solid matter is removed leaving only the product of the herbs mixed with the solvent.

The extract may be made from fresh, dried or otherwise processed plant material.

In the context of the present invention, the extract is a combination of substances obtained by extraction of Cistus (dried or fresh material). For this, Cistus (preferably leaves) may be disintegrated (e.g. broken, chopped, ground etc.) and than contacted with a solvent. A preferred solvent is water.

In the present invention the Cistus extract is comprised in a pharmaceutical composition. The pharmaceutical composition of the present invention may be formulated in any suitable manner. The pharmaceutical composition of the present invention may further encompass pharmaceutically acceptable carriers and/or excipients. The pharmaceutically acceptable carriers and/or excipients useful in this invention are conventional and may include buffers, stabilizers, diluents, preservatives, and solubilizers. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the extracts disclosed herein. Depending on the type of application, the concentration of the extract will vary in the form of use. Normally, the amount of the extract is between 1 to 1,000 mg per dosing unit in solid application forms. Preferably, the amount of extract is between 5 to 500 mg per unit. In fluid application forms, the extract may be present in a concentration of 1 μ /ηι1 to 100 mg/ml, preferably of 25 μ§/ηι1 to 50 mg/ml. In semisolid application forms, the content of extract is 1 to 90 wt %, preferably 5 to 75 wt %.

In general, the nature of the carrier or excipients will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e. g. powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the carrier to render the formulation isotonic. Examples of the carrier include but are not limited to saline, Ringer's solution and dextrose solution. Preferably, acceptable excipients, carriers, or stabilisers are preferably non-toxic at the dosages and concentrations employed, including buffers such as citrate, phosphate, and other organic acids; salt-forming counter- ions, e.g. sodium and potassium; low molecular weight (> 10 amino acid residues) polypeptides; proteins, e.g. serum albumin, or gelatine; hydrophilic polymers, e.g. polyvinylpyrrolidone; amino acids such as histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates including glucose, mannose, or dextrins; monosaccharides; disaccharides; other sugars, e.g. sucrose, mannitol, trehalose or sorbitol; chelating agents, e.g. EDTA; non-ionic surfactants, e.g. Tween, Pluronics or polyethylene glycol; antioxidants including methionine, ascorbic acid and tocopherol; and/or preservatives, e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, e.g. methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol.

The pharmaceutical composition of the present invention is used in a method of preventing and/or treating an infection. Treatment or treating is the attempted remediation of a health problem, usually following a diagnosis. A treatment deals with an existing medical problem, and may lead to its cure, but often ameliorates a problem only for as long as the treatment is continued, especially in chronic diseases. Cures are a subset of treatments that reverse illnesses completely or end medical problems permanently. Prevention or preventing is a way to avoid an injury, sickness, or disease in the first place, and generally it will not help someone who is already ill (though there are exceptions). A treatment or cure is applied after a medical problem has already started, whereas prevention is applied before the medical problem is detectable. The treatment or prevention may be in any subject, particularly a mammal such as cat, dog, rat, mouse, cow, horse, rabbit, or primate, especially in a human.

Prevention or preventing refers to measures taken to prevent diseases, rather than curing them or treating their symptoms. The term contrasts in method with curative and palliative medicine. In the context of the present invention, prevention of AIDS means that the development of AIDS is prevented before any signs or symptoms of the disease are detectable, i.e. at a stage in which the subject is still free of AIDS.

In the context of the HIV infection of individuals, such as humans, preventing the HIV infection relates to a situation in which transmission and/or the entry of the virus into a human is inhibited or prevented, whereas treatment relates to a situation in which the virus has entered the individual and replicated in cells, but in which further replication of the virus is inhibited. In the context of AIDS, prevention relates to a situation in which the outbreak of the disease in an infected subject or patient is prevented or inhibited, whereas treatment relates to a situation in which the subject or patient shows signs and symptoms of AIDS, which are intended to be ameliorated.

The pharmaceutical composition of the present invention is used in a method of preventing and/or treating an HIV infection. An infection is the invasion of body tissues by disease- causing microorganisms, their multiplication and the reaction of body tissues to these microorganisms. In the context of HIV, infection requires the entry of the virus into a cell in order to allow for its multiplication. HIV belongs to the family of Retroviridae, subfamily Orthoretrovirinae, genus Lentivirus. There are two types of HIV: type 1 and type 2. HIV type 1 (HIV-1) is the predominant HIV type. HIV-2 causes milder forms of disease than HIV-1. Viral genomes in virions (= virus particles) consist of single-stranded mR A-like molecules (i.e. positive polarity, 5' cap, 3' polyadenylated sequence). Within the host cell, the viral reverse transcriptase uses the viral RNA genome to synthesize a double-stranded DNA copy, which is integrated into the genome of the host cell as provirus. The HIV provirus is about 9.75 kbp in length. Long terminal repeats (LTRs) at both ends of the provirus contain sequences that direct and control generation of HIV transcripts, with the 5' LTR directing initiation and the 3' LTR termination of transcription. In addition the provirus contains gag, pol and env segments that encode for the major proteins of all retroviruses. The gag (group specific antigen) region encodes for the matrix (pi 7), capsid (p24) and nucleocapsid (p7) proteins, the pol region for the protease, reverse transcriptase/RNAse H, integrase and the env region for the envelope glycoproteins gpl20 and gp41. In addition the HIV pro viral genome contains reading frames that encode for regulatory proteins (Tat, Rev) and accessory proteins, (Vif, Vpu, Vpr, Nef).

The mature HIV virion is about 100-120nM in diameter and is enveloped by a double lipid membrane that is derived from the host cell in which the virion was produced. The viral envelope contains two glycoproteins, the surface glycoprotein (gpl20) and the transmembrane protein (gp41). Noncovalently associated homotrimers of gpl20 and gp41 molecules form "spikes" on the viral envelope which mediate the entry of the virus into the host cell. The matrix proteins (Gagpl7) associate with the viral membrane and stabilize the membrane of mature HIV particles. The capsid protein (p24) forms a conical shell that harbors two copies of the viral RNA genome, which are coated by nucleocapsid proteins (p7), and the viral enzymes reverse transcriptase, protease and integrase and a cellular tRNA primer for reverse transcription. The capsid is attached to the envelope via the linker protein (p6). Virus particles also contain several accessory proteins (Vpr, Nef, Vif) but no regulators of viral gene expression (Tat, Rev). These are the first viral proteins produced in the host cell.

The replication cycle of HIV is a complex process and will be summarized only briefly here. For more details the reader is referred to comprehensive review articles in the scientific literature e.g. (Freed 2001) The major route of entry of HIV into immune cells involves recognition and attachment of the "spikes" on the viral envelope to the receptor (CD4) and co-receptors (CXCR5 or CCR5) on the surface of the host cell. A series of conformational changes of gpl20 and gp41 allows fusion of the viral membrane with the cellular membrane and entry of the viral capsid into the host cell. Reverse transcription takes place in the cytoplasm of the host cell. Subsequently, the viral DNA is transported to the nucleus as part of a preintegration complex in which it associates with viral (integrase, Vpr, matrix) and host proteins (e.g. the SET complex). In the nucleus at least one copy of the double stranded HIV DNA genome must be integrated into the host genome as provirus for successful HIV replication. Low- level transcription of the integrated provirus leads to production of transcripts that are initially completely spliced and encode for the viral regulatory proteins Tat (trans-activator of transcription) and Rev (regulator of expression of virion proteins). Tat greatly increases the production of full-length transcripts, from which numerous different mRNA species are produced by alternative splicing. Rev binds to a specific structure (RRE = Rev response element) in full-length and incompletely spliced mRNAs and recruits factors for the transport of these viral mRNAs from the nucleus to the cytoplasm. Rev-dependent mRNA species encode for the virion proteins (i.e. core and virus envelope proteins) and also include the full-length viral RNA genome. In addition to nuclear export, Rev facilitates many other post-transcriptional steps of viral protein expression and promotes packaging of HIV RNA genomes into virus particles. Since Tat and Rev are the first viral proteins produced, all events preceding and including the production of Tat and Rev comprise the early phase of the HIV-1 replication cycle, whereas the subsequent events that culminate in the production of mature infectious virus particles comprise the late phase of the replication cycle.

The viral envelope proteins are produced by ribosomes associated with the endoplasmic reticulum (ER) and are processed by cellular proteases in the ER. Proteins that form the viral core and the viral enzymes are produced by cytoplasmic ribosomes in the form of Gag and Gag-Pol polyproteins. Production of the Gag-Pol polyprotein requires ribosomal frameshifting during translation, resulting in the production of lower amounts of Gag-Pol (-10%) than the Gag protein. The Gag and Gag-Pol polyproteins associate with the plasma membrane of the host cell. Gag mediates assembly and budding of virus particles by various domains that direct Gag-Gag interactions, binding to viral RNA and recruitment of host factors (e.g. ESCRT). The virus is initially assembled and buds from the cells as an immature virus particle which is not infectious. Maturation to full infectivity requires autocleavage of the protease domain from the Gag-Pol precursor polyprotein. The viral protease then excises the individual Gag proteins and the remaining viral enzymes (integrase, reverse transcriptase/R Ase H) from the precursor polypeptides, resulting in major rearrangements and the formation of mature, infectious virions.

In a very preferred embodiment of the present invention, the pharmaceutical composition of the present invention is extracted from Cistus incanus, Cistus creticus or Cistus villosus, especially Cistus incanus ssp. Ί amicus or Cistus incanus ssp. PANDALIS. Cistus incanus (also referred to Cistus creticus or Cistus villosus) is known for its high content of polyphenols which are thought to be responsible for the inhibitory effects of Cistus. Especially preferred are Cistus incanus ssp. Tauricus or Cistus incanus ssp. PANDALIS. A plant variety right has been granted to the breeder of a new variety Cistus incanus ssp. PANDALIS (EU 27578).

In accordance with the present invention, the extract is obtainable or obtained by extracting a part of a Cistus plant, particularly root, stem, seed, flower and/or leaf of Cistus, especially leaf of Cistus. It is known for Cistus that secondary plant substances, particularly polyphenols, are present in all parts of the plant, including but not limited to the root, stem, seed, flower and/or leaf. Accordingly, any of these may be used in the preparation of the extract. However, leaves are particularly preferred due to the high portion in volume of the plant, the easy accessibility and the option of repeated harvest. Additionally, the leaves allow for convenient extraction.

More particularly, the extract is obtainable or may be obtained by a method comprising a) extracting Cistus parts with an extractant selected from the group consisting of water, methanol, ethanol, 1-propanol, 2-propanol and mixtures thereof, particularly water.

For the extracting of step a) any part of Cistus is contacted with a solvent. Suitable parts of Cistus as well as solvents are specified above. For example, fresh, dried or processed parts of Cistus, especially leaves or root or the combination thereof, optionally disaggregated by grinding or by cutting into pieces, is contacted with a solvent, preferably a small alcohol as defined above or especially water, for a time and under conditions allowing extracting of Cistus parts, i.e. allowing dissolving the components of interest from the Cistus matrix. It is evident to the skilled person that an increase in temperature accelerates extraction. Usually, the time for extraction may be reduced, if extraction temperature is increased. However, suitable extraction times will be from 1 minute to 2 days, especially from 1 minute to 1 day or 10 hours, such as from 5 minutes to 1, 2, 3, 4, 5, 6, 7, or 8 hours (depending on the temperature). The temperature will be typically in the range of from 2°C to about 150°C, such as about 4°C (cooling), room temperature or 80°C to 100°C (boiling).

Prior to the extracting of step a), the plant material may be cleaned, e.g. by rinsing with water or alcohol.

The extraction is carried out with a suitable solvent as detailed above. In order to achieve as high a yield as possible, the plant material may be extracted multiple times. Here, different solvents may be used in the different extraction steps, or an extraction with a solvent can be followed by an extraction with a fat, wax or oil, and vice- versa. A liquid or semisolid crude product is obtained by the extraction, which may be used in this form for the preparation of a medicament for the prophylaxis and/or treatment of HIV. The crude product may also be concentrated and/or dried and/or worked up further prior to the processing to a pharmaceutical composition. For example, the workup may include purification steps known to one of ordinary skill in the art, such as centrifugation, filtration, and decanting in order to remove suspended materials from the extract.

The present invention thus further relates to the use of a dry extract. For the preparation of the dry extract, the solvent can be removed from the liquid crude extract, the concentrated extract, or the purified extract, for example by spray drying, freeze drying or vacuum drying.

In a preferred embodiment, the method further comprises the following steps:

a) extracting the extract obtained in step a) by solid phase extraction using

i) a solid phase suitable for extraction of non-polar phases, particularly a solid phase with functional group selected from the group consisting of octadecyl, octyl, phenyl and cyanopropyl, especially octadecyl; and

ii) an eluent selected from the group consisting of water, methanol, ethanol, 1- propanol, 2-propanol and mixtures thereof, particularly methanol;

b) optionally at least partially removing the eluent; and c) preparing a pharmaceutically acceptable composition.

For this, Cistus (preferably incanus spp. Ί amicus or PANDALIS) may be obtained, prepared, extracted and optionally further purified as detailed above. The resulting solution may be loaded on a solid phase (such as a column, e.g. a C18 SPE column (Varian Bond Etute). After loading the solid phase with the extract a washing-step (e.g. using 1 ml water with 1% formic acid) may be carried out. Afterwards the solid phase-bound components are eluted (e.g. with methanol 1% formic acid). The elute is captured and then may be evaporated and/or dissolved in order to prepare the pharmaceutical composition for use in the present invention.

In a preferred embodiment the extract is obtainable or obtained by a method comprising a) extracting a Cistus part with water;

b) optionally filtrating the extract of step a);

c) optionally extracting the extract obtained in step a) or b) by solid phase extraction using a solid phase with octadecyl as functional group and methanol;

d) at least partially removing the water of step a) and/or the methanol of step c); and e) preparing a pharmaceutical composition. For this, Cistus (preferably incanus spp. Tauricus or PANDALIS) may be obtained, prepared, extracted and optionally further purified as detailed above. The extract may be filtered in order to remove solid contaminants. Suitable filters are known to the skilled person. Exemplarily, a 0.10 to 0.50 μιη filter, such as a 0.22 μιη filter, may be used. Thereafter, a solid phase extraction as detailed above may be carried out. In order to prepare the pharmaceutical composition for use in the present invention, the water of step a) and/or the methanol of step c) at least partially removed.

Particularly preferred methods of preparing the extract according to the invention are detailed in the following. The skilled person will understand that these are exemplary illustrations and there may be variations within the scope of the present invention:

For the preparation of decoction, dried leaves (e.g. the tea marketed by Dr. Pandalis Urheimische Medizin GmbH und Co. KG, Glandorf, DE) may be heated to 50 to 100 °C (e.g. boiled) in water for a short period, e.g. up to 5 minutes. Thereafter, the matrix (solid parts) are removed, e.g. by centrifugation. The remaining extract may be sterile filtered (e.g. using a 0.22 μιη filter) and concentrated (e.g. by reduction of volume by boiling). The resulting concentration may be ready-made as detailed in the context of the pharmaceutical composition of the present invention.

Alternatively, the tea may be pulverized and the resulting powder may be suspended, e.g. in water, or methanol or mixtures thereof such as 50% methanol und 50% water. The suspension may be heated to 50 to 100 °C (e.g. boiled) for a short period, e.g. up to 5 minutes. Alternatively, the suspension may be kept at room temperature, e.g. for one or more hours. Extracting may be accelerated by vortexing or sonicating. Thereafter, the matrix (solid parts) are removed, e.g. by centrifugation. The remaining extract may be sterile filtered (e.g. using a 0.22 μιη filter) and concentrated (e.g. by boiling). The resulting concentration may be ready-made as detailed in the context of the pharmaceutical composition of the present invention.

In a further alternative, fresh parts of Cistus plant may be cleaned (e.g. by removing soil and washing with e.g. water and/or ethanol). Parts (e.g. leaves and/or stems) are cut into pieces and heated to 50 to 100 °C (e.g. boiled) in water for a short period, e.g. up to 1 hour. Alternatively, parts may be also incubated in methanol for several hours (e.g. overnight) at cooling (e.g. 4°C) and stirring. Thereafter, the matrix (solid parts) are removed, e.g. by centrifugation. The remaining extract may be sterile filtered (e.g. using a 0.22 μιη filter) and concentrated (e.g. by reduction by boiling). The resulting concentration may be ready- made as detailed in the context of the pharmaceutical composition of the present invention. In a still further alternative, Cistus incanus plants are harvested and cleaned with water and ethanol. The whole fresh plant is then cut into pieces (e.g. roots, stem and leafs) and the cooked for 1 to 5 hours, especially 1 hour, in water. Then the supernatant is filtered sterile (e.g. using a 0.22 μιη filter). The resulting solution is then loaded on a C18 SPE column (Varian Bond Etute). After loading the column with the extract a washing-step using water with e.g. 1% formic acid is carried out. Afterwards the column-bound components are eluted with e.g. methanol 1% formic acid. The elute is captured and may be evaporated. The resulting concentrate may be ready-made as detailed in the context of the pharmaceutical composition of the present invention. In a preferred embodiment, the extract according to the present invention is obtainable or obtained by a method comprising

a') providing a Cistus extract obtainable or obtained as defined above;

b') contacting the extract of step a') with polyvinylpolypyrrolidone (PVPP) as solid phase to allow binding of polyphenols to PVPP;

c') isolating the solid phase; and

d') eluting polyphenols from the solid phase.

Further details on this method are given below in the context with the method of the present invention.

In the methods described herein, the extracting of step a) is preferably performed at 50°C to 120°C, particularly at 80°C to 110°C, especially at 90°C to 100°C, especially wherein the solvent is water, methanol or ethanol or mixtures thereof.

HIV can infect a variety of cells such as CD4+ helper T-cells and macrophages that express the CD4 molecule on their surface. HIV-1 entry into macrophages and T-helper cells is mediated through interaction of the virion envelope glycoproteins (gpl20, gp41) with the CD4 molecule and with chemokine coreceptors on the target cells. Macrophage (M-tropic) strains of HIV-1 use the beta-chemokine receptor CCR5 for entry and are thus able to replicate in macrophages and CD4+ T-cells. This CCR5 coreceptor is used by many primary HIV-1 isolates regardless of viral genetic subtype. T-tropic isolates, replicate in primary CD4+ T-cells as well as in macrophages and use the alpha-chemokine receptor, CXCR4, for entry. These strains are now called X4 viruses. Viruses that use only the CCR5 receptor are termed R5 or CCR5-tropic strains, those that only use CXCR4 are termed X4 or CXCR4-tropic strains, and those that use both, X4R5 or CCR5- and CXCR4- tropic (dual tropic) strains. As detailed in above and in Example 2, Cistus extract has an inhibitory effect on a CCR5 -tropic strain and a CXCR4-tropic strain. Accordingly, the HIV is preferably a CCR5 -tropic strain, a CXCR4-tropic strain or a CCR5- and CXCR4-tropic (dual tropic) strain.

As a retrovirus, HIV uses the enzyme reverse transcriptase to synthesize DNA from its RNA genome and lacks a mechanism for correcting errors made while reproducing its genome. As a result, HIV replicates its genome with the highest known mutation rate of any living organism. This creates an ideal situation for natural selection to act on the HIV population, as genetic variation is the raw material for natural selection. Since HIV has a high mutation rate it can quickly form resistance against already existing HIV inhibitors, thus creating the constant need for new inhibitors. Inhibitors inhibiting infection and therefore replication of the virus are particularly beneficial. Accordingly, the pharmaceutical composition of the present invention is particularly suitable for use in a method of preventing and/or treating an HIV infection, wherein the HIV is a drug-resistant HIV, i.e. resistant to at least one drug used in HIV treatment. However, the virus may also be resistant to two or more HIV medicaments or may not be resistant to conventional drugs.

The extract can be used in any galenic application form known to one of ordinary skill in the art, for example as tablets, film tablets, capsules, powder, granulates, dragees, ointments, creams, gels, solutions, or sprays. The extract can also be used in the form of a powder for admixing into food, in particular into animal food. Here, the extract can be processed with the common galenic adjuvants, such as tablet binders, fillers, preservatives, tablet degradation agents, flow regulators, softeners, wetting agents, dispersion agents, emulsifiers, solvents, retarding agents, antioxidants, consistency- conferring agents, agents for improving penetration and/or propellants. The extract can also be mixed with other plant extracts, in particular with plant extracts with similar or synergetic effect.

Particularly, the pharmaceutical composition may be formulated as solid dosage form or as liquid dosage form, particularly as a capsule, tablet, pill, powder, granule, emulsion, microemulsion, solution, suspension, syrup, elixir, suppository, ointment, paste, cream, lotion, gel, powder, spray, inhalant or patch.

The pharmaceutical composition of the present invention may be manufactured in any suitable manner. Particularly, the pharmaceutical composition may be manufactured for oral, nasal, rectal, parenteral, topic, vaginal, subcutaneous, intracutaneous, intramuscular, intraarterial, intravenous or intraperitoneal administration.

The pharmaceutical composition of the present invention may be administered in any suitable manner and in any suitable regimen. Particularly, the pharmaceutical composition may to be administered after presumed HIV infection, particularly within 7 days, more particularly within 6, 5, 4, 3 days, especially within 2 days or 1 day from the presumed HIV infection. As detailed above, the pharmaceutical composition of the present invention is particularly suitable in inhibiting HIV entry into the cell. Accordingly, it could be used analogously to the morning-after pill. The morning-after pill is taken in a situation in which a suspected or assumed pregnancy is to be avoided. Analogously, the pharmaceutical composition of the present invention may be used in a situation, which it is not known, whether HIV was incorporated, but in which this could be the case (e.g. after unprotected sex), in order to avoid HIV entry into cells and virus replication. Furthermore, the pharmaceutical composition may be administered alone or in combination with an additional HIV medicament. As detailed above, the pharmaceutical composition of the present invention may be used as sole HIV medicament in order to inhibit HIV. However, it may be also used in combination with one or more additional HIV medicament. Particularly, it may be either used concomitantly and/or consecutively. By combining HIV medicaments, different steps of HIV entry and replication are inhibited, inhibiting virus spreading more effectively.

In a preferred embodiment, the Cistus extract is used in the absence of components or medicaments derived from plants of the genus Ribes and/or Ravensara. More preferably, the Cistus extract may be used alone or in combination with other HIV medicaments, preferably in combination with HAART (see also below), but is the only anti-HIV plant extract used in preventing and/or treating an HIV infection according to the present invention. A possible application of Ci extracts could be as a food supplement ingested by HIV- infected individuals, optionally during HAART or during intervals of HAART interruption. Ingestion of Ci extracts may allow lengthening of therapy interruption intervals and may help reduce the adverse effects of HAART. Ingestion of Ci extracts during HAART may allow a reduction in the dosage of conventional anti-HIV drugs during HAART, since Ci extracts inhibit entry of HIV into cells and thus interfere with infection at an earlier stage of the virus replication cycle than the reverse transcriptase and protease inhibitors most frequently used during HAART. Dosage reduction of these drugs would also reduce the risk of adverse effects that include, but are not limited to, hallucinations, dizziness, peripheral neuropathies, nausea, lipodystrophy, insomnia, insulin resistance and Diabetes mellitus. These considerable adverse effects of drugs used in HAART may prevent HIV-infected individuals from initiating HAART or to cause these individuals to discontinue HAART, resulting in rapid viral increase. Thus coingestion of Ci extracts may improve the HAART compliance by reducing adverse effects of HAART drugs. Furthermore, Ci coingestion may decrease the cost of HAART by reducing the required dosage of HAART drugs. Finally, HIV-infected individuals should also benefit from the antioxidant, anti- inflammatory, antimicrobial and antifungal effects of polyphenol compounds, leading to a better overall level of health and quality of life.

The additional HIV-medicament may be any medicament used to treat or prevent HIV infection. Presently, the common treatment is referred to as highly active antiretroviral therapy, or HAART. This has been highly beneficial to many HIV-infected individuals since its introduction in 1996, when the protease inhibitor-based HAART initially became available. Current HAART options are combinations (or "cocktails") consisting of at least three drugs belonging to at least two types, or "classes," of antiretroviral agents. Classes of antiretroviral drugs include entry inhibitors, nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs, NtRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors and integrase inhibitors. The majority of approved anti-HIV drugs are NRTIs, NtRTIs, NNRTIs and protease inhibitors. Other classes of drugs such as entry inhibitors provide treatment options for patients infected with viruses already resistant to common therapies, although they are not widely available and not typically accessible in resource-limited settings. Preferably, the at least one HIV-medicament is selected from the group consisting of an entry or fusion inhibitor, a nucleoside/nucleotide reverse transcriptase inhibitor, an integrase inhibitor, and a protease inhibitor, especially T20, AZT, Efavirenz, Raltegravir or Darunavir or any anti-HIV drug, e.g. those approved by the Food and Drug Administration.

Another aspect of the present invention relates to a product suitable for the prevention of HIV transmission selected from the group consisting of a condom, a lubricant, such as a personal lubricant, an anal-specific lubricant or a fertility lubricant, a diaphragm, a female condom (femidom), a medical glove and a laboratory glove, wherein a Cistus extract as defined in the context of the present invention is present in or on the product. The products mentioned above, are suitable for preventing transmission of HIV by acting as a physical barrier. The safety of the products could be further increased by applying a Cistus extract to the product so that an effective amount of the extract is present in or on the product. For this, the product may be sprayed with a liquid extract and dipped in the same and dried or a power form of the extract may be applied to the products. As a result the product may not only act as a physical barrier but also by inhibiting HIV entry into cells.

Still another aspect of the present invention relates to a piece of victuals, particularly for use in the prevention and/or treatment of an HIV infection, wherein an extract as defined in the context of the present invention is present in or on the product, particularly wherein the victuals is food product, especially baby food or children food, particularly for an HIV- infected baby or child. A piece of victuals is food to provide nutritional support for the body. It is usually of plant or animal origin, and contains essential nutrients, such as carbohydrates, fats, proteins, vitamins, or minerals. The substance is ingested by an organism and assimilated by the organism's cells in an effort to produce energy, maintain life, or stimulate growth. Liquids not providing nutritional support for the body such as water or tea and the pure Cistus preparations (e.g. dried or fresh leaves) are excluded from the above definition. The piece of victuals may also be used in a method for the prevention and/or treatment of an HIV infection. The food may be any raw or cooked food preparation. Particularly preferred is baby food or children food including without limitation infant formula that is made specifically for infants, roughly between the ages of four to six months to 2 years. The food comes in multiple varieties and tastes, can be produced by many manufacturers, or may be table food that the rest of the family is eating, mashed up. Because infants lack teeth, many different baby foods are designed for ease of eating; they are either a soft, liquidy paste or an easily chewed food. Pureed vegetables and fruits are an example of liquid style baby food. Then, as the baby is better able to chew, small, soft pieces or lumps may be included. Care should be taken, as babies with teeth have the ability to break off pieces of food but they do not possess the back molars to grind, so concerned parents should carefully mash or break baby food into manageable pieces for their baby. Evidently, the food is particularly suitable for an HIV-infected baby or child in order to inhibit HIV entry into cells. The Cistus extract in the piece of victuals should be sufficient in an amount effective in the inhibition of HIV infection.

Yet another aspect of the present invention relates to a kit comprising at least two HIV medicaments, wherein one HIV medicament is a pharmaceutical composition as defined in the context of the present invention and the other HlV-medicament(s) is/are at least one drug used in HAART and/or selected from the group consisting of a entry or fusion inhibitor, a nucleoside/nucleotide reverse transcriptase inhibitor, an integrase inhibitor, and a protease inhibitor, especially T20, AZT, Efavirenz, Raltegravir or Darunavir or any anti- HIV drug, e.g. those listed by the Food and Drug Administration. The above definitions and embodiments are also to be applied for this aspect of the invention. The kit may be designed for the simultaneous, separate or sequential use in of the at least two HIV medicaments. In addition to the medicaments, the kit may comprise further elements such as instructions for use. Another aspect of the present invention relates to a method of producing a polyphenol- enriched Cistus extract, the method comprising

a') providing a Cistus extract obtainable or obtained as described above;

b') contacting the extract of step a') with polyvinylpolypyrrolidone (PVPP) as solid phase to allow binding of polyphenols to PVPP;

c') isolating the solid phase; and

d') eluting polyphenols from the solid phase, thereby producing a polyphenol-enriched

Cistus extract.

Polyphenols (also known as polyhydroxyphenols) are a structural class of organic chemicals characterized by the presence of multiple phenol structural units (aromatic benzenoid (phenyl) ring with a hydroxyl (-OH) group). The number and characteristics of these phenol structures underlie the unique physical, chemical, and biological (metabolic, toxic, therapeutic, etc.) properties of particular members of the class. In nature, the most abundant polyphenols are the condensed tannins, found in virtually all families of plants, and comprising up to 50% of the dry weight of leaves. Some polyphenols produced by plants in case of pathogens attacks are called phytoalexins and high levels of polyphenols in some woods can explain their natural preservation against rot. In Cistus three main groups of compounds were found, i.e. ellagitannins, flavonoids and phenolic acids derivatives. The polyphenolic profile slightly varies between different Cistus species.

In the context of the present invention, the feature "polyphenol-enriched Cistus extract" relates to a Cistus extract, in which the content or concentration of intrinsic polyphenols is increased relative to other Cistus components. In accordance with the present invention this may be achieved by contacting a (liquid) Cistus extract with PVPP in order to bind polyphenols of the extracts thereto, isolating PVPP having bound the polyphenols and eluting the polyphenols from PVPP. Furthermore, PVPP may be separated from the polyphenols. Preferably, the method of the present invention is further defined in that

i) the solid phase obtained in step c') is washed, particularly with water;

ii) the eluting of step d') is by addition of a caustic solution, particularly NaOH, and optionally followed by neutralization, particularly with HC1; and/or

iii) the eluting of step d') is followed by further purification, particularly with solid phase extraction.

Accordingly, in an exemplary method of producing a polyphenol-enriched Cistus extract, the method may be carried out as follows: A Cistus extract is provided. The extract may be prepared as described above, e.g. by extracting Cistus parts with an extractant such as water, methanol, ethanol, 1-propanol, 2- propanol and mixtures thereof, particularly water. The extract is then contacted with PVPP under conditions conducive to the binding of polyphenols to PVPP. Exemplary conditions may be by adding 2 g of PVPP 50 ml extract of Cistus incanus and incubating the mixture at room temperature under stirring for 15 minutes. After binding, PVPP particles may be isolated, e.g. by centrifugation or filtration. The supernatant/filtrate will be discarded and the PVPP particles with bound compounds are maintained. For further purification or enrichment, PVPP particles may be washed, e.g. 2 times with pure water. Thereafter, bound compounds may be eluted, e.g. by addition of a caustic solution such as NaOH (exemplary concentration 0.5 mol/1). PVPP particles may be isolated again, e.g. by centrifugation or filtration. Next, caustic solution with dissolved compounds may be equilibrated, e.g. by addition of an acid such as HC1 to pH 7.

In case further purification should be desired, an exemplary method could be by solid phase extraction (SPE) e.g. with a CI 8 SPE-column. For this, the SPE column may be cleaned e.g. with MeOH and equilibrated e.g. with 1% formic acid in water. The polyphenol-containing solution will be loaded onto the column. After one or more optional washing step(s) (e.g. with 1% formic acid in water), the column-bound compounds are eluted (e.g. with an organic solvent such as 100 % MeOH). The solvent may or may not be evaporated and/or may or may not be dissolved (e.g. preferably in water optional with pharmaceutically acceptable carriers and/or excipients). The dissolved polyphenol-enriches extract or dried remainders may be stored until further use. Still another aspect of the present invention relates to a polyphenol-enriched Cistus extract obtainable or obtained by the method of the present invention.

In other aspects the polyphenol-enriched Cistus extract of the present invention may be for use as a medicament or for use in a method of preventing and/or treating an HIV infection, as defined and/or specified above in the context of the pharmaceutical composition of the present invention comprising a Cistus extract for use in a method of preventing and/or treating an HIV infection. Further details on pharmaceutical compositions and medical uses are given above. The invention is not limited to the particular methodology, protocols, and reagents described herein because these may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Similarly, the words "comprise", "contain" and "encompass" are to be interpreted inclusively rather than exclusively.

Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods, and materials are described herein.

The invention is further illustrated by the following examples, although it will be understood that the examples are included merely for purposes of illustration and are not intended to limit the scope of the invention unless otherwise specifically indicated. FIGURES

Figure 1. Effects of the commercial CYSTUS052® extract on HIV infection of cells and viability of LC5-RIC cells. The commercial CYSTUS052® extract inhibits HIV infection at concentrations that do no effect cell viability. Effects of CYSTUS052® extract on HIV infection were measured using the EASY-HIT assay with LC5-RIC cells and HIV- I_LAI ·^' Relative infection /relative cell viability (%) (y-axis) represents fluorescent/MTT signal intensities of cultures exposed to CYSTUS052® extracts during infection normalized to those of control cultures infected by HIV in the absence of extracts (= 100%). The concentrations of CYSTUS052® extracts (dry weight of extract per ml cell culture volume) are plotted on the x-axis. The dotted line represents the results of the first step of the EASY-HIT assay (IC₅₀ = 4.85 ± 0.41 μg/ml), the dashed line the results of the second step of the EASY-HIT assay (IC₅₀ = 3.78 ± 0.28 μg/ml) and the solid line the results of the MTT assay (LD₅₀ > 400 μg/ml). Each data point represents the mean results of triplicate tests, and standard deviations are indicated.

Figure 2. Effects of extracts of different parts of Cistus incanus plants on HIV infection (A, B) and cell viability (C, D). Extracts from different parts of Cistus incanus plants and prepared with different solvents (water or methanol) show anti-HIV activity at concentrations that do not affect cell viability. Extracts were prepared from leaves (white columns), stems (checkered columns) and roots (black columns) of fresh plants, using either water (A,C) or methanol (B,D) as solvent. Anti-HIV activities were determined by the EASY-HIT assay (i.e. first step) using LC5-RIC cells and HIV- I_LAI and cell viability by the MTT assay. Relative infection /relative cell viability (%) (y-axis) represents fluorescent/MTT signal intensities of cultures exposed to Cistus incanus extracts during infection normalized to those of control cultures infected by HIV in the absence of extracts (= 100%). Concentrations of extracts in treated cultures ^g dry weight of extract per ml cell culture volume) are plotted below the columns. Each column represents the mean results of triplicate tests, and standard deviations are indicated.

Figure 3. Effects of Cistus incanus (Ci) extracts on infection by HIV-1 variants with different envelope proteins. Ci extracts inhibit infection by HIV variants with R5 and X4 tropisms. Inhibition of infection depends on HIV envelope proteins. (A) Inhibitory activities of Cistus incanus extracts generated from the herbal tea were determined by the EASY-HIT assay (first step), using a subline of LC5-RIC (i.e. LC5-RIC-R5) that is permissive for infection by X4 and R5 tropic variants of HIV- 1 and VSV-G pseudotyped HIV-1 particles. Inhibitory activities of Ci extracts are shown for infections with the R5- tropic HIV-1_AD8 (solid curve; triangles) and the X4-tropic HIV- I_LAI (dotted curve; squares). (B) Effect of Ci extracts on infection by HIV pseudotypes that contain the core of HIV-1_NL4-₃ and envelope proteins either from the R5 -tropic JRFL (dotted curve; squares) or from the heterologous Vesicular stomatits virus (G-protein) (solid curve; triangles). Relative infection (%) (y-axis) represents fluorescent signal intensities of cultures exposed to Ci extracts during infection normalized to those of control cultures infected by HIV in the absence of extracts (= 100%). Each data point represents the mean results of triplicate tests, and standard deviations are indicated.

Figure 4. Comparison of the activity profiles of Cisius incanus (Ci) extracts with those of approved entry inhibitors in time-of-addition (TO A) assays. The results demonstrate that Cistus incanus extracts inhibit entry of HIV. For time-of-addition assays, LC5-RIC cells were exposed to preparations of R5-tropic HIV-1 virus particles (HIV- 1_NL4._3AEIWJRFL) and Ci extracts added to cultures either simultaneously with the virus (time point 0) or at different time points after addition of the virus (x-axis). TOA profiles are shown for Ci extracts (continuous curve; squares) and for entry inhibitors that target fusion of viral membranes with cell membranes (T20; dotted curve; diamonds), or recognition of the CCR5 molecule by the virus particle (maraviroc; dashed curve; triangles). Final concentrations of Ci extracts and reference drugs in TOA assays were 5-10-fold > IC50. Relative infection (%) (y-axis) represents fluorescent signal intensities of cultures exposed to Ci extracts during infection, normalized to those of control cultures infected by HIV in the absence of extracts (= 100%). Each data point represents the mean results of triplicate tests, and standard deviations are indicated.

Figure 5. Effects of preincubation of virus or cells with Cistus incanus extracts on HIV infection. Preincubation of virus preparations with Cistus incanus extracts (3h) before addition to LC5-RIC cells increases inhibition of HIV infection (continuous curve), compared to simultaneous addition of virus and Ci extracts to the cells (dashed curve). No antiviral activity is observed when cells are preincubated with Ci extracts (3h), followed by infection in the absence of extracts (dotted curve). Anti-HIV activities were determined by the EASY-HIT assay (i.e. first step), using LC5-RIC cells and HIV- I_LAI virus. The x-axis indicates concentrations of extracts in cultures either during infection (virus preincubation, no preincubation) or prior to infection (cell preincubation). Relative infection (%) (y-axis) represents fluorescence signal intensities of cultures exposed to Cistus incanus extracts normalized to those of control cultures infected by HIV in the absence of extracts (= 100%). Each data point represents the mean results of triplicate tests, and standard deviations are indicated.

Figure 6. Effects of enrichment of polyphenolic ingredients of extracts of Cistus incanus plants on the anti-HIV activity and in vitro cytotoxicity. To divide the crude extract of Cistus incanus (Ci) a precipitation of polyphenols was performed using polyvmylpolypyrrolidone (PVPP). The resulting fractions were enriched Ci polyphenols and a polyphenol-free fraction. For both fractions anti-HIV activities were determined by the EASY-HIT assay. Relative infection (%) (y-axis) represents fluorescent signal intensities of cultures exposed to respective fractions during infection normalized to those of control cultures infected by HIV in the absence of extracts (= 100%). Concentrations of fractions in treated cultures ^g dry weight of fraction per ml cell culture volume) are indicated (x-axis). Each data-point represents the mean results of triplicate tests, and standard deviations are indicated. Figure 7. Effects of extracts (A, B) and polyphenol-enriched fractions (C, D) of Cistus incanus plants on HIV infection and cell viability of primary human HIV-target cells (PBMCs). To test anti-HIV activities of extracts of Cistus incanus and the enriched polyphenols on primary cells, primary human cells (PBMCs) were treated with crude extracts or the polyphenol-enriched fractions fraction and were then exposed to HIV-1 (LAI). After 48 hours, parts of the supernatant were transferred to LC5-RIC indicator cells to analyze the production of new infectious virus by the extract-treated PBMCs (A, C). PBMC cell viability was determined by MTT-assay (B, D). Relative infection (%) (y-axis) represents fluorescent signal intensities of LC5-RIC cultures exposed to supernatant of PBMCs that were treated with respective extracts/fractions during infection normalized to those of control cultures infected by HIV in the absence of extracts (= 100%). Concentrations of fractions in treated cultures ^g dry weight of fraction per ml cell culture volume) are indicated (x-axis). Each data-point represents the mean results of triplicate tests, and standard deviations are indicated. Figure 8. Effects of extracts of Cistus incanus plants on HIV infection of primary human target cells (PBMCs) on DNA-level. To test anti-HIV activities of extracts of Cistus incanus primary human cells on DNA-level, PBMCs were treated with extracts (concentrations indicated on x-axes) and were then exposed to an HIV-1. After 48 hours, the cells were tested for the presence of proviral HIV DNA-copies by Real-Time PCR. The results were normalized to HIV-infected, untreated cells (100%). Each column represents the mean results of triplicate tests, and standard deviations are indicated.

Figure 9. Effects of extracts of Cistus incanus plants on HIV infection and cell viability of primary human macrophages (MDM). To test anti-HIV activities of extracts of Cistus incanus on primary human macrophages (MDM), MDM were treated with extracts (concentrations indicated on x-axes) and were then exposed to an HIV-1 virus that carries a gene for the expression of GFP upon established infection of a cell. The GFP-signal positive cells (%) (Y-axis) were measured by FACS. Living cells were determined by forward/sideward scatter. Each data-point represents the mean results of triplicate tests, and standard deviations are indicated

Figure 10. Effects of extracts of Cistus incanus plants (Ci) and polyphenol-enriched fractions (CiPP) thereof on HIV attachment to target cells. To investigate the mode-of- action of Ci and CiPP in further detail, an HIV-1 virus that is tagged with a green fluorescent protein (GFP) was added to cells (LC5RIC-R5) that were either treated with Ci or CiPP. After an incubation time of 4 hours, the cells were washed, fixed with paraformaldehyde and nuclei were stained with DAPI. Using fluorescence microscopy, the GFP-spots on the cells were counted and the ratio of GFP-spots per Ci/CiPP -treated cell (as a count for virus particles bound to cells) was compared to that of untreated cells (control; virus only) and na^'ive cells (mock; no Ci/CiPP and no virus). Cell numbers analyzed: control 52; mock 33; Ci 66; CiPP 59. Standard deviations are indicated for each bar. EXAMPLES

1. Various extracts of Cistus incanus inhibit HIV infection. Various extracts of Cistus incanus were tested for inhibition of HIV infection. Tests were performed with EASY-HIT technology, which is described in detail in Kremb et al 2010. Briefly, the EASY-HIT technology comprises HIV-reporter cells (LC5-RIC) and assay procedures established and validated for the identification, quantification and characterization of anti-HIV activities. LC5-RIC cells efficiently replicate HIV-1 and produce a red fluorescent protein upon infection (LC5-RIC = LC5-Red Infected Cells). Expression of the reporter gene in LC5-RIC cells requires both HIV-1 Tat and Rev proteins and is induced during the early phase of HIV-1 replication that precedes production of viral structural proteins and virions. The LC5-RIC cells support all steps of the HIV-1 replication cycle, from virus attachment to the release of virions from the cell surface. LC5-RIC cells express CD4 and the chemokine receptor CXCR4, permitting entry of X4-tropic viruses. Cells of the LC5-RIC-R5 subline additionally express the chemokine receptor CCR5 on their surface, permitting infection by both X4-tropic and R5-tropic HIV- 1 variants. The EASY-HIT assay evaluates the effects of compounds on the HIV-infection of LC5-RIC cells in two steps that measure parameters of the early (i.e. production of Tat and Rev) and late (i.e. release of infectious virus particles) phases of the HIV-1 replication cycle, respectively (Kremb et al, 2010). Effects of test compounds on cell viability in test cultures are determined by a colorimetric assay that measures the reduction of the yellow tetrazole MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a purple formazan by mitochondrial enzymes (Mosmann,T. 1983).

First, the commercial decoction CYSTUS052® (Dr. Pandalis Urheimische Medizin GmbH und Co. KG, Glandorf, DE) was tested for anti-HIV activity. For testing, the CYSTUS052® decoction was sterilized by filtration (pore size 0.22 μΜ) and concentrated by centrifugal vacuum evaporation at 60°C (Eppendorf concentrator). The dry mass was dissolved in sterile, bidistilled water at a concentration of 10 mg/ml. This stock solution was used in all experiments with CYSTUS052®.

Antiviral activity of the CYSTUS052® concentrate was analysed with EASY-HIT technology by exposing LC5-RIC cells to the X4-tropic HIV-1 strain HIV- I_LAI in the presence of various concentrations of CYSTUS052®. Each concentration of CYSTUS052® was evaluated in triplicate. Controls consisted of cultures infected with HIV- 1 LAI in the absence of CYSTUS052® and served as reference values for 100% infection and cell viability. Effects of CYSTUS052® on the early phase of HIV-1 replication were evaluated by measuring the fluorescence signals of cultures incubated with extracts and virus for 48 hours (Step 1 of the EASY-HIT assay). Cell viability was determined by MTT assays of the treated cultures. To evaluate the effects of CYSTUS052® on the late phase of HIV-1 replication (i.e. production of virus), aliquots of supernatants of treated cultures were transferred to uninfected LC5-RIC cultures and fluorescence signals measured 72 hours after transfer (Step 2 of the EASY-HIT assay). For both steps, values of treated cultures were normalized to those of control cultures.

As shown in Fig. 1, CYSTUS052® extracts showed dose-dependent inhibition of HIV-1 infection in both steps of the assay. Mean IC₅₀ values of inhibitory effects calculated from these experiments were 4.85 ± 0.41 μg/ml for Step 1 and 3.78 ± 0.28 μg/ml for Step 2 of the EASY-HIT assay. The calculation was done using the equation for sigmoidal dose- response with variable slope and the constraints for top and bottom set to either 100 and 0, respectively. We previously demonstrated that early-phase inhibitors of HIV-1 replication yield similar IC50 values in both steps of the EASY-HIT assay (Kremb et al. 2010). This indicates that CYSTUS052® extracts interferes with HIV-1 replication at an early phase. Cell death by treatment with CYSTUS052® extracts occurred at much higher concentrations than inhibition of infection, with LD₅₀ at >400 μg/ml, yielding a selectivity index > 80. After having established that CYSTUS052® has anti-HIV activity, we investigated whether Cistus incanus plants contain HIV-inhibitory activity. For preparation of plant extracts, Cistus incanus spp. tauricus seedlings were obtained from a commercial vendor and allowed to grow for several weeks (final height ~20 cm). Plants were then divided into three parts (leaves, stalks and roots). Each part was washed thoroughly, minced and extracted either with water at 100°C for 1 hour or with 100% methanol at 4°C on a rotator overnight. Extracts were sterilized by filtration and concentrated by centrifugal vacuum evaporation at 60°C (Eppendorf concentrator). The dry mass was dissolved in sterile, bidistilled water at a concentration of 10 mg/ml. Subsequently, the effects of various concentrations of the plant extracts on HIV infection and on cell viability were investigated in step 1 of the EASY-HIT assay as described above. Fig. 2 shows that extracts from all three parts of the plant inhibited HIV-1 infection in a dose-dependent manner. Anti-HIV activities were observed for extracts prepared with either water or ethanol as solvent. The IC₅₀ values for the extract generated with water from leaves was 15.6 ± 0.94 μg/ml; from stems, 12.15 ± 0.60 μg/ml and from roots 11.15 ± 0.75 μg/ml. The IC₅₀ value for the extract generated with methanol from leaves was 22.67 ± 1.15 μg/ml; from stems, 15.21 ± 1.63 μg/ml and from roots 10.47 ± 0.52 μg/ml. None of the extracts exhibited cytotoxic activity at the HIV-inhibitory concentrations, confirming that anti-HIV activity is not linked to cell death.

Since we detected anti-HIV activity in various parts of the Cistus incanus plants we next tested a commercial mixture of dry minced Cistus incanus plants sold as a herbal tea (Cystus® Bio Teekraut Zistrosentee; Naturprodukte Dr. Pandalis GmbH & Co. KG) for anti-HIV activity in both steps of the EASY-HIT assay, using HIV- I_LAI for infection. lOg of the dry tea was boiled for lhour and a stock solution of extracts containing lOmg dry mass/ml prepared, as described above. This tea-based extract was designated as Ci. As expected, the Ci extract showed anti-HIV activity, yielding IC₅₀ values of 11.76 ± 0.6 μg/ml in the first step of the assay and 11.10 ± 1.3 μg/ml in the second step of the assay. Cytotoxic effects of the Ci extracts were observed at much higher concentrations, with LD₅₀ >400 μ^πιΐ.

These results show that aequeous Cistus incanus extracts inhibit HIV infection and act at the early phase of replication. Anti-HIV activity of Cistus incanus extracts is resistant to desiccation of Cistus incanus plants, methanol extraction and to boiling (100°C) for at least one hour.

All further examples described here were established with the Ci extracts, i.e. the extracts produced from the herbal tea.

2. Cistus incanus extracts inhibit X4 and R5 tropic HIV-1 strains

We then evaluated the capacity of Ci extracts to inhibit infection by different HIV-1 variants in the EASY-HIT assay (first step). To this end we investigated antiviral activities of the Ci extract against a prototypical X4-tropic virus (HIV- I_LAI) and a prototypical R5- tropic virus (HIV-1 _ADS), using LC5-RIC-R5 cells which are permissive for both X4 and R5 HIV-1 variants. Fig. 3 A shows that Ci extracts inhibited infection by both viruses. In addition to these HIV-1 variants, we also investigated the efficacy of Ci extracts to inhibit infection by pseudotyped viruses that contain the core of HIV-1_NL4-3 and envelope proteins either from a different HIV-1 variant (i.e. the R5-tropic JRFL) or from a heterologous virus (G-protein from Vesicular stomatits virus). Fig 3B shows that the profile obtained for inhibition of HIV-1 pseudotyped with the envelope proteins of the R5-tropic JRFL (HIV- 1_NL4.3_AEIIVJRFL) closely resembled the profile for wildtype HIV- I_ADS- In contrast, the Ci extract showed much weaker inhibitory activity against the HIV-1 pseudotype containing the VSV-G envelope protein (HIV-l_NL4.3AEnvVSVG).

Together these results indicate that Ci displays inhibitory activity against both X4- and R5- tropic viruses and that the inhibitory activity of the Ci extracts involves the HIV envelope proteins.

3. Cistus incanus extracts inhibit HIV-entry

As described above, Cistus incanus extracts inhibit HIV-1 replication at the early phase of the replication cycle by a mode of action involving HIV-1 envelope proteins (Figures 1, 2 and 3). This suggests that Cistus incanus extracts inhibit HIV-1 entry.

Time-of-addition (TOA) assays yield information about the mode-of-action of viral inhibitors with respect to the step of the replication cycle at which they act. TOA assays monitor the activity of test compounds at various timepoints of infection. The compound retains antiviral activity when added to the virus-exposed cells at a timepoint preceding the targeted replication step but loses activity when added at later timepoints. We previously demonstrated that TOA assays with LC5-RIC cells and reference drugs allows the discrimination of compounds from different anti-HIV drug classes, yielding distinct inhibitory profiles for each drug class (Kremb et al. 2010). To analyse whether Ci extracts inhibit HIV-1 entry, we compared the inhibitory profiles of Ci with those of two approved drugs from the class of entry inhibitors (the fusion inhibitor T20 and the CCR5 antagonist maraviroc) in TOA assays. TOA asssays were performed with LC5-RICR5 cells and R5- tropic HIV-1 (HIV-1_NL4.3_AEI JRFL) using concentrations of inhibitory compounds exceeding 5 - 10 fold the corresponding IC₅₀. The results are shown in Figure 4. A clear decrease in antiviral activity of Ci extracts was observed when addition of Ci extracts to virus-exposed cells was delayed for only a few hours, with 50% loss of activity occurring at 3 hours post infection for Ci extracts. The TOA profiles of the Ci extracts resembled those of the reference entry inhibitors T20 and Maraviroc, which showed 50% loss of activity at 3.5 hours. These results confirm that Ci extracts inhibit HIV entry.

4. Cistus incanus extracts reduce infectivity of virus particles The results presented so far show antiviral activity of Cistus incanus extracts in the presence of both LC5-RIC cells and virus particles. To determine whether Ci extracts can interfere with the infectivity of virions in the absence of the host cells, we examined the effects of pre-incubating virus particles with Ci extracts before addition to the host cells on the infectivity of the virus particles.

HIV- 1 LAI virus preparations were incubated with Ci extracts at different concentrations for 3 hours and the mixtures then added to LC5-RIC cells and infection analysed in step 1 of the EASY-HIT assay. We also investigated the effect of pre-incubation of the LC5-RIC cells with the same concentrations of Ci extracts for 3 hours, followed by gently removing the Ci extracts without washing of cells and addition of the virus. The results from both pre-incubation experiments were compared with the antiviral effects obtained under standard conditions, i.e. simultaneous addition of Ci extracts and virus to the cells.

As shown in Figure 5, preincubation of the virus preparation with the Ci extracts reduced infectivity of the virus ~2.5-fold, compared to standard conditions. In contrast, no antiviral effect was observed after pre-incubation of the cells with Ci extracts.

These results indicate that Ci extracts can reduce the infectivity of HIV- 1 virus particles in the absence of host cells and therefore has virucidal activity. Lack of antiviral effects of pre-incubation of host cells with Ci extracts suggests that antiviral activity of Ci extracts is not mediated by high affinity binding of components of Ci extracts to cell surface molecules, a mode-of-action exemplified by the CCR5 antagonist maraviroc. However, these results do not rule out the possibility that antiviral activity of Ci extracts may involve binding of extract components to both viral and cellular partners during virus entry. 5. Extracts of Cistus incanus (Ci) also inhibit HIV infection in primary human HIV target cells (PBMCs and macrophages) Next inventors tested the inhibitory effect of Ci on the HIV infection of primary human cells. For this, they treated PBMCs (isolated from buffy coats) with Ci and infected them with HIV- I_LAI- 2 days later they took some supernatant and transferred it to LC5-RIC reporter cells. In this way they quantify the production of new infectious virus by the Ci- treated and HIV-infected PBMCs as a measure for the rate of infection. As shown in figure 7A, the level of infectious virus could be decreased dose-dependent by treatment of the PBMCs with Ci. They also evaluated cell-toxicity of Ci for PBMCs by MTT assay (Fig. 7B). As can be seen, within the range of activity shown in Fig. 7A, no toxicity could be observed. Besides PBMCs, they also tested the inhibitory effect in primary human macrophages. For this, macrophages were treated with Ci as described above and then infected with an HIV- 1 virus that expresses a GFP together with its own proteins. The number of GFP expressing cells was measured by FACS analysis. In Figure 9 the GFP -positive cells represent the number of infected cells relative to Ci-untreated cells. Again a clear dose-dependent reduction of GFP -positive - and thus HIV-positive cells - could be observed with rising amounts of Ci.

Finally, the inhibition of infection of PBMCs was measured by analyzing the total amount of HIV-DNA in Ci-treated cells. PBMCs were treated and infected as described above. After 2 days, DNA was extracted and the relative amount of HIV provirus as a measure of infection was quantified by qRT-PCR. In Figure 8 can be seen that the treatment of PBMCs with 50 or 100 μg/ml of Ci almost totally inhibits the establishment of infection by decreasing the number of HIV DNA-copies/cell below 0.05% relative to Ci-untreated cells (100%).

Taken together, these results show that the anti-HIV effect observed in LC5-RIC and LC5- RIC-R5 cells could be reproduced with primary human cells including PBMCs and macrophages that represent the natural HIV target-cells. 6. Polyphenol-enrichment retains anti-HIV activity and decreases cellular toxicity of Cistus incanus extracts

To investigate the role of polyphenols in HIV-inhibition, an extraction of the polyphenolic substances from Ci was performed by precipitation with polyvinylpyrrolidone (PVPP). PVPP-precipitation of polyphenols is an accepted method in beverage industry to decrease the amount of polyphenolic compounds in brewages. For this, 2 g of PVPP were added to 50 ml extract of Cistus incanus (obtained as described in Example 1). The mixture was incubated at room temperature under stirring for 15 minutes. Afterwards the PVPP particles were pelleted by centrifugation. The supernatant was discarded and the PVPP- particles with bound compounds were washed 2 times with pure water. To elute the bound compounds, 0.5 N NaOH was added. After pelleting the PVPP-particles, the NaOH with dissolved compounds was equilibrated to pH 7 with HCL. The resulting solution was cleaned by solid phase extraction (SPE) with a C18 SPE-column. For this, the SPE column was cleaned with MeOH and equilibrated with 1% formic acid in water. The compound containing solution was loaded onto the column. After a washing step with 1% formic acid in water, the column-bound compounds were eluted with 100 % MeOH. MeOH was evaporated, the dried remains were weighed and dissolved in water to a final concentration of 10 mg/ml and stored at -20°C until usage.

The fractions obtained by SPE were either polyphenol-free (or drastically reduced in the amount of total polyphenols) or enriched in polyphenols. Both fractions were tested in LC5-RIC cells for anti-HIV activity and the results are shown in Fig. 6. The polyphenol- free fraction exhibited a complete loss of inhibitory activity, whereas the enriched Ci polyphenols retained it completely.

The enriched fraction was also tested in primary human cells (PBMCs). The results in Fig. 7C show, that the activity was retained here, too. The cellular toxicity was decreased for the enriched polyphenols (Fig 7D) by more than factor 5 in contrast to the original extract (Fig 7B).

These results indicate the possibility to modify an extract of Cistus incanus plants by e.g. reduction by depletion of distinct substances (or enrichment of others) which can be used to design an optimized anti-HIV agent as described in this application. 7. Extracts of Cistus incanus plants (Ci) and polyphenol-enriched fractions (CiPP) thereof inhibit HIV-attachment to HIV-target cells.

To investigate the effect of virus-attachment to HIV-target cells, LC5RIC-R5 cells were exposed to an HIV-virus that carries green-fluorescent proteins. These particles can be visualized and counted directly by fluorescence microscopy. For this experiment cells were seeded on glass cover slips and exposed to GFP-carrying virus either treated with Ci, CiPP or culture medium only. After an incubation period of 4 hours, the cover slips were washed 3 times with culture medium (2x) and PBS (lx). Then the cells were fixed in paraformaldehyde and nuclei were stained with DAPI. The analysis was performed by fluorescence microscopy. All GFP-spots in the taken pictures were counted by a software and have been set in ratio to the number of cells (DAPI-positive) seen on the picture. The results presented in figure 10 clearly demonstrate that an exposure of viral particles to Ci or CiPP respectively reduce the amount of virus-particles bound to cells in contrast to Ci/CiPP-untreated cells.

This observation accounts for the previous observations, that the anti-HIV effect of Ci/CiPP is an inhibition of HIV entry at the stage of HIV attachment by disturbing the ability of viruses to attach to the target cells.

REFERENCES

US 2008/0274214

Bravo, L. (1998). "Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance." Nutrition reviews 56(11): 317-333.

Droebner, K., C. Ehrhardt, et al. (2007). "CYSTUS052, a polyphenol-rich plant extract, exerts anti-influenza virus activity in mice." Antiviral research 76(1): 1-10.

Freed, E. (2001). "HIV-l replication." Somatic cell and molecular genetics 26(1): 13-33. Kalus, U., H. Kiesewetter, et al. (2010). "Effect of CYSTUS052® and green tea on subjective symptoms in patients with infection of the upper respiratory tract."

Phytotherapy Research 24(1): 96-100.

Kremb, S., M. Heifer, et al. (2010). "EASY-HIT: HIV full-replication technology for broad discovery of multiple classes of HIV inhibitors." Antimicrobial agents and chemotherapy 54(12): 5257-5268.

Mosman, T.J. (1983) Rapid colorimetric assay for cellular growth and survival:

Application to proliferation and cytotoxic assays. J. Immunol. Methods 65, 55. Pan, M. H., C. S. Lai, et al. (2010). "Anti-inflammatory activity of natural dietary flavonoids." Food Funct. 1(1): 15-31.

Petereit, F., H. Kolodziej, et al. (1991). "Flavan-3-ols and proanthocyanidins from Cistus incanus." Phytochemistry 30(3): 981-985.

Claims

Claims

1. A pharmaceutical composition comprising a Cistus extract for use in a method of preventing and/or treating an HIV infection.

2. The pharmaceutical composition for use of claim 1, wherein Cistus is Cistus ssp., particularly Cistus incanus, Cistus creticus or Cistus villosus, especially Cistus incanus ssp. Tauricus or Cistus incanus ssp. PANDALIS.

3. The pharmaceutical composition for use of claim 1 or 2, wherein the extract is obtainable by extracting a part of a Cistus plant, particularly root, stem, seed, flower and/or leaf of Cistus, especially leaf of Cistus.

4. The pharmaceutical composition for use of any of claims 1 to 3, wherein the extract is obtainable by a method comprising

a) extracting Cistus parts with an extractant selected from the group consisting of water, methanol, ethanol, 1-propanol, 2-propanol and mixtures thereof, particularly water.

5. The pharmaceutical composition for use of claim 4, wherein the method further comprises

b) extracting the extract obtained in step a) by solid phase extraction using

i) a solid phase suitable for extraction of non-polar phases, particularly a solid phase with functional group selected from the group consisting of octadecyl, octyl, phenyl and cyanopropyl, especially octadecyl; and

ii) an eluent selected from the group consisting of water, methanol, ethanol, 1- propanol, 2-propanol and mixtures thereof, particularly methanol; c) optionally at least partially removing the eluent; and

d) preparing a pharmaceutically acceptable composition.

6. The pharmaceutical composition for use of any of claims 1 to 5, wherein the extract is obtainable or obtained by a method comprising

a) extracting a Cistus part with water;

b) optionally filtrating the extract of step a);

c) optionally extracting the extract obtained in step a) or b) by solid phase extraction using a solid phase with octadecyl as functional group and methanol;

d) at least partially removing the water of step a) and/or the methanol of step c); and e) preparing a pharmaceutical composition.

7. The pharmaceutical composition for use of any of claims 1 to 6, wherein the extract is obtainable or obtained by a method comprising

a') providing a Cistus extract obtainable or obtained as defined in any of claims 1 to 6; b') contacting the extract of step a') with polyvinylpolypyrrolidone (PVPP) as solid phase to allow binding of polyphenols to PVPP;

c') isolating the solid phase; and

d') eluting polyphenols from the solid phase.

8. The pharmaceutical composition for use of any of claims 1 to 7, wherein the extracting of step a) is performed at 50°C to 120°C, particularly at 80°C to 110°C, especially at 90°C to 100°C.

9. The pharmaceutical composition for use of any of claims 1 to 8, wherein the HIV is a CCR5-tropic strain, a CXCR4-tropic strain or a CCR5- and CXCR4-tropic (dual tropic) strain or wherein the HIV is a drug-resistant HIV.

10. The pharmaceutical composition for use of any of claims 1 to 9, wherein the pharmaceutical composition is formulated as solid dosage form or as liquid dosage form, particularly as a capsule, tablet, pill, powder, granule, emulsion, microemulsion, solution, suspension, syrup, elixir, suppository, ointment, paste, cream, lotion, gel, powder, spray, inhalant or patch.

11. The pharmaceutical composition for use of any of claims 1 to 10, wherein the pharmaceutical composition is manufactured for oral, nasal, rectal, parenteral, topic, vaginal, subcutaneous, intracutaneous, intramuscular, intraarterial, intravenous or intraperitoneal administration.

12. The pharmaceutical composition for use of any of claims 1 to 11, wherein the pharmaceutical composition is to be administered after presumed HIV infection, particularly within 7 days, more particularly within 6, 5, 4, 3 days, especially within 2 days or 1 day from the presumed HIV infection.

13. The pharmaceutical composition for use of any of claims 1 to 12, wherein the pharmaceutical composition is to be administered alone or in combination with an additional HIV medicament, particularly concomitantly and/or consecutively and/or particularly wherein the additional HIV-medicament at least one drug used in Highly Active Antiretroviral Therapy (HAART) is selected from the group consisting of a entry or fusion inhibitor, a nucleoside/nucleotide reverse transcriptase inhibitor, an integrase inhibitor, and a protease inhibitor, especially T20, AZT, Efavirenz, Raltegravir or Darunavir.

14. A product suitable for the prevention of HIV transmission selected from the group consisting of a condom, a lubricant, such as a personal lubricant, an anal-specific lubricant or a fertility lubricant, a diaphragm, a female condom (femidom), a medical glove and a laboratory glove, wherein an extract as defined in claims 1 to 1 1 present in or on the product.

15. A piece of victuals for use in the prevention and/or treatment of an HIV infection, wherein an extract as defined in claims 1 to 1 1 present in or on the product, particularly wherein the piece of victuals is a food product, especially baby food or children food, particularly for an HIV-infected baby or child.

16. A kit comprising at least two HIV medicaments, wherein one HIV medicament is a pharmaceutical composition as defined in any of claims 1 to 13 and the other HIV- medicament(s) is/are at least one drug used in HAART and/or selected from the group consisting of a entry or fusion inhibitor, a nucleoside/nucleotide reverse transcriptase inhibitor, an integrase inhibitor, and a protease inhibitor, especially T20, AZT, Efavirenz, Raltegravir or Darunavir.

17. A method of producing a polyphenol-enriched Cistus extract, the method comprising a') providing a Cistus extract obtainable or obtained as defined in any of claims 1 to 6; b') contacting the extract of step a') with polyvinylpolypyrrolidone (PVPP) as solid phase to allow binding of polyphenols to PVPP;

c') isolating the solid phase; and

d') eluting polyphenols from the solid phase, thereby producing a polyphenol-enriched Cistus extract.

18. The method of claim 17, wherein

i) the solid phase obtained in step c') is washed, particularly with water;

ii) the eluting of step d') is addition of a caustic solution, particularly NaOH, and optionally followed by neutralization, particularly with HC1; and/or

iii) the eluting of step d') is followed by further purification, particularly with solid phase extraction.

19. A polyphenol-enriched Cistus extract obtainable or obtained by the method of claim 17 or 18.

20. The polyphenol-enriched Cistus extract of claim 19 for use as a medicament.

21. The polyphenol-enriched Cistus extract of claim 19 for use as defined in claims 1 to 13.