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WO2010076730A1 - Méthode de traitement d'une infection par le vih - Google Patents

Méthode de traitement d'une infection par le vih Download PDF

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Publication number
WO2010076730A1
WO2010076730A1 PCT/IB2009/055840 IB2009055840W WO2010076730A1 WO 2010076730 A1 WO2010076730 A1 WO 2010076730A1 IB 2009055840 W IB2009055840 W IB 2009055840W WO 2010076730 A1 WO2010076730 A1 WO 2010076730A1
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Prior art keywords
composition
monoterpenes
patient
oil
hiv
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English (en)
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Max Reynolds
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DONALD JENNY
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DONALD JENNY
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Priority claimed from AU2009900005A external-priority patent/AU2009900005A0/en
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Publication of WO2010076730A1 publication Critical patent/WO2010076730A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/61—Myrtaceae (Myrtle family), e.g. teatree or eucalyptus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • the present invention relates to a method of treatment or management of an HIV infection.
  • HIV human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • Treatment and strategies for controlling viral infections are generally targeted towards steps in the virus replication cycle. It will be appreciated that different types of viruses have different replication mechanisms which means that distinct treatment strategies must be developed for each type of virus.
  • Retroviruses are RNA viruses that upon entry into a host cell, transchbess its viral RNA into cellular DNA.
  • the first antiretroviral drugs concentrated on inhibiting reverse transcriptase activity whereby viral RNA is formed from viral RNA by reverse transcription.
  • Nucleoside and nucleotide reverse transcriptase inhibitors inhibit reverse transcription be being incorporated into the newly synthesized viral DNA and preventing it further elongation.
  • Non-nucleoside reverse transcriptase inhibitors directly bind to the reverse transcriptase enzyme, thereby interfering with its function.
  • the next class of antiretrovirals to be developed were the protease inhibitors that target viral assembly within the cell, lntegrase inhibitors block the action of integrate, a viral enzyme that inserts the viral genome into the DNA of the host cell. Other agents inhibit entry of the retrovirus into the cell.
  • Combinations of retroviral agents are used in order to target the retrovirus at different stages in its replication cycle.
  • individual therapies do not suppress infection for long, which is at least partly due to the rapid mutation of the virus.
  • Such types of combination therapies are generally known as HAART (Highly Active Anti-Retroviral Therapy). Although such therapies may slow down replication of the virus they do not completely eradicate the virus.
  • Treatment of an HIV infection is also dependent upon the stage of the infection.
  • the first stage of infection there is a large amount of HIV in the blood and the immune system responds by producing HIV antibodies.
  • the viral level in the blood drops to very low levels and there are no clinical symptoms.
  • the immune system becomes compromised and deteriorates. Deterioration of the immune system is accompanied by mild symptoms.
  • This third stage is referred to as symptomatic HIV infection.
  • the status of the immune system may be monitored by measuring the CD4 cell count.
  • Anti-HIV therapy is generally started when the CD4 levels drop below about 500 cells/mm 3 .
  • Another factor to consider when to begin anti-HIV therapy is the viral load level. Both CD4 and viral load levels are used to monitor progression of the disease. At present, there is no treatment of management of HIV in the viral stage. In other words, a person must wait until they become symptomatic before treatment is begun.
  • a method for the treatment of an HIV infection in a patient comprising administering to the patient a therapeutically effective amount of a liquid oil composition that has been produced from a Melaleuca oil, wherein the composition contains 10% or less monoterpenes.
  • a method for post exposure prophylaxis of an HIV infection in a patient comprising administering to the patient an antivirally effective amount of a liquid oil composition that has been produced from a Melaleuca oil wherein the composition contains 10% or less monoterpenes.
  • a method for the treatment of a latent HIV infection in a patient comprising administering to the patient a therapeutically effective amount of a liquid oil composition that has been produced from a Melaleuca oil, wherein the composition contains 10% or less monoterpenes.
  • a method for the prophylaxis of an HIV infection in an at risk patient comprising administering to the patient a therapeutically effective amount of a liquid oil composition that has been produced from a Melaleuca oil, wherein the composition contains 10% or less monoterpenes.
  • a method for treating an HIV infection in a patient comprising administering in combination with an antiretroviral agent an effective amount of a liquid oil composition that has been produced from a Melaleuca oil, wherein the composition contains 10% or less monoterpenes.
  • compositions that has been produced from a Melaleuca oil, wherein the composition contains 10% or less monoterpenes for manufacture of a medicament for the treatment of an HIV infection in a patient.
  • Melaleuca oil refers to oil extracted from a Melaleuca plant that complies with International Standard ISO 4730-2004 or Australian Standard AS 2782-1997, which are both incorporated herein by reference.
  • the conventional Melaleuca is extracted from Melaleuca alternifolia.
  • Melaleuca or tea tree oil must comply with International standards (ISO 4730:2004 for Oil of Melaleuca (tea tree oil)). There is also an Australian standard AS 2782-1997. In order to comply with these standards, the oil must be obtained by steam distillation of the foliage and terminal branches of a Melaleuca plant and must contain levels of compounds in the compositional limits set by the standards. High quality oil is considered to be an oil having high levels of terpinen-4-ol and low levels of 1 ,8-cineole.
  • the source plant is not limited to M. Alternifolia but can be obtained from other species such as M. dissitiflora and M. linarifolia.
  • Tea tree oil Conventional steam distilled oil extracts from Melaleuca Alternifolia are commonly known as "tee tree oil”. Tea tree oil is well characterized and is described at page 1622 of the Thirteenth edition of the Merck Index 2001 and has been allocated CAS number 68647-73-4.
  • Melaleuca oi l can com prise u p to about 50% monoterpenes and typically between about 30% and 40%.
  • Monoterpenes found in Melaleuca oil include ⁇ -pinene, sabinene, ⁇ terpinene, ⁇ terpinene and limonene with ⁇ -terpinene being the predominant monoterpene at levels in the order of 17-20%.
  • the standards require the oil to be analysed by gas chromatography using a flame ionization detector.
  • the proportions of components in % are indicated by the integrator.
  • the % proportions of components of the oil unless otherwise stated, will also represent the proportion indicated by an integrator using a flame ionization detector.
  • the term "monoterpene” refers to compounds derived from an isoprene unit that have the formula C10H-16 Monoterpenes found in the essential oil from Melaleuca Alternifolia include ⁇ -pinene, ⁇ -thujene, ⁇ -pinene, sabinene, oc-phellandrene, ⁇ - terpinene, limonene, ⁇ -terpinene, ⁇ -phellandrene, ⁇ -terpinene and terpinolene.
  • Oxygenates or oxygenated terpenes Another class of compounds commonly found in essential oils are known as oxygenates or oxygenated terpenes. These compounds have a terpene skeleton and an oxygen containing functional group. Examples are aldehydes, phenol alcohols, carboxylic acids, ketones and esters. Terpinen-4-ol, having the formula, doH-isO is a major constituent of Melaleuca oil and can constitute up to
  • 1 ,8 cineole is another oxygenated terpene found in Melaleuca oil.
  • Terpinen-4-ol is considered to be the major active constituent of Melaleuca oil.
  • other oxygenated products and the monoterpenes are also believed to have some antimicrobial activity.
  • Oxygenated terpenes are sometimes loosely referred to as monoterpenes, whereas others use the term monoterpene to refer only to compounds having the formula Ci 0 Hi 6 . As the latter is considered to be the more accurate nomenclature, this is the nomenclature that will be used in the present specification and claims.
  • Monoterpenoid is another term that is understood to include the monoterpenes and other related compounds having the monoterpene skeleton.
  • the term "contains 10% or less” refers to the total monoterpene content in the oil composition and is not intended to refer to less than 10 wt% of a single monoterpene.
  • treatment of an HIV infection in a patient refers to any one or more of improving or alleviating symptoms caused by the infection, lowering viral load, maintaining or increasing CD4 counts, preventing progression of infection and the like.
  • post exposure prophylaxis refers to treatment of a patient immediately after exposure or suspected exposure to the HIV virus.
  • treatment of latent infection refers to treatment of an HIV infection during the latent period in which the virus remains dormant in infected cells.
  • At risk patient refers to a person that may be at risk from becoming infected with the HIV virus.
  • antiretroviral agent refers to any agent that inhibits viral replication and includes a combination of antiretroviral drugs.
  • antiretroviral drugs include efavirenz, zidovudine, lamivudine, stavudine, lamivudine and nevirapine.
  • terapéuticaally effective amount means that amount of the composition that elicits the biological or medicinal response in a patient that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • the precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition being treated, recommendations of the treating physician, and the therapeutics or combination of therapeutics selected for administration.
  • essential oils derived from Melaleuca species comprise a monoterpene fraction, an oxygenate fraction and a sesquiterpene fraction, although it will be appreciated that different species may contain different relative amounts of each fraction.
  • the monoterpenes are generally the most volatile and have the lowest molecular weight. Thus, they may be removed by techniques known to those of skill in the art including vacuum low temperature techniques, such as inert gas flushed distillation; fractional distillation, molecular weight separation techniques including chromatographic techniques and selective solvent extraction techniques.
  • the monoterpenes are removed under reduced pressure and at a temperature that does not exceed 5O 0 C, preferably 4O 0 C. Suitable chromatographic techniques have been described in U.S. Patent No.
  • the exact amount of monoterpenes required to be removed to provide a maximum content of 10% may vary, depending upon the monoterpene content of the parent conventional oil. This amount may be readily calculated by a skilled person. For example, a typical Melaleuca oil contains about 40% monoterpenes. Removal of just over 80% i.e. 83.5% of the monoterpenes yields a final extract having a total remaining monoterpene content of 10%. It will also be appreciated that by removing a significant proportion of the total content of the oil, the compositional profile of other non- monoterpene components will be changed.
  • the extract contains less than 10%, 7.5%, 5%, 4%, 3%, 2% and most preferably less than 1 % or 0.5% monoterpenes. Where there are less than 1% monoterpenes, the levels of any one or more of the monoterpenes may fall below the detectable limit of the gas chromatograph used for analysis. This would be understood by a person of skill in the art.
  • a preferred composition of the invention is derived from the essential oil of Melaleuca alternifolia and typically comprises from between about 40% to about 70%, preferably between about 50% to about 65% most preferably between about 60% to about 65% terpinen-4-ol and between about 1 to about 30%, preferably between about 2% to about 25% sesquiterpenes. These ranges include all intermediate subranges and values.
  • the sesquiterpene fraction may include aromadendrene, viridiflorene, delta cadinene, globulol and/or viridiflorol.
  • Table 2 shows a profile of a typical composition of the present invention (MAC) and for comparison the profile of the tea tree oil (TTO) before removal of the monoterpenes.
  • composition of the invention is substantially different to that of the standard tea tree oil.
  • the present inventor has sought to remove these proposed active agents from the composition. Still further, the present inventor has surprisingly discovered that the composition as used in the present invention has a high anti-viral activity against HIV. It will be appreciated that it is well known in the field that an agent that works for one type of virus may not have any activity against a completely different form of virus and that although many candidate compounds may show in vitro activity, this may not necessarily correspond to in vivo activity.
  • compositions for use in the methods of the present invention have a low oral and dermal toxicity.
  • tests conducted on a preferred 10 wt% composition of the present invention with male Wistar rats shows an LD50 values of 21 g/kg which is considered a practically non-toxic compound.
  • the oral dose of 208.87g (2.98 g/kg) is found.
  • compositions comprising the modified Melaleuca oil for use in the methods of the present invention can be manufactured by methods well known in the art such as conventional mixing, dissolving, encapsulating, lyophilizing or emulsifying, among others.
  • the compositions can be in the form of, for example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions.
  • the instant compositions can be formulated for various routes of administration, for example, by oral administration, by transmucosal administration, by rectal administration, or subcutaneous administration as well as intrathecal, intravenous, intramuscular, intraperitoneal, intranasal, intraocular or intraventricular injection.
  • the compound or compounds of the instant invention can also be administered in a local rather than a systemic fashion, such as injection as a sustained release formulation.
  • the following dosage forms are given by way of example and should not be construed as limiting the instant invention
  • powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets are acceptable as solid dosage forms. These can be prepared, for example, by mixing one or more compounds of the instant invention, or pharmaceutically acceptable salts or tautomers thereof, with at least one additive or excipient such as a starch or other additive.
  • Suitable additives or excipients are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, sorbitol, starch, agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycehdes, methyl cellulose, hydroxypropylmethyl- cellulose, and/or polyvinylpyrrolidone.
  • oral dosage forms can contain other ingredients to aid in administration, such as an inactive diluent, or lubricants such as magnesium stearate, or preservatives such as paraben or sorbic acid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or perfuming agents. Additionally, dyestuffs or pigments may be added for identification. Tablets and pills may be further treated with suitable coating materials known in the art.
  • Li q u id dosage forms for oral adm i nistrati on may be i n the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, slurries and solutions, which may contain an inactive diluent, such as water.
  • Pharmaceutical formulations may be prepared as liquid suspensions or solutions using a sterile liquid, such as, but not limited to, an oil, water, an alcohol, and combinations of these.
  • Pharmaceutically suitable surfactants, suspending agents, emulsifying agents may be added for oral or parenteral administration.
  • the pharmaceutical formulations may be a spray or aerosol containing and appropriate solvents and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations of these.
  • a propellant for an aerosol formulation may include compressed air, nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.
  • the compound or compounds of the instant invention are conveniently delivered in the form of an aerosol spray presentation from a nebulizer or the like.
  • Injectable dosage forms generally include aqueous suspensions or oil suspensions which may be prepared using a suitable dispersant or wetting agent and a suspending agent.
  • Injectable forms may be in solution phase or in the form of a suspension, which is prepared with a solvent or diluent.
  • Acceptable solvents or vehicles include sterilized water, Ringer's solution, or an isotonic aqueous saline solution.
  • sterile oils may be employed as solvents or suspending agents.
  • the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri-glycehdes.
  • the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.
  • the compounds may be formulated for parenteral administration by injection such as by bolus injection or continuous infusion.
  • a unit dosage form for injection may be in ampoules or in multi-dose containers.
  • the pharmaceutical formulations may be in the form of a suppository, an ointment, an enema, a tablet or a cream for release of compound in the intestines, sigmoid flexure and/or rectum.
  • Rectal suppositories are prepared by mixing one or more compounds of the instant invention, or pharmaceutically acceptable salts or tautomers of the compound, with acceptable vehicles, for example, cocoa butter or polyethylene glycol, which is present in a solid phase at normal storing temperatures, and present in a liquid phase at those temperatures suitable to release a drug inside the body, such as in the rectum. Oils may also be employed in the preparation of formulations of the soft gelatin type and suppositories.
  • Water, saline, aqueous dextrose and related sugar solutions, and glycerols may be employed in the preparation of suspension formulations which may also contain suspending agents such as pectins, carbomers, methyl cellulose, hydroxypropyl cellulose or carboxymethyl cellulose, as well as buffers and preservatives.
  • the compounds may also be administered dermally. It has been observed that the compounds are readily absorbed through the skin such that dermal uptake directly into the lymphatic system by dermal application about the lymph nodes is possible.
  • Dosage levels for treatment typically depend upon the stage of the disease.
  • dosages for the treatment of a latent infection may be between about 100 to about 500mg, typically about 200 to about 400mg per day for an adult 70kg human.
  • Dosages during symptomatic stages of the disease may increase to up to about 1200mg per day, typically between about 250 to about 750mg per day.
  • Post prophylactic doses may be a one of very high dose or may be continued daily for up to about a week.
  • composition for use in the method of the present invention may also be used in a combination therapy with one or more other anti-viral agents.
  • composition of the present invention kills or inactivates the viral particle on contact. This offers an advantage over current therapies, in that the present composition is relatively insensitive to viral mutations. It will be appreciated that the compositions of the invention my find use as an adjunct or in combination therapies with conventional ant-viral agents.
  • HDAC histone deacetylase 1
  • the enzyme HDAC1 is known to help HIV to persist in a latent phase of infection. Inhibition of this enzyme can stimulate production of infectious virus, thereby eliminating the store of latent HIV.
  • the activated virus particles can inactivate released particles.
  • a composition of the present invention was prepared by removing essentially all the monoterpene fraction from Melaleuca alternifolia essential oil .
  • the composition of the oils before and after removal of the monoterpene fraction was analysed by gas chromatography.
  • Table 3 shows a profile of a typical composition of the present invention following monoterpene removal and for comparison the profile of the Melaleuca oil (TTO, tea tree oil) before removal of the monoterpenes.
  • TTO Melaleuca oil
  • the area percentages in Table 2 were measured by total ion chromatography and are not directly comparable with the flame ionization detection used according to the International and Australian standards. However, they are comparable between each other and significant changes in composition of the monoterpenes can be seen. Although, the compositions are not directly comparable with flame ionization detection, the changes in the monoterpene levels are so dramatic that the inventor believes that it is reasonable to conclude that the levels of each monoterpene in the composition represented in Table 2 lies well outside of the minimum levels required by the Australian and International standards.
  • the physical properties of the extract of the invention are significantly different from that of a conventional Melaleuca oil.
  • the relative density is significantly higher and it is considerably more viscous, reflecting the significant drop in the content of relatively light monoterpenes.
  • an extract of the invention has a minimum relative density of 0.9400, which effectively distinguishes it from a conventional Melaleuca oil extract.
  • the extract of the invention does not fall within either the chemical or physical requirements to comply with the Australian standard for Melaleuca or tea tree oil.
  • conventional Melaleuca oil and the extract of the invention are chemically, compositionally and physically distinct.
  • the extract of the invention does not comply with the standard, it cannot be classified as tea tree oil. 2.
  • the virucidal activity and cellular cytotoxicity of 10% MAC in DMSO or Vitamin E was tested against HIV-1 92UG021 and 93BR021 in PBMCs and HIV-1 Ba-L and IMB in MAGI-CCR5 cells.
  • MAC 100% MAC, 10% MAC in Vitamin E and 100% Vitamin E were used as stock solutions.
  • Each stock solution was diluted 1 :5 (0.2ml_ stock + 0.8ml_ tissue culture medium) to achieve a 2X working solution.
  • Equal volumes of the 2X working solution and diluted virus were incubated for 2 or 4hr as described below.
  • the final concentrations of MAC are expressed as 10, 1.0 and 0.1%. This reflects 10, 1.0 and 0.1 % of the original 10% stock solution.
  • Aldhthiol was used as a virucidal control at a final working concentration of 200 ⁇ g/ml_.
  • MAC in DMSO or Vitamin E was tested at three (3) concentrations (10, 1.0, and 0.1 % of 10% stock solutions) in virucidal assay against one (1 ) CXCR4-tropic HIV-1 isolate (92UG021 ) and one (1 ) CCR5-tropic HIV-1 isolate (93BR021 ) in PBMCs using a reverse transcriptase (RT) endpoint, and against one (1 ) CXCR4-tropic HIV-1 isolate (IMB) and one (1 ) CCR5-tropic HIV-1 isolate (Ba-L) in MAGI-CCR5 cells using a chemiluminescence endpoint. Two (2) virus-drug exposure times were evaluated: 2 and 4 hr. The test compound was evaluated in both the presence of Vitamin E or DMSO. Vitamin E and DMSO were also included as controls. The study design is summarized in the table below.
  • HIV-I92UG021 CXCR4-tropic, Group M, Subtype D
  • H IV-1 93B RO2I CCR5- tropic, Group M, Subtype B
  • Pre-titered aliquots of virus were removed from the freezer (LN2) and thawed rapidly to room temperature in a biological safety cabinet immediately before use.
  • Fresh human PBMCs, seronegative for HIV and HBV, were isolated from screened donors (Biological Specialty Corporation, Colmar, PA). Cells were pelleted/washed 2-3 times by low speed centrifugation and re-suspension in PBS to remove contaminating platelets.
  • the Leukophoresed blood was then diluted 1 :1 with Dulbecco's Phosphate Buffered Saline (DPBS) and layered over 14 ml_ of Lymphocyte Separation Medium (LSM; Cellgro® by Mediatech, Inc.; density 1.078+/-0.002 g/ml; Cat.# 85-072-CL) in a 50 mL centrifuge tube and then centhfuged for 30 minutes at 600 X g. Banded PBMCs were gently aspirated from the resulting interface and subsequently washed 2X with PBS by low speed centrifugation.
  • DPBS Dulbecco's Phosphate Buffered Saline
  • cells were enumerated by trypan blue exclusion and re- suspended at 1 x 10 6 cells/mL in RPMI 1640 supplemented with 15 % Fetal Bovine Serum (FBS), and 2 mM L-glutamine, 4 ⁇ g/mL Phytohemagglutinin (PHA, Sigma). The cells were allowed to incubate for 48-72 hours at 37°C.
  • FBS Fetal Bovine Serum
  • PHA Phytohemagglutinin
  • PBMCs were centrifuged and re-suspended in RPMI 1640 with 15% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, and 20 U/mL recombinant human IL-2 (R&D Systems, Inc). IL-2 was included in the culture medium to maintain the cell division initiated by the PHA mitogenic stimulation.
  • PBMCs were maintained in this medium at a concentration of 1-2 x 10 6 cells/mL with biweekly medium changes until used in the assay protocol. Cells were kept in culture for a maximum of two weeks before being deemed too old for use in assays and discarded. MDMs were depleted from the culture as the result of adherence to the tissue culture flask.
  • PHA stimulated cells from at least two normal donors were pooled (mixed together), diluted in fresh medium to a final concentration of 1 x 10 6 cells/mL, and plated in the interior wells of a 96 well round bottom microplate at 50 ⁇ L/well (5 x 10 4 cells/well) in a standard format developed by the Infectious Disease Research department of Southern Research Institute. Pooling (mixing) of mononuclear cells from more than one donor was used to minimize the variability observed between individual donors, which results from quantitative and qualitative differences in HIV infection and overall response to the PHA and IL-2 of primary lymphocyte populations.
  • the HIV-1 isolates were incubated with the test material at 4 0 C for two (2) or four (4) hours. Following this incubation the virus plus test material was serially diluted and then plated onto the PBMC target cells.
  • PBMC viability remains high throughout the duration of the incubation period (no virus-induced cytopathic effect is observed). Therefore, infected wells were used in the assessment of both virucidal activity and cytotoxicity.
  • the PBMC cultures were maintained for seven days following infection at 37 0 C, 5% CO 2 After this period, cell-free supernatant samples were collected for analysis of reverse transcriptase activity and compound cytotoxicity was then measured by addition of MTS to the plates for determination of cell viability. Wells were also examined microscopically and any abnormalities were noted.
  • RT reverse transcriptase
  • Poly rA:oligo dT template:phmer (Pharmacia) was prepared as a stock solution by combining 150 ⁇ l_ poly rA (20 mg/mL) with 0.5 ml_ oligo dT (20 units/mL) and 5.35 ml_ sterile dH 2 O followed by aliquoting (1.0 ml_) and storage at -20°C.
  • the RT reaction buffer was prepared fresh on a daily basis and consisted of 125 ⁇ L 1.0 M EGTA, 125 ⁇ l_ dH 2 O, 125 ⁇ l_ 20% Triton X100, 50 ⁇ L 1.0 M Tris (pH 7.4), 50 ⁇ L 1.0 M DTT, and 40 ⁇ L 1.0 M MgCI 2 .
  • the final reaction mixture was prepared by combining 1 part 3 H-TTP, 4 parts dh ⁇ O, 2.5 parts poly rA:oligo dT stock and 2.5 parts reaction buffer. Ten microliters of this reaction mixture was placed in a round bottom microtiter plate and 15 ⁇ L of virus containing supernatant added and mixed. The plate was incubated at 37°C for 60 minutes.
  • reaction volume was spotted onto DE81 filter-mats (Wallac), washed 5 times for 5 minutes each in a 5% sodium phosphate buffer or 2X SSC (Life Technologies). Next they were washed 2 times for 1 minute each in distilled water, 2 times for 1 minute each in 70% ethanol, and then dried. Incorporated radioactivity (counts per minute, CPM) was quantified using standard liquid scintillation techniques.
  • assay plates were stained with the soluble tetrazolium- based dye MTS (CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity.
  • MTS CellTiter 96 Reagent, Promega
  • the mitochondrial enzymes of metabolically active cells metabolize MTS to yield a soluble formazan product. This allows the rapid quantitative analysis of cell viability and compound cytotoxicity.
  • the MTS is a stable solution that does not require preparation before use.
  • 20 ⁇ L of MTS reagent was added per well.
  • the microtiter plates were then incubated 4-6 hrs at 37°C. The incubation intervals were chosen based on empirically determined times for optimal dye reduction.
  • Adhesive plate sealers were used in place of the lids, the sealed plate inverted several times to mix the soluble formazan product and the plate read spectrophotomethcally at 490/650 nm with a Molecular Devices Vmax or SpectraMaxPlus plate reader.
  • MAGI-CCR5 cells, HIV-WL (CCR5-tropic, Group M, Subtype B), and HIV-1 m B (CXCR4-tropic, Group M, Subtype B) were obtained from the NIAID AIDS Research and Reference Reagent Program (Bethesda, MD).
  • Pre-titered aliquots of HIV-1 Ba-L and HIV-1 IMB were removed from the freezer (-80°C) and thawed rapidly to room temperature in a biological safety cabinet immediately before use.
  • the virucidal assay was performed using MAGI-CCR5 cells as target cells; these cells naturally express the CXCR4 co-receptor and are engineered to express CD4 (HIV-1 cell surface receptor), the CCR5 co-receptor and also to contain an LTR- ⁇ -galactosidase reporter construct. Infection of the cells with HIV-1 results in expression of the H IV-1 Tat protein, leading to transactivation of the LTR- ⁇ -galactosidase reporter construct, which serves as a simple measure for virus infectivity and replication.
  • MAGI-CCR5 cells are routinely cultured with the required selection antibiotics to maintain their expression constructs. Twenty- four hours prior to initiation of the assay the cells were trypsinized, counted and 2 x 10 4 cells/well placed in 96-well plates in media without selection antibiotics.
  • toxicity controls included cells treated with test compound in the absence of virus.
  • Tables A1-A5 provide a combined summary of the virucidal activity and cytotoxicity of MAC in DMSO or Vitamin E against HIV-1 92UG021 and 93BR021 in PBMCs. Overall, at dilutions beyond the cytotoxic levels, 10% MAC demonstrated virucidal effects on these viruses. As shown in Table A6 there was a marked reduction in the calculated TCID50 for HIV-1 exposed for 2 or 4 hours to MAC in DMSO or Vitamin E. Table A7 expresses these results as percent virus killing, and the virucidal effect of MAC was generally comparable to that of the virucidal control, Aldrithiol.
  • Tables B1 -B5 provide a combined summary of the virucidal activity and cellular toxicity of MAC in DMSO or Vitamin E against HIV Ba-L and IMB in MAGI-CCR5 cells.
  • 10% MAC in DMSO or Vitamin E clearly exhibited virucidal effects at dilutions beyond the cytotoxic effects.
  • DMSO alone exerted some cytotoxicity but did not exert virucidal effects.
  • Twenty nine subjects were divided into three equal dosing groups of 5 males and 4-5 females. The subjects were aged between 19 to 53 years of age with normal body weight. The three groups were subjected to the following dosage regimes of an oral capsular does of MAC; Group 1 - a single 600mg dose after a meal; a single 750mg dose after a meal and three 300 mg doses at 8 hour intervals for 5 days.
  • Urine chemistry, urine sediments, blood chemistry and haematological profiles were measured prior to dosing and at 14 days post dosing. The results showed that after all dosage regines, urine sediments, levels of urine, blood chemistries and haematological profiles were in normal values . These findings show that there was no apparent harm to the liver where the total levels of total and direct biliruben, SGOT and alkaline phopshatase in the blood were in normal ranges and there was no biliruben nor urobilin found in urine.
  • T1 9-23 December 2008 (about 4 weeks from TO)
  • T2 9 February 2009 (about 51/2 weeks from TO)
  • T3 14 February 2009) (about 6 weeks from TO)
  • T4 16 March 2009 (about 16 weeks from TO)
  • T5 18 September 2009 (about 36 weeks from TO)
  • Table D shows the change in CD4 count with time.
  • Tables E1 and E2 shows the change in viral load with time.
  • treatment according to the method of the invention has resulted in a general decrease in viral load and a concomitant increase in CD4 cells. Still further, the patients are experiencing fewer symptoms and opportunistic infections and have an improved quality of life. Still further, the patient's are experiencing if any, side affects with treatment according to the present invention.

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Abstract

Méthode de traitement d'une infection par le VIH d'un patient, la méthode comprenant l'administration au patient d'une quantité thérapeutiquement active d'une composition d'huile liquide produite à partir d'une huile de Melaleuca, la composition contenant 10 % de monoterpènes ou moins.
PCT/IB2009/055840 2009-01-02 2009-12-18 Méthode de traitement d'une infection par le vih Ceased WO2010076730A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2009900005A AU2009900005A0 (en) 2009-01-02 A patent for the use of Melaleuca Alternifolia Concentrate (M.A.C.) in the treatment of HIV patients both active and latent infections
AU2009900005 2009-01-02

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WO2010076730A1 true WO2010076730A1 (fr) 2010-07-08

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012104568A1 (fr) * 2011-02-02 2012-08-09 GILHOLM, Stephen Philip Composition de monoterpénoïdes pour stimuler le système immunitaire

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2709964A1 (fr) * 1993-09-14 1995-03-24 Dupont Paul Compositions pharmaceutiques antivirales proposées dans le traitement du SIDA et de l'Herpès.
FR2830198A1 (fr) * 2001-09-28 2003-04-04 Jean Pierre Willem Compositions pharmaceutiques comprenant des huiles essentielles et leurs utilisations
WO2004076561A1 (fr) * 2003-02-27 2004-09-10 Novasel Australia Pty Ltd Preparations sous forme d'emulsions a base de poloxameres
US20060068044A1 (en) * 2004-09-30 2006-03-30 Max Reynolds Antimicrobial compositions and applications therefore

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2709964A1 (fr) * 1993-09-14 1995-03-24 Dupont Paul Compositions pharmaceutiques antivirales proposées dans le traitement du SIDA et de l'Herpès.
FR2830198A1 (fr) * 2001-09-28 2003-04-04 Jean Pierre Willem Compositions pharmaceutiques comprenant des huiles essentielles et leurs utilisations
WO2004076561A1 (fr) * 2003-02-27 2004-09-10 Novasel Australia Pty Ltd Preparations sous forme d'emulsions a base de poloxameres
US20060068044A1 (en) * 2004-09-30 2006-03-30 Max Reynolds Antimicrobial compositions and applications therefore

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012104568A1 (fr) * 2011-02-02 2012-08-09 GILHOLM, Stephen Philip Composition de monoterpénoïdes pour stimuler le système immunitaire
WO2012104567A1 (fr) * 2011-02-02 2012-08-09 GILHOLM, Stephen Philip Méthode pour le traitement d'une infection à vih

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