WO2010076730A1 - Method for the treatment of an hiv infection - Google Patents

Abstract

A method for the treatment of an HIV infection in a patient, the method comprising administering to the patient a therapeutically effective amount of a liquid oil composition produced from a Melaleuca oil wherein the composition contains 10% or less monoterpenes.

Description

METHOD FOR THE TREATMENT OF AN HIV INFECTION

FIELD OF THE INVENTION

The present invention relates to a method of treatment or management of an HIV infection.

BACKGROUND OF THE INVENTION

The human immunodeficiency virus (HIV) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). Since the first reported cases of HIV and AIDS in about 1981 there have been intensive efforts, first to understand the virus and its life cycle and second to develop therapies and strategies for treatment and control.

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. In practice, 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. In the first stage of infection there is a large amount of HIV in the blood and the immune system responds by producing HIV antibodies. After this initial stage the viral level in the blood drops to very low levels and there are no clinical symptoms. However, during this time, 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.

As the damage to the immune system becomes more severe, patients experience opportunistic infections and leads to late stage HIV or AIDS.

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/mm³. 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.

There are many recognized disadvantages associated with the use of HAART therapy. Many patients experience undesirable side effects, the combination therapy dosage regimes require strict adherence to protocols that require taking multiple pills at regular time intervals. Patient compliance is critical to therapy success. Still further, such therapies are expensive and are of the reach in many countries. However, it is those counties where the HIV pandemic is most severe.

There are of course chemical disinfectants and virucides that are can destroy or inactivate retroviruses upon contact. Such agents are considered toxic to animals and are wholly unsuitable for in vitro use as a method of treatment.

There is clearly a need for an alternative to existing strategies and methods for the treatment, control and/or management of an HIV infection.

SUMMARY OF THE INVENTION

According to a first broad form of the invention, there is provided a method for the treatment of an HIV infection in a patient, the method 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.

According to a second broad form of the invention, there is provided a method for post exposure prophylaxis of an HIV infection in a patient, the method 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.

According to a third broad from of the invention, there is provided a method for the treatment of a latent HIV infection in a patient, the method 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.

According to a fourth broad from of the invention, there is provided a method for the prophylaxis of an HIV infection in an at risk patient, the method 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.

According to a fifth broad form of the invention there is provided a method for treating an HIV infection in a patient, the method 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.

According to a sixth broad form of the invention there is provided use of an a composition 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.

The term "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. Preferably, the conventional Melaleuca is extracted from Melaleuca alternifolia.

Commercially available 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. 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.

Commercially available 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 <χ-pinene, sabinene, γ terpinene, ∞ terpinene and limonene with γ-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. In the present specification and claims, 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 main constituents of tea tree oil from the International (ISO 4730:2004) and Australian (AS 2782-1997) standards, together with an indication of the typical amounts found in commercial tea tree oil are shown in the following Table 1. The standards are similar but contain small differences in the maximum values for some components.

TABLE 1

^* monoterpenes + sesquiterpenes # oxygenated terpenes

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 <χ-pinene, <χ-thujene, β-pinene, sabinene, oc-phellandrene, ∞- terpinene, limonene, γ-terpinene, β-phellandrene, γ-terpinene and terpinolene.

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

48% of the oil. 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. However, 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 Ci₀Hi₆. 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.

The term "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.

The term "post exposure prophylaxis" refers to treatment of a patient immediately after exposure or suspected exposure to the HIV virus.

The term "treatment of latent infection" refers to treatment of an HIV infection during the latent period in which the virus remains dormant in infected cells.

The term "at risk patient" refers to a person that may be at risk from becoming infected with the HIV virus.

The term "antiretroviral agent" refers to any agent that inhibits viral replication and includes a combination of antiretroviral drugs. Examples of antiretroviral drugs include efavirenz, zidovudine, lamivudine, stavudine, lamivudine and nevirapine.

The term "therapeutically effective amount" as used herein 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.

As discussed above, 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. Of these fractions, 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. Preferably, the monoterpenes are removed under reduced pressure and at a temperature that does not exceed 5O⁰C, preferably 4O⁰C. Suitable chromatographic techniques have been described in U.S. Patent No. 4,605,783 and by Hayashi, et al., Bulletin of the Chemical Society of Japan, vol 42, 3026-3028 (1969), both of which are incorporated by reference). The use of fractional distillation of essential oils is well known for obtaining fractions of different boiling points. The monoterpenes found in Melaleuca oil have boiling points ranging from about 155°C for <χ-pinene to about 185°C for terpinolene. Terpinen-4-ol has a boiling point of 212°C. The sesquiterpenes aromadendrene and alloaromadendrene have boiling points of 257°C to 258°C. It will be appreciated that 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.

Generally between about 80% and about 99% of the monoterpenes are removed, typically between about 90% and about 99%. Preferably, 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.

It may be seen that the combined minimum and maximum contents for all of the monoterpenes in Melaleuca oil defined by both the Australian and International standards provides a range of monoterpene content from 18% to 59.5%. It may therefore be clearly appreciated that the composition of the present invention that has a maximum total monoterpene content of 10% falls well outside the standard range and therefore cannot be considered to have the same chemical profile as conventional Melaleuca or tea tree oil.

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. TABLE 2

nd - not detected + - outside the range of ISO 4730:2005 * - outside the range of AS 2782-1997 It will be appreciated that the exact composition may vary between batches. It may also be appreciated that the chemical profile of the composition of the invention is substantially different to that of the standard tea tree oil.

Antiviral activity of tee tree oil against HSV-1 has been reported. Tea tree oil has been proposed for use as a topical medication for herpes lesions. A recent article in Phytotherapy Research published on-line 3 August 2009by Astani et al "comparative study on the antiviral activity of selected monoterpenes derived from essential oils", reports a comparative study of the antiviral activity of selected monoterpenes derived from essential oils, γ-terpinene, <χ-pinene, terpine-4-ol, oc-terpineol, thymol, citral and 1 ,8-cineole were examined for their antiviral activity against HSV-1 in vitro. Only modest antiviral activity was detected when the drugs were added to host cells prior to infection or after entry whereas all tested drugs interacted with virus particles. This suggests inactivation of virus particles rather than interfering with viral replication. The monoterpenes were slightly superior in activity to the alcohols and the monoterpene mixtures present in natural tea tree oil revealed a tenfold higher selectivity index than isolated monoterpenes.

Contrary to this study, 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.

Published data indicates that tea tree oil is toxic if ingested in higher doses and can also cause skin irritation at higher concentrations. (A review of the toxicity of

Melaleuca alternifolia (tea tree) oil, Hammer et al Food Chem Toxicol. 2006 May;44(5):616-25. Epub 2005 Oct 21 ). Again, in contrast to the expectations based upon the parent oil, the present inventor has discovered that the compositions for use in the methods of the present invention have a low oral and dermal toxicity. In particular, 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. When this is converted to a 70 kg human, using a body surface area method, then the oral dose of 208.87g (2.98 g/kg) is found.

In light of these toxicity concerns, tee tree oil is not recommended for internal use. However, again contrary to these recommendations, the modified oil of the present compositions has been shown to be virtually non-toxic.

Pharmaceutical 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.

For oral, buccal, and sublingual administration, 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. Optionally, 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.

For nasal or buccal 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. Alternatively, sterile oils may be employed as solvents or suspending agents. Preferably, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri-glycehdes.

For injection, 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.

For rectal administration, 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. For example 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.

The 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. Whilst not wishing to be bound by theory, it is believed that contrary to the activity of conventional ant-viral agents that target various stages of the HIV replication cycle, the 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.

Another therapy in which the present invention may be suitable is on combination with histone deacetylase 1 (HDAC) inhibitors. 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. In combination with the composition of the invention, the activated virus particles can inactivate released particles.

DETAILED DESCRIPTION OF THE INVENTION Example 1

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.

monoterpenes ^* sesquiterpenes÷ compounds falling outside ISO 4730 are shown in italics

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.

It may be seen that a significant proportion of all compounds of the parent tea tree oil having a retention time of less than 6 minutes have been removed. The total area% of compounds having a retention time of less than 6 minutes in the tea tree oil is 32.86% as compared to 2.11 % in the extract. There has also been an increase in the concentration and number of compounds detected having a retention time of over 30 minutes. Another significant difference is the almost complete removal of γ-terpinene which dropped from 16.65% to 0.57%. The overall monoterpene content has been reduced from 35.91 % to 1.18%.

Physical parameters of the oils were also compared with each other and that of the Australian standard. The samples were analysed in accordance with that standard. The results are shown in Table 4.

TABLE 4

^*- outside the standard range

Clearly, the physical properties of the extract of the invention are significantly different from that of a conventional Melaleuca oil. In particular, the relative density is significantly higher and it is considerably more viscous, reflecting the significant drop in the content of relatively light monoterpenes. In practice, it has been observed that an extract of the invention has a minimum relative density of 0.9400, which effectively distinguishes it from a conventional Melaleuca oil extract. As shown by the analysis above, 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. Thus, it is quite clear from these results that conventional Melaleuca oil and the extract of the invention are chemically, compositionally and physically distinct. Further, as the extract of the invention does not comply with the standard, it cannot be classified as tea tree oil. 2. In vitro experiments

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.

I. Materials and Methods

Drug preparation

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 200μg/ml_.

Scope of work

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.

Basic Virucidal Assay Design

On the day of assay set-up HIV-1 was incubated with the test material at 4⁰C for two and four hours. Following this incubation the virus plus test material was serially diluted (9 dilutions) and then plated onto the target cells. The following table summarizes the experimental setup:

Virucidal Evaluation in PBMCs Anti-HIV Efficacy Evaluation in Fresh Human PBMCs

HIV-I92UG021 (CXCR4-tropic, Group M, Subtype D), and H IV-1_93BRO2I (CCR5- tropic, Group M, Subtype B) were obtained from the NIAID AIDS Research and Reference Reagent Program (Bethesda, MD). 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. After the final wash, cells were enumerated by trypan blue exclusion and re- suspended at 1 x 10⁶ cells/mL in RPMI 1640 supplemented with 15 % Fetal Bovine Serum (FBS), and 2 mM L-glutamine, 4 μg/mL Phytohemagglutinin (PHA, Sigma). The cells were allowed to incubate for 48-72 hours at 37°C. After incubation, PBMCs were centrifuged and re-suspended in RPMI 1640 with 15% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/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 10⁶ 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.

For the PBMC assay, PHA stimulated cells from at least two normal donors were pooled (mixed together), diluted in fresh medium to a final concentration of 1 x 10⁶ cells/mL, and plated in the interior wells of a 96 well round bottom microplate at 50 μL/well (5 x 10⁴ 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.

On the day of assay set-up the HIV-1 isolates were incubated with the test material at 4⁰C 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.

For each experimental variable, matched cell and virus control samples were run in parallel. In this in vitro assay, 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 37⁰C, 5% CO₂ 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.

Reverse transcriptase activity assay

A microtiter plate-based reverse transcriptase (RT) reaction was utilized (Buckheit et al., AIDS Research and Human Retroviruses 7:295-302, 1991 ). Tritiated thymidine triphosphate (³H-TTP, 80 Ci/mmol, NEN) was received in 1 :1 dH₂O:Ethanol at 1 mCi/mL. Poly rA:oligo dT template:phmer (Pharmacia) was prepared as a stock solution by combining 150 μl_ poly rA (20 mg/mL) with 0.5 ml_ oligo dT (20 units/mL) and 5.35 ml_ sterile dH₂O 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 μL 1.0 M EGTA, 125 μl_ dH₂O, 125 μl_ 20% Triton X100, 50 μL 1.0 M Tris (pH 7.4), 50 μL 1.0 M DTT, and 40 μL 1.0 M MgCI₂. The final reaction mixture was prepared by combining 1 part ³H-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 μL of virus containing supernatant added and mixed. The plate was incubated at 37°C for 60 minutes. Following incubation, the 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.

MTS staining for PBMC viability to measure cytotoxicity

At assay termination, assay plates were stained with the soluble tetrazolium- based dye MTS (CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. 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. At termination of the assay, 20 μL 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.

Data Analysis

Using an in-house computer program, the titer of virus remaining in each sample (TCID₅₀) was calculated and the fold-reduction in titer resulting from compound exposure was calculated. Virucidal Evaluation in MAGI-CCR5 Cells

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-β-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-β-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 10⁴ cells/well placed in 96-well plates in media without selection antibiotics.

On the day of assay set-up HIV-1 isolates were incubated with the test material at 4⁰C for two (2) or four (4) hours. Following this incubation the virus plus test material was serially diluted and then plated onto the MAGI-CCR5 cells in order to determine its infectivity, which was assayed by induction of the β- galactosidase reporter gene 48 hours post infection using a chemiluminescence detection system (Tropix Gal-screen™, Bedford, MA). In addition to the conditions described, toxicity controls included cells treated with test compound in the absence of virus. Compound cytotoxicity was assessed in separate plates by incubating test compounds in media without virus for 2 hours prior to adding to MAGI-CCR5 cells to mimic the conditions used for the virucidal assay. Cytotoxicity was determined as described above after addition of 15 μL of MTS reagent per well and incubation for 1.5hr. Data analysis was performed as described above for the PBMC experiment.

II. Results

A. PBMC Summary Tables

Tables A1-5 Summar of C totoxic and Virucidal Activit in PBMCs

(2)=2HR only (4)=4HR only

(2)=2HR only (4)=4HR only

A3

Effect of MAC in Vitamin E on HIV-1 in PBMCs

C=Cytotoxicity Observed V=Virucidal Activity Observed

(2)=2HRonly (4)=4HRonly

(2)=2HRonly (4)=4HRonly

(2)=2HR only (4)=4HR only Table A6. Virucidal Effects on HIV-1 92UG021 and 93BR021 in PBMCs

Exposure Virus Titer (TCID₅₀/mL) Log₁₀

Treatment Time

HIV-1 HIV-1

(hr) 92UG021 93BR021

10% MAC in DMSO 3.509 3.709

1% MAC in DMSO 4.913 5.315

0.1% MAC in DMSO 4.913 4.713

10% DMSO 4.913 5.515

1% DMSO 4.311 4.713

0.1% DMSO 5.315 5.515

10% MAC in Vitamin E 2.104 2.907

1% MAC in Vitamin E 4.913 4.913

0.1% MAC in Vitamin E 4.913 4.913

10% vitamin E 4.913 5.114

1% Vitamin E 4.713 5.315

0.1% Vitamin E 4.713 5.315

Aldrithiol 3.308 2.907

Virus Control 5.315 5.114

10% MAC in DMSO 3.308 3.308

1% MAC in DMSO 4.913 4.512

0.1% MAC in DMSO 5.515 5.114

10% DMSO 4.713 5.515

1% DMSO 4.913 5.114

0.1% DMSO 4.913 4.713

10% MAC in Vitamin E 2.304 3.509

1% MAC in Vitamin E 5.114 4.713

0.1% MAC in Vitamin E 4.512 4.512

10% vitamin E 4.913 3.910

1% Vitamin E 5.114 4.512

0.1% Vitamin E 4.913 4.512

Aldrithiol 3.709 3.107

Virus Control 4.713 4.713

Table A7. %Virucidal Effects on HIV-1 92UG021 and 93BR021 in PBMCs

Tables A8-12: % Cell Toxicity in PBMCs

MAGI-CCR5 Summary Tables

Tables B1-5. Summary of Cytotoxic and Virucidal Activity in MAGI-CCR5 cells

(2)=2HRonly (4)=4HRonly

(2)=2HRonly (4)=4HRonly

(2)=2HRonly (4)=4HRonly

(2)=2HRonly (4)=4HRonly

(2)=2HRonly (4)=4HRonly

Table B6. Virucidal Effects on HIV-1 Ba-L and IMB in MAGI-CCR5 cells

1% DMSO 3.960 4.161

0.1% DMSO 3.960 4.161

10% MAC in Vitamin E 1.452 1.452

1% MAC in Vitamin E 1.452 2.856

0.1% MAC in Vitamin E 3.960 4.161

10% vitamin E 3.258 3.759

1% Vitamin E 3.860 4.161

0.1% Vitamin E 3.759 4.161

Aldrithiol 1.953 2.054

Virus Control 3.960 4.161

10% MAC in DMSO 1.452 1.853

1% MAC in DMSO 3.258 3.759

0.1% MAC in DMSO 3.860 4.161

10% DMSO 3.258 3.559

1% DMSO 3.860 4.161

0.1% DMSO 3.759 4.161

10% MAC in Vitamin E 1.452 1.452

1% MAC in Vitamin E 1.452 3.358

0.1% MAC in Vitamin E 3.559 4.161 10% vitamin E 2.555 3.860

1% Vitamin E 3.860 4.161

0.1% Vitamin E 3.860 4.161

Aldrithiol 1.953 2.254

Virus Control 4.161 4.161

Table B7. % Virucidal Effects on HIV-1 Ba-L and NIB in MAGI-CCR5 cells

Table B8-12. % Cell Toxicity in MAGI-CCR5 cells

1 :4 90 91

1 :8 91 54

1 :16 91 16

1 :32 91

1 :64 12

1 :128

1 :256

1 :512

III. Discussion PBMC results

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.

10% MAC in DMSO exerted cytotoxic effects on PBMCs (Table A8). A similar effect was observed for 10% MAC in Vitamin E (Table A10). Cytotoxic effects >20% are considered to be outside of the inherent error associated with biological assay data and thus significant. DMSO alone was only cytotoxic at the highest concentration tested (Table A9) and this effect was not observed when the DMSO was diluted. Vitamin E alone exhibited a similar cytotoxicity profile (Table A11)

MAGI-CCR5 results 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. Here it was observed that 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. There also was some cytotoxicity associated with Vitamin E and slight amounts of virucidal activity.

As summarized in Tables B6 and B7, 10% and 1 % dilutions of MAC in DMSO resulted in significant virucidal activity. DMSO itself at 10% exhibited some virucidal activity, but this effect was lower than when combined with MAC. The 10% and 1 % dilutions of the 10% MAC in Vitamin E also were virucidal, and 10% and 1 % dilutions of Vitamin E exhibited some virucidal effects that were less than that observed when combined with MAC. Finally, MAC in DMSO (Table B8) was more cytotoxic than DMSO alone (Table B9) and MAC in Vitamin E (Table B10) was more cytotoxic than Vitamin E alone (Table B11). Overall, the data indicate that MAC exhibited a virucidal effect against all four viruses evaluated in the two experimental systems.

1. Phase 1 Trials Study Design

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.

Results

The effect of a single dose on the vital body parameters, systolic and diastolic blood pressures and heart rate at one and two hours after administration was observed. No clinically significant changes were observed.

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. Siliar results were obtained for the kidneys where the levels of total protein albunmin, blood urea nitrogen (BUN) and creatinine in blood were normal and no prtoetin, blood cells nor leucocytes were found in the urine,. The abscence of glucose and ketone bodies in the urine also indicted that 600mg of MAC did not affect glycogeniesis as well. Hematological profiles (particularly the levels of white and red blood cells, haemoglobin, hematocrtite, thrombocyte (platelets) and coagulation time of the subjects were also unaffected by the doses. Further there was no production of Ca oxalate, triphosphate or uric acid crystals in the urine.

The conclusion that can be made from this trial is that MAC is well tolerated in subjects and is not likely to affect vital organs such as liver, kidney and hematology and it did not cause crystaluria.

2. Formulations

The following formulations will be prepared Oral Formulation 2

300mg oil composition

500mg microcrystalline cellulose

Total - 800mg

Oral Formulation 3

150 mg oil composition

500 mg microcrystalline cellulose

Total - 650mg

3. In vivo trials

11 patients with an HIV diagnosis, a high viral load of over 10000 and a CD4 count of less than about 1000 were selected for study. The patients had been on a range of anti-HIV medications and had shown to be unresponsive.

All patients commenced treatment of a twice daily dose of 150mg MAC (total 300mg) and patients 6 and 10 commenced on a thrice daily dose of 150mg MAC. CD4 counts and viral loads were measured at the following intervals:

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)

Base line data is shown in Table C.

TABLE C

Table D shows the change in CD4 count with time.

Table D

Tables E1 and E2 shows the change in viral load with time.

Table E1

Table E2

Clinical symptoms at T1 and T5 may be summarized in Table F.

It may seen that 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.

It will be appreciated that various change and modifications may be made to the inventions as described and claimed herein without departing from the spirit and scope thereof.

Claims

1. A method for the treatment of an HIV infection in a patient, the method comprising administering to the patient a therapeutically effective amount of a liquid oil composition produced from a Melaleuca oil wherein the composition contains 10% or less monoterpenes.

2. The method of claim 1 , wherein the composition comprises less than

5wt% monoterpenes.

3. The method of claim 1 , wherein the composition comprises less than 1wt% monoterpenes.

4. The method of any one of claims 1 to 3, wherein the composition contains trace amounts or less than detectable amounts of each of <χ-pinene, sabinene and ∞ - terpinene.

5. The method of any one of claims 1 to 3, wherein the composition has the following composition:

wherein nd means not detectable.

6. The method of any one of claims 1 to 5, wherein the composition is formulated for oral administration.

7. The method of claim 6, wherein the composition is administered in an amount of about 150mg to about 900mg per day.

8. A method for post exposure prophylaxis of an HIV infection in a patient, the method comprising administering to the patient a therapeutically effective amount of a composition that has been produced from a Melaleuca oil, wherei n the com positi on contai ns 1 0% or less monoterpenes.

9. A method for the treatment of a latent HIV infection in a patient, the method comprising administering to the patient a therapeutically effective amount of a composition a produced from a Melaleuca oil wherein the composition contains 10% or less monoterpenes.

10. A method for the prophylaxis of an HIV infection in an at risk patient, the method comprising administering to the patient an therapeutically effective amount of a composition that has been produced from a Melaleuca oil, wherein the composition contains 10% or less monoterpenes.

11. A method for treating an HIV infection in a patient, the method comprising administering in combination with an antiretroviral agent an effective amount of a composition that has been produced from a Melaleuca oil, wherein the composition contains 10% or less monoterpenes.

12. Use of a composition that has been produced from a Melaleuca oil, wherein the composition contains 10% or less monoterpenes for the manufacture of a medicament for the treatment or prophylaxis of an HIV infection in a patient.

13. The use of claim 12, wherein the composition comprises less than 5wt% monoterpenes.

14. The use of claim 12, wherein the composition comprises less than 1wt% monoterpenes.

15. The use of any one of claims 12 to 14, wherein the composition contains trace amounts or less than detectable amounts of each of <χ-pinene, sabinene and ∞ - terpinene.

16. The use of any one of claims 1 to 3, wherein the composition has the following composition:

wherein nd means not detecable

17. The use of any one of claims 12 to 16, wherein the composition is formulated for oral administration.

18. The use of any one of claims 12 to 17, wherein the composition is administered in an amount of about 150mg to about 900mg per day.

19. The use of any one of claims 12 to 18, wherein the prophylaxis is post exposure prophylaxis.

20. The use of any one of claims 12 to 18, wherein the prophylaxis is post exposure prophylaxis

21. The use of any one of claims 12 to 17 wherein the HIV infection is a latent HIV infection.