CA2582559A1 - Treatment of hpv infections and cancer - Google Patents

Abstract

The present invention concerns the use of agents that inhibit the chymotryptic activity of the 26s unit of the proteosome for the prevention of infection by, or to effect elimination of, human papilloma virus (HPV) from tissues of subjects infected with, or susceptible to, such viruses. The agents are particularly useful for preventing the development of cancers caused by HPV.

Description

Treatment of HPV infections and Cancer The present invention relates to prevention and treatment of human papilloma virus infections and also to the prevention and treatment of cancer.

Many different forms of cancer exist, and it is believed that there are many different causes of the disease. The incidence of cancer varies, but it represents the second highest cause of mortality, after heart disease, in most developed countries.
Current estimates suggest that one in three Americans alive at present will suffer from some form of cancer. Methods of treatment for cancer exist, although there is a well recognised need to develop new and improved techniques. Furthermore, there is also a requirement to develop chemopreventative agents that could be used to inhibit the development of cancer in the general population, susceptible high-risk individuals or as an agent to prevent re-occurrence of disease in individuals already affected.

Human tumour viruses are emerging as a major cause of human cancer and there is now a great deal of evidence which supports the contention that these viruses cause cancer by inducing geiletic instability in infected cells. For example, the E6 protein from high risk forms of the human papilloma virus such as type 16 (HPV16) is known to induce genetic instability producing abnormal numbers of centrosomes, multinucleation and nuclear atypia although the mechanisms underlying this process are poorly understood.

According to a first aspect of the invention there is provided the use of an agent that inhibits the chymotryptic activity of the 26s unit of the proteosome in the manufacture of a topical medicament to prevent infection,, or to effect, eliznination, of human papilloma virus (HPV) from tissues of subjects infected with, or susceptible to, such viruses.

According to a second aspect of the preserit invention there is provided a method for the prevention of infection, or to effect elimination, of human papilloma virus (HPV) from tissues of subjects infected with, or susceptible to, such viruses comprising topically administering to a patient in need of such treatment an effective amount of an agent that inhibits the chymotryptic activity of the 26s unit of the proteosome.

The inventors have found to their surprise that agents which inhibit the chymotryptic activity of the 26s unit of the proteosome (some of which have been proposed for use as orally ingested medicaments for the systemic clinical management of retroviral infections such as HIV) are also clinically useful for topical administration to tissues to prevent or treat malignancies caused by human papilloma virus.

The invention has been based on the inventors findings relating to the effects of agents according to the first aspect of the invention, such as indinavir, on HPV
infections and particularly cancers associated with such infections. It has been recognised that high-risk human papilloma virus type 16 (HPV 16) or human papilloma virus type 18 (HPV18) are particularly associated with the aetiology of cervical carcinoma whereas low-risk viruses such as HPV6/11 are associated with genital warts. Low and high risk HPVs produce different forms of the viral E6 oncoprotein with HPV 16 E6 or HPV 18 E6 producing effects, which are thought to more readily promote malignant conversion than, for example, HPV6 E6. Clearly there is a great deal of interest in the cellular targets of such viral oncoproteins since it is realised that these may be proteins with a pivotal role in the transformation process.
HPV 16 E6 is known to associate with a cellular protein E6 accessory protein (E6AP) that is an E3 ubiqitin ligase. This interaction causes a gain of function activation of the E3 ligase which inappropriately labels (ubiquitinylates) several important cellular proteins for degradation by the 26S proteasome eg p53. Since p53 is the most widely studied of all tumour suppressor proteins, this interaction is believed to be important in the development of HPV related malignant disease.

The inventors conducted experiments that demonstrated that both HPV16 and 6 E6 proteins interacted very strongly with the tissue, serine protease inhibitor alpha-1-antichymotrypsin (AACT). This interaction is likely to inhibit the activity of AACT
thereby implying that activation of chymotryptic activity may: be a consequence of HPV infection. Furthermore, since this interaction was observed with both low and high risk HPV E6 proteins this implies that it is necessary for HPV related disease per se.

However the proteasome is very complex and the inventors are the first to realise that the chymotryptic activity of the proteasome may be the preferred target for treatment of HPV infections. It is known that Indinavir and related inhibitors can suppress the chymotryptic activity of the 26S proteasome but have little effect on the petidyl-glutamyl-peptide hydrolysing activity of this complex or the three peptidase activities of the 20S proteasome. Thus the inventors are the first to propose that the anti-chymotryptic activity of indinavir and related compounds may be useful for the treatment of HPV infections Since the E6 protein from low risk virus also interact with AACT the inventors results indicate that agents according to the invention are also useful for the treatment of low risk HPV infections (e.g. genital warts caused by HPV6/11) as well as high risk H.PV infections (HPV 16, HPV 18 etc) that can lead to the development of cancer (eg. in the cervix).

Furthermore they have realised that these effects make indinavir, and related compounds, particularly useful for topical application to a site of infection (or site of potential infection) and thereby act as a treatment or prophylactic against HPV
infection and the development of HPV related cancers.

It is preferred that the agents according to the first aspect of the invention are used to prevent the development of cancer or treat cancers. The inventors have found that the agents are particularly useful for preventing the development of cancers caused by HPV. Accordingly asymptomatic HPV infected individuals (i.e. those with a viral infection but no evidence of malignant disease) or HPV infected individuals with pre malignant cells may be treated according to the invention by topical administration of the agents with a view to treating the viral infection and thereby preventing the development of cancer.

The invention, to the extent that it is applicable to the prevention and treatment of cancer, may be applied to range of cancers, in which HPV is implicated, such as cervical, vulval, anal and oral carcinomas The invention is applicable particularly, but by no means exclusively, to pre-cancerous conditions and cancers caused by oncogenic viruses, e.g. transforming human papilloma viruses (HPVs).

The inventors have further established that agents as defined herein are useful for eliminating HPV from tissues and thereby reverse the predisposition state of "HPV positivity". It will be appreciated that the elimination of this virus will reduce the risk of malignancies associated with infection as well as being useful for reducing/preventing other conditions/diseases associated with this virus.

According to a preferred embodiment of the invention the agents may be topically applied to the vulva, anus or penis to treat, or prevent, genital warts (e.g.
caused by the abovementioned low risk HPV6/11) According to another preferred embodiment of the invention the agents may be topically applied to the larynx or oral cavity to treat, or prevent, HPV
related oral conditions such as recurrent respiratory papillomatosis.

According to another preferred embodiment of the invention the agents may be topically applied to skin to treat, or prevent cutaneous HPV related warts caused by HPV type 2 and others.

According to a further preferred embodiment of the invention the inhibitors may be topically applied to the vulva, anus, cervix or penis to treat, and particularly prevent, the development of cancer caused by high risk HPVs (e.g. HPV 16 or HPV
18).

Examples of agents that may be used according to the invention include amprenavir, abacavir, CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir, tipranavir, saquinavir, ABT-378, AG 1776, and BMS-232,632. Other agents inhibitors which may be used according to the present invention are disclosed in EP 0 541 168.

The inventors have found that certain HIV protease inhibitors (e.g.
ritonavir) do not have the same activity as agents according to the first aspect of the invention. Accordingly ritonavir and related compounds do not fall with the scope of the present invention.

A list of preferred agents (and their sources) that may be used according to the inventions is given in Table 1.

Table 1:
Brand name INN Lab code no. Company/Source Lexiva fosamprenavir GSK/Vertex Agenerase amprenavir GSK
Reyataz atazanavir BMS
Crixivan Indinavir Merck Viracept Nelfinavir Agouron Invirase/Fortovase saquinavir Roche tipranavir PNU-140690 Beohringer Ingelheim RO-033-4649 Roche TMC-114 Tibotec Virco L-756,423 Merck mozenavir DMP-450 Triangle velcade PS-341 Millenium A most preferred agent is indinavir and salts thereof. Functional derivatives of indinavir are also preferred inhibitors for use according to the invention.
Examples of such derivatives are known to the art. For instance US 5,413, 999 discloses a number of molecules related to indinvar that may be usefully employed according to the present invention. By way of example only, the molecules defined by Claim 1 of US

5,413, 999 may be used according to the first or second aspects of the present invention.

A preferred salt of indinavir is indinavir sulphate (see the formula 1).
Indinavir sulphate is a white to off-white, hygroscopic, crystalline powder with a molecular formula of C36H47N504.H2SO4 and a molecular weight of 711.88.
Indinavir sulphate is soluble in both water, preferably at acidic pHs, and methanol.

N OH HO = H2SO4 H
N N, N

~ 0 O'" NHC(CH3)3 Formula I
Indinavir sulphate is marketed by Merck & Co. as CRIXNAN .
CRIXNAN comprises indinavir sulphate, anhydrous lactose and magnesium stearate within a capsule (consisting of gelatin, dyes, titanium dioxide, silicon dioxide and sodium lauryl sulphate. Such capsules may be used according to the invention.
However CRIXIVAN is formulated for oral ingestion and it is preferred that indinavir sulphate is formulated such that it is better suited for topical application to the target tissue as discussed in more detail below.

The inventors have found that the agents according to the first aspect of the invention are not only useful for treating HPV related cancer but are also surprisingly useful for preventing the development of HPV related cancer. Accordingly compounds such as indinavir have a most preferred use as a prophylactic.

The agents may be given to subjects with a genetic disposition to developing cancer (most particularly cervical carcinoma) or even those facing environmental risk (e.g. people exposed to carcinogens). In a preferred embodiment the inhibitors may be given to women who are at risk of developing cancer. Such women can include those who have been diagnosed as having a high risk HPV infection (e.g.. HPV16 or HPV18) of the urino-genital tract (and particularly the cervix). There may not be any symptomatic evidence that such women have a HPV infection, cervical carcinoma or even precancerous cells of the cervix. However women with such infections are believed by the inventors to be at risk of developing cervical cancer.
Therefore, according to a preferred embodiment of the invention, the agents may be topically applied to the cervix, vulva or anus of women with a viral infection at these sites with a view to preventing the development of cancer at a future date.

The agents may be used to prevent or treat cancer as a monotherapy (i.e. use of the inhibitor alone) or in combination witli other compounds or treatments used in cancer therapy (e.g. chemotherapeutic agents, radiotherapy).

It is most preferred that the agents are used to treat liumans (e.g. women at risk of developing cervical, vulval or anal cancer or at risk of HPV infection).
However it will be appreciated that the agents may also have some veterinary use.

The medicaments used according to the invention may take a number of different forms depending, in particular on the manner in which the medicament is to be applied topically. Thus, for example, the medicament may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle, liposome or any other suitable form that may be administered to a person or animal. It will be appreciated that the vehicle of the medicament of the invention should be one which is well tolerated by the subject to whom it is given and enables delivery of the inhibitors to the effected or target site.

It is most preferred that the agents are formulated for topical use (e.g as creams or ointments). For instance, when used to treat (or prevent the development of) cervical cancer, it is preferred that they are formulated as creams or ointments that may be applied directly to the vulva, penis or cervix by techniques known to the art.
Alternatively the agents may be formulated in a pessary according to techniques known to the art.

Examples of preferred formulations for use according to the invention are gels or hydro-gels containing the active agent and which are specifically formulated for application to the target tissue (e.g. the cervix). A more preferred hydro-gel may be composed of: polyoxyethylene or polyoxypropylene block copolymers.
Alternatively it may comprisetive ethylene oxide, styrene oxide di-block copolymers or poly(ethylene oxide)-block-poly(DL-lactide) copolymers.

Most preferred hydro-gels for use for vaginal delivery according to the invention may comprise monoolein (e.g. the Myverol 18-99)/water lyotrophic liquid crystalline gels. These gels show good bioadhesive characteristics and will bind to the cervix when delivered into the vagina and thereby represent a good means of delivering the active agent to a target tissue. Furthennore such gels are able to absorb water when delivered to the vagina until they reach an equilibrium water content of approximately 40% in the vagina but still maintain there physical integrity.
Such gels can delivery (to the cervix) the active agent over a time period of approximately 18 hours and will follow square route of time kinetics (i.e. the rate of release of the agent is diffusion controlled).

Alternatively the hydrogel may comprise a triblock copolymer of pol(epsilon-caprolactone) and poly (oxyethylene). Such gels also exhibit good release properties of agents according to the first aspect of the invention within the vagina (e.g. for use in the prevention of cervical cancer).

It will be appreciated that the amount of the agent required is determined by biological activity and bioavailability which in turn depends on the mode of administration, the physicochemical properties of the compound employed and whether the compound is being used as a monotherapy or in a combined therapy.
The frequency of administration will also be influenced by the abovementioned factors and particularly the half-life of the coinpound within the subject being treated.

Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular compound in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition.
Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to establish specific formulations of compositions and precise therapeutic regimes (such as daily doses of the compounds and the frequency of administration).

Generally, a daily dose of between 0.01 g/kg of body weight and 1.0 g/kg of body weight of an agent (e.g. indinavir) may be used for the prevention or treatment of cancer - depending upon which specific compound is used. More preferably, the daily dose is between 0.01 mg/kg of body weight and 100 mg/kg of body weight.

When given topically, it is preferred that a hulnan adult is given a sufficient amount of inhibitor to ensure that a dose of between about 0.1 M and 10mM
reaches the target cells. Accordingly an ointment or cream should comprise at least between about 0.1 M and 10mM of an inhibitor according to the invention and preferably a greater concentration that will be effective for delivering an effective concentration of inhibitor to the tissue being treated by the ointment or cream. However, it will be appreciated that the required dose may be varied since it is known that agents according to the first aspect of the invention (such as indinavir) can be substrates for multi-drug resistance gene product p-glycoprotein. The agent may therefore be co-administered with a p-glycoprotein inhibitor such as verapamil, tariquidar XR9576, zosuquidar LY335979, laniquidar R101933 and OBT-093.

Daily doses may be given as a single administration (e.g. as a pessary).
Alternatively, the agent used may require administration twice or more times during a day. As an example, indinavir for preventing the development of cervical cancer may be administered as a hydrogel or cream, once, twice, three or more times a day in an amount sufficient to ensure between about 0.1 M and 10mM of the inhibitor reaches the target cells.

This invention further provides a pharmaceutical composition comprising a therapeutically effective amount of an agent according to the first aspect of the invention and a pharmaceutically acceptable vehicle. In one embodiment, the amount of the agent is an amount from about 0.01 mg/ml to about 100 mg/ml. In another embodiment, the amount is from about 0.1 mg/ml to about 100 mg/ml. When the agent is indinavir sulphate, the amount of indinavir sulphate may be an amount from about 0.01 mg/ml to about 20 mg/ml; and more preferably about 1 mg/ml to about 10 mg/ml.

In one embodiment, the vehicle is a liquid and the composition is a solution.
In another embodiment, the vehicle is a gel and the composition is a suppository or pessary. In a further embodiment the vehicle is an emulsion (or other pharmaceutically acceptable base) and the composition is a cream.

This invention provides a process for making a pharmaceutical composition comprising combining a therapeutically effective amount of an agent according to the first aspect of the invention and a pharmaceutically acceptable vehicle.

By "therapeutically effective amount" we mean any amount of an agent or composition which, when administered to a subject suffering from a disease against which the agent is effective, causes reduction, remission, or regression of the malignant disease or HPV infection or prevents the development of malignant disease or HPV infection.

A "subject" is a vertebrate, mammal, domestic animal or preferably a human being.

The "pharmaceutically acceptable vehicle" may be any physiological vehicle known to those of ordinary skill in the art useful in formulating pharmaceutical compositions.

Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators.

Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g.
fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellent.

The invention will be further described, by way of Example and with reference to the following figures:-Figure 1(A) illustrates a Western Blot, referred to in Example 1, of proteins isolated from indinavir treated vector control transfected C33A-V and HPV16 E6 transfected C33A-E6 Cells which were immuniprobed with anti-p53 antibody and for which the lane order was as follows:
1- 5= C33AE6 plus 1mM, 0.5mM, 0.1mM, 0.05mM and 0 Indinavir 6 - 9= Wild type C33A plus 0.05mM, 0.1mM, 0.5mM and 1mM Indinavir;
Figure 1(B) and (C) illustrate the effects of different concentrations of Indinavir, as used in Fig 1(A), on the growth of C33A-E6 and C33A-V cells;

Figure 2 illustrate the effects of different concentrations of the HIV
protease inhibitor ritonaviron the growth of C33A parental, C33A-E6 and C33A-V cells;
and Figure 3 illustrates a Western Blot, referred to in Example 2, of proteins isolated from C33A-E6 Cells treated with (A) increasing amounts of ritonavir and (B) 30 M ritonavir wherein both blots were immunoprobed with an anti p53 antibody.

Experiments were conducted to demonstrate that Indinavir, an inhibitor according to the invention, was useful for preventing or treating malignant conditions caused by HPV infections.

C33A-E6 (wildtype C33A cells transfected with E6) and C33A-V (wildtype C33A cells transfected with vehicle for E6 only) cervical carcinoma cells were inoculated at a density of 4.0 x 105 cells per well into six well multiwell dishes, cultured in 5% CO2 at 37 C and incubated with various concentrations of Indinavir for 48 hours (0 - 1mM). Cells were counted and lysed in 2x Laemli buffer and the equivalent of 106 cells per track applied to 12 % PAGE followed by electroblotting onto Hybond C Extra. The blot was blocked with skimmed milk and immunoprobed with an anti p53 antibody followed by a horse radish peroxidase conjugated secondary. The signals were visualised by the use of an ECL detection system and exposure to Hyperfilm for various time intervals up to a maximum of 3 minutes (see Figure 1A). Cell counts obtained were plotted and are shown in Figures 1B and 1C.

The data illustrated in Figure 1 show that the expression of the HPV 16 E6 protein in C33A cervical carcinoma cells activates the 26S proteasome to induce the degradation of the tumour suppressor p53 protein. Lanes 6-9 are loaded with proteins from C33A-V control cells and have a much stronger signal than lanes 1-5.
However, treatment of E6 expressing cells with increasing amounts of Indinavir inhibits the E6 mediated proteasomal degradation of p53 and at 1mM the p53 protein is seen to accumulate in the E6 expressing C33A cells. C33A parent cells were not originally dependent on continued expression of E6 although the inventors observed selective toxicity with indinavir against E6 expressing cells when compare to C33A-V
control cells (see Figures 1B and 1C). These data indicate that expression of HPV16 E6 in C33A cells produced the well known effect of oncogene addiction. Thus the ability of indinavir to block the function of E6 in these cells inhibits both cell growth and the chymotryptic activity of the 26S proteasome, which produces an increase in the levels of the p53 protein.

These results indicate that Indinavir and Indinavir like compounds will be useful as a topically applied treatment for genital and non-genital HPV
infections. It is particularly important to note that indinavir is protective for non-transformed cells, This surprisingly indicates that agents according to the invention are useful as a prophylactic and can prevent HPV infection. It will be appreciated that the prevention of such infections will be protective against the development of cancers.

Taking all these observations together with our observation on E6 interaction with AACT, this implies that Indinavir may be useful for the treatment of HPV
related disease. Indeed there is evidence to suggest that high risk HPVs remain associated with and contribute to the growth of HPV related tumours such as cervical carcinomas. Therefore Indinavir and related compounds are useful for the treatment of non-malignant HPV infection and HPV related malignancies.

Experiments were conducted to compare the activity of an inhibitor according to the invention (i.e indinavir) with the activity of an HIV protease inhibitor (ritonavir) which falls outside the present invention.

Growth curves were conducted on parental HPV -ve cervical C33A cells, HPV16 E6 expressing C33A-E6 cells and C33A-V cells (see previous Example 1) in the presence of increasing concentrations of ritonavir. Cells were seeded into a 96 well plate at a density of 2.0 x 103 cells per well and left to attach overnight in 5% CO2 at 37 C prior to adding ritonavir. Viable cell numbers were determined at each time point by addition of Cell AQ 96 reagent (Promega), incubated for 4 hours in 5%

at 37 C, after which the optical density was measured at 490nm wavelength.

Figure 2 shows that ritonavir is highly toxic to all the cell types tested with minimal selectivity for the presence or absence of the HPV16 E6 protein. Total cell protein was harvested from C33A-E6 cells treated with increasing amounts of ritonavir upto 15 M, western blotted as described previously (Example 1) and the results shown in Figure 3A. Despite the toxicity observed at 15 M and higher concentrations, unlike indinavir treated C33A-E6 cells, immunoprobing this blot showed no increase in levels of the p53 indicating that the toxicity observed with ritonavir was non-specific and not related to blocking the function of the E6 protein.

C33A E6 cells were also treated with 30 gM of ritonavir for a time course of 0-8 hours, protein harvested and western blotted with exposure times of greater than 30 minutes. Again in contrast to the results obtained with indinavir (Example 1) the p53 signal did not show any increase in activity (Figure 3B) indicating that ritonavir exhibits non-specific toxicity and is less likely to be useful as a specific antiviral agent for the topical treatment of HPV infections. Accordingly it is preferred that ritonavir is not included as an inhibitor for use according to the present invention.
Indeed ritonavir treatment actually reduces the levels of p53 found in C33A-E6 cells.

These data clearly show that not all HIV protease inhibitors are effective for the treatment, or prevention of HPV infections and cancer. This illustrates the selective, and surprising, anti HPV activity of indinavir and other agents that inhibit the chymotryptic activity of the 26s unit of the proteosome compared to more general HIV protease inhibitors.

Claims (14)

1. The use of an agent that inhibits the chymotryptic activity of the 26s unit of the proteosome in the manufacture of a topical medicament to prevent infection, or to effect elimination, of human papilloma virus (HPV) from tissues of subjects infected with, or susceptible to, such viruses.

2. The use according to claim 1 wherein the agent is selected from amprenavir, abacavir, CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir, tipranavir, saquinavir, ABT-378, AG 1776, BMS-232,632 and velcade.

3. The use according to claim 1 wherein the agent is selected from those listed in Table 1.

4. The use according to claim 1 wherein the agent is indinavir or a functional or structural derivative thereof.

5. The use according to any preceding claim wherein the medicament is for treating, or preventing the development of, cancer associated with human papilloma virus (HPV).

6. The use according to claim 5 wherein the cancer is cervical, vulval, anal or penile carcinoma.

7. The use according to claim 5 or 6 wherein the cancer is associated with a high risk HPV virus

8. The use according to claim 7 wherein the high risk HPV virus is HPV16 or HPV18.

9. The use according to any one of claims 1- 4 for the treatment or prevention of non-malignant ano-genital warts caused by low risk HPV.

10. The use according to claim 9 wherein the low risk HPV is HPV6 or HPV11.

11. The use according to any one of claims 1- 4 for the treatment or prevention of cutaneous warts caused by low risk HPV.

12. The use according to claim 11 wherein the low risk HPV is a type 2 virus.

13. The use according to any one of claims 1- 4 for the treatment or prevention of HPV related oral conditions.

14. The use according to claim 13 wherein the condition is recurrent respiratory papillomatosis.