WO2004062659A1

WO2004062659A1 - Methods and compositions for preventing/reducing the severity of side effects of chemotherapy and/or radiotherapy

Info

Publication number: WO2004062659A1
Application number: PCT/AU2004/000041
Authority: WO
Inventors: Kerrie Setiawan; Ian Keith Smith
Original assignee: Mayne Pharma Pty Ltd
Current assignee: Mayne Pharma Pty Ltd
Priority date: 2003-01-13
Filing date: 2004-01-13
Publication date: 2004-07-29
Anticipated expiration: 2005-07-13
Also published as: WO2004062659A8

Abstract

A method of inhibiting cell cycle progression by administering to a subject an effective amount of a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor is disclosed. More particularly, the specification discloses the use of a HMG-CoA reductase inhibitor to prevent or reduce the severity of epithelial cytotoxicity side-effects, such as alopecia, mucositis and/or plantar-palmar syndrome in a subject receiving chemotherapy and/or radiotherapy. The HMG-CoA reductase inhibitor is preferably a statin or a derivative, analogue or prodrug thereof.

Description

METHODS AND COMPOSITIONS FOR PREVENTING/REDUCING THE SEVERITY OF SIDE EFFECTS OF CHEMOTHERAPY AND/OR RADIOTHERAPY

FIELD OF THE INVENTION

This invention relates to a method of inhibiting cell cycle progression by administering to a subject an effective amount of a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor such as a statin or a derivative, analogue or prodrug thereof. More particularly, the invention relates to the use of a HMG-CoA reductase inhibitor to prevent or reduce the severity of epithelial cytotoxicity side-effects, such as alopecia, mucositis and/ or plantar-palmar syndrome in a subject receiving chemotherapy and/ or radiotherapy.

BACKGROUND TO THE INVENTION

Chemotherapeutic techniques and radiotherapy techniques are well-established in the treatment of neoplastic conditions of various types. As concomitant side-effects to the administration of chemotherapy and /or radiotherapy, patients commonly experience severe host epithelial cell toxicity. The consequences of damage to the proliferating epithelium induced by chemotherapy frequently include hair loss (alopecia), mucositis and plantar-palmar syndrome; such side-effects, especially mucositis, are also known to occur as a result of radiotherapy. These side-effects may be of varying severity, depending on the type, dosages and dosing schedule of the respective chemotherapy and/ or radiotherapy involved. However, mucositis is often a dose-limiting side-effect of antineoplastic therapy and has the potential to compromise the clinical outcome. While not life-threatening, alopecia is a distressing side-effect of antineoplastic therapy and it is a side-effect that causes a high level of stress to cancer patients.

Several preventative non-drug methods have been proposed to prevent or reduce the severity of alopecia caused by epithelial cell toxicity arising from chemotherapy and/ or radiotherapy. These methods include scalp tourniquet, scalp hypothermia, or a combination of both, the rationale of which is to reduce the blood circulation during chemotherapy or radiotherapy. However, none of these methods has been shown to have a definite protective effect, although undesirable effects, such as headaches, may arise.

Many chemotherapeutic agents cause hair loss because they are aimed at rapidly dividing cells, which is one of the defining characteristics of cancer cells. Alopecia occurs when these chemotherapeutic drugs (eg cyclophosphamide) kill, harm or inhibit the growth of cells of the hair follicle which are dividing more rapidly than the majority of normal cells in the body. Such damage to the hair follicle can lead to abnormally slow growth of the hair or may lead to frank loss. While various attempts have been made to protect against alopecia and /or abnormally slow rates of hair growth during such treatments, there remains a significant need for an agent that prevents damage to hair follicles in a safe and effective manner.

A number of patent specifications have been published disclosing methods utilising a variety of pharmacological agents for preventing and treating chemotherapy and radiotherapy-induced alopecia. These include Bol etal (WO 01/80813), Fahl etal(VIO 01 / 85142), Jimenez et al (US 6,291,443), Atwal (US 2001 / 0020038), Kim et al (US

6,214,852), Davis etal (WO 00/78229), Moran etal (US 5,962,523), Holick (US 5,958,384), Wong etal(XJS 5,843,422), and Giguere etal (WO 97/05866). However, the active agents disclosed in these patent specifications are structurally distinct from the compounds utilised in the present invention.

The use of minoxidil in cancer patients to decrease the duration of baldness caused by chemotherapy is disclosed by Duvic, M. etal, "A Randomised Trial of Minoxidil in Chemotherapy-Induced Alopecia", fAm Acad Dermatol 35:74-78, 1996. Duvic etal disclose that in patients treated with fluorouracil, doxorubicin and cyclophosphamide, a 2% topical solution of minoxdil administered during chemotherapy and 4 weeks thereafter, did not prevent alopecia but did decrease the period of baldness. On the other hand, Rodriguez, R. etal, "Minoxidil as a Prophylaxis of Doxorubicin-Induced Alopecia", Annals of Oncology 5:769-770, 1994 disclose that a 2% topical solution of minoxidil was not effective in preventing doxorubicin-induced alopecia. In another report, Hussein, A. M., "Protection Against Cytosine Arabinoside-Induced Alopecia By Minoxidil In A Rat Animal Model", Intf Dermatol 34(7);470-473, 1995 discloses that minoxidil, when injected locally, offered good local prevention against 1-B-D-arabinofurano-sylcytosine but not cyclophosphamide-induced alopecia. Also, a compound designated GW8510, when administered topically, has been reported to prevent alopecia by arresting the cell cycle and reducing the sensitivity of the epithelium to many cell cycle-active anti-tumour agents; Davis, S. T. etal, "Prevention of Chemotherapy-Induced Alopecia in Rats by CDK- Inhibitors", Science 291:134-137, 2001. GW8510 arrests the cell cycle by inhibiting cyclin- dependent kinase II. While some of the methods mentioned in the preceding two paragraphs have achieved limited success, there is an ongoing need to identify and develop new and /or improved methods for chemotherapy and /or radiotherapy-induced alopecia treatments.

SUMMARY OF THE INVENTION

Thus, in a first aspect, the present invention provides a method of preventing or reducing the severity of epithelial cell toxicity side-effects of chemotherapy and /or radiotherapy treatment(s) of a subject, said method comprising administering to said subject an effective amount of a HMG-CoA reductase inhibitor optionally in combination with a pharmaceutically acceptable carrier, excipient and /or diluent.

In a second aspect, the present invention provides the use of a HMG-CoA reductase inhibitor in the preparation of a medicament for preventing or reducing the severity of epithelial cell toxicity side-effects of chemotherapy and /or radiotherapy treatment(s).

In a third aspect, the present invention provides a medicament for preventing or reducing the severity of epithelial cell toxicity side-effects of chemotherapy and /or radiotherapy treatment(s), said medicament comprising a HMG-CoA reductase inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 provides a schematic diagram of a microtitre plate layout used for conducting a chemoprotection study for lovastatin in accordance with Study (B) of Example 1.

Figure 2 provides graphical results for lovastatin (5 μM) protection of CCL64 cells from paclitaxel mediated growth inhibition.

Figure 3 provides graphical results for lovastatin (5 μM) protection of CCL64 cells from cytarabine mediated growth inhibition.

Figure 4 provides graphical results for lovastatin (5 μM) protection of CCL64 cells from cisplatin mediated growth inhibition.

Figure 5 provides a schematic diagram of a microtitre plate layout used for conducting a chemoprotection study for atorvastatin in accordance with Study (A) of Example 3.

Figure 6 provides graphical results for atorvastatin (5 μM) protection of CCL64 cells from paclitaxel mediated growth inhibition. Figure 7 provides graphical results for atorvastatin (5 μM) protection of CCL64 cells from cytarabine mediated growth inhibition.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of preventing or reducing the severity of epithelial cell toxicity side-effects of chemotherapy and/ or radiotherapy treatment(s) of a subject, said method comprising administering to said subject an effective amount of a HMG-CoA reductase inhibitor optionally in combination with a pharmaceutically acceptable carrier, excipient and/ or diluent.

The method may prevent or reduce the severity of alopecia, mucositis and/ or plantar- palmar syndrome induced by chemotherapy and/ or radiotherapy.

The HMG-CoA reductase inhibitor is any agent capable of inhibiting the activity of this enzyme, namely inhibiting the conversion of HMG-CoA to mevalonic acid. Preferably, the HMG-CoA reductase inhibitor is a statin, or derivative, analogue or prodrug thereof. A statin is a drug that blocks cholesterol production in the body by inhibiting the enzyme HMG-CoA reductase in the early steps of its synthesis in the mevalonate pathway.

Preferred statins for use in the present invention are atorvastatin, cerivastatin, dalvastatin, fluvastatin, lovastatin, mevastatin, pravastatin, simvastatin, pitavastatin and rosuvastatin and mixtures thereof. Most preferred are lovastatin, atorvastatin and pravastatin.

Generally, these HMG Co-A reductase inhibitors are known to lower serum cholesterol in humans. However, the present invention is not so limited. That is, an inhibitor of HMG Co-A reductase or one of its analogues or prodrugs may work in the method of the present invention without necessarily lowering serum cholesterol. The present invention focuses not on the compound's ability to lower cholesterol, but rather on the compound's ability to prevent/ reduce the severity of side-effects of chemotherapy and/ or radiotherapy.

As used herein, a "derivative" of a statin refers to any compound derived from a statin by chemical or enzymatic modification, which retains the ability to inhibit the activity of HMG-CoA reductase in converting HMG-CoA to mevalonic acid. In contrast, an "analogue" of a statin refers to any compound that differs from a statin in chemical structure but retains similarity to a statin in one or more features that provide the activity characteristic of a statin (ie the ability to inhibit the activity of HMG-CoA reductase in converting HMG-CoA to mevalonic acid). An analogue may therefore have substantial overall structural similarity with a statin or only structural similarity with one or more regions of a statin for the provision of the activity characteristic of a statin.

The term "prodrug", as used herein, refers to a compound which represents an inactive form of a statin, which upon administration to a subject is hydrolysed or otherwise metabolised by the body to an active statin compound (ie a statin with the ability to inhibit the activity of HMG-CoA reductase in converting HMG-CoA to mevalonic acid).

The HMG-CoA reductase inhibitor may be administered before, concurrently and /or after the chemotherapy and /or radiotherapy treatment(s) of the subject. Preferably, administration of the HMG-CoA reductase inhibitor is prior to, or commences prior to, the chemotherapy and / or radiotherapy treatment(s) of the subject.

The HMG-CoA reductase inhibitor may also be administered in combination (ie in co- administration or alternating administration) with other known agents for treatment of alopecia, mucositis or plantar-palmar syndrome, for more effective treatment of the subject undergoing chemotherapy and/ or radiotherapy.

For the prevention or reduction of severity of alopecia induced by chemotherapy and /or radiotherapy, the HMG-CoA reductase inhibitor is preferably administered topically to a corporal locus susceptible to alopecia. For example, an effective amount of the HMG-CoA reductase inhibitor may be formulated into a medicament adapted for topical administration, and the medicament administered to the subject's scalp and /or facial areas (eg eyebrow, beard and /or moustache regions). More preferably, the medicament is periodically administered (eg every 1 to 6 hours) to the subject's scalp and/ or facial areas commencing approximately 24 hours prior to chemotherapy or radiotherapy and finishing approximately 1 to 6 hours after the chemotherapy or radiotherapy. Most preferably, for the prevention or reduction of severity of alopecia induced by chemotherapy and/ or radiotherapy, the medicament is a topical solution comprising about 5 to 25 mg/ml of a statin and is administered to the subject's scalp and/or facial areas every 6 hours, four time prior to chemotherapy or radiotherapy and once after chemotherapy (ie a total of five applications).

For the prevention or reduction of severity of mucositis induced by chemotherapy and /or radiotherapy, the HMG-CoA reductase inhibitor is preferably administered locally to the mucosa of the mouth, throat and /or gastrointestinal (GI) tract. For the prevention or reduction of the severity of plantar-palmar syndrome induced by chemotherapy and /or radiotherapy, the HMG-CoA reductase inhibitor is preferably administered topically to the plantar and/ or palmar regions susceptible to lesioning.

It is well within the skill of persons skilled in the art to determine an effective amount of a HMG-CoA reductase inhibitor in accordance with the present invention. In this regard, it is to be understood that the effective amount will vary depending upon known factors, such as: the nature of the antineoplastic or radiotherapy intervention; the pharmacodynamic characteristics of the particular HMG-CoA reductase inhibitor being administered and its route of administration; the age, health, height and weight of the recipient; the total surface area to be treated; the nature and extent of the symptoms; the frequency of administration of the HMG-CoA reductase inhibitor; and the result desired.

In another aspect of the present invention, there is provided the use of a HMG-CoA reductase inhibitor in the preparation of a medicament for preventing or reducing the severity of epithelial cell toxicity side-effects of chemotherapy and /or radiotherapy treatment(s).

Preferably, the HMG-CoA reductase inhibitor is administered directly to the epithielial cells to be protected or treated, and as a result the medicament may be formulated in any suitable dosage form.

Where the medicament is intended for the prevention or reduction of severity of alopecia induced by chemotherapy and /or radiotherapy, the medicament is preferably adapted for topical administration. The medicament may therefore be in the form of a solution, suspension, lotion, gel, cream, shampoo, ointment, mousse, paste, wax, powder, liniment, tincture, aerosol, patch or other transdermal drug delivery system. Such a medicament will preferably comprise a pharmaceutically acceptable carrier, excipient and /or diluent and, optionally, an agent which enhances the percutaneous absorption of the HMG-CoA reductase inhibitor. Such agents include dimethylsulfoxide, dimethylacetamide, dimethylformamide, surfactants, azone, alcohol, acetone, propylene glycol, polyethylene glycol and mixtures thereof. Physical methods such as iontophoresis or sonophoresis may also be used to enhance the percutaneous absorption of the HMG-CoA reductase inhibitor.

A medicament intended for the prevention or reduction of severity of alopecia induced by chemotherapy and /or radiotherapy, may further comprise an agent which promotes hair growth such as potassium channel openers, minoxidil (Upjohn) and /or diazoxide (Shiseido and Schering-Plough), as well as cromakalim and pinacidil; a 5-alpha-reductase inhibitor such as finasteride (Merck's Proscar.RTM.), terazosin HC1 (Abbott's Hytrin.RTM.), or doxazosin mesylate (Pfizer's Cardura.RTM.); and/ or an androgen blocker such as 4-(5-methoxyheptyl)-hex-ahydro-2(lH)-pentalenone as disclosed in WO 92/09259 or cyproterone acetate, corticosteroids such as hydrocortisone and betamethasone dipropionate, and an antimuscarinic agent such as scopolamine.

Where the medicament is intended for the prevention or reduction of severity of mucositis induced by chemotherapy and /or radiotherapy, the medicament is preferably adapted for local administration to the mucosa of the mouth, throat and /or GI tract via a mouthwash, lozenge, tablet (including buccal and sublingual), capsule (including timed release and sustained release formulations), troche, pastille, pill, lollipop, chewing gum, powder, granule, solution, elixir, tincture, suspension, syrup, emulsion, or spray. Such a medicament will preferably comprise a pharmaceutically acceptable carrier, excipient and /or diluent and, optionally, other agents which may, for example, enhance retention of the HMG-CoA reductase inhibitor on the mucosa. Such agents may include bioadhesive agents, viscosity-enhancing agents (eg polymers), complexation agents (eg cyclodextrins) and mixtures thereof.

In a further aspect, the present invention provides a medicament for preventing or reducing the severity of epithelial cell toxicity side-effects of chemotherapy and /or radiotherapy treatment(s), said medicament comprising a HMG-CoA reductase inhibitor, and wherein said medicament is adapted for topical administration. The present invention may be used for preventing or reducing the severity of epithelial cell toxicity side-effects arising from any chemotherapy and/ or radiotherapy treatments.

Thus, the present invention may be used with chemotherapy treatments utilising any of the antineoplastic agents listed in Martindale The Extra Pharmacopoeia or the Physician's Desk Reference or with antineoplastic agents in development. Such antineoplastic agents prevent cancer cells from multiplying, generally: by interfering with the cell's ability to replicate DNA; or inducing apoptosis in the cancerous cells. Examples of suitable antineoplastic agents include microtuble-stabilising agents such as the taxanes, for example, paclitaxel (also known as Taxol.RTM.), docetaxel (also known as

Taxotere.RTM.), 7-O-methylthio-methylpaclitaxel (disclosed in US Patent No.5,646,176), 3'-tert-butyl-3'-N-tert-butyloxycarbonyl-4-deacetyl-3'-de- phenyl-3'-N-debenzoyl-4-O- methoxycarbonyl-paclitaxel (disclosed in US Serial No. 60/179,965 filed on Feb.3, 2000), C-4 methyl carbonate paclitaxel (disclosed in WO 94/14787), the epothilones, such as epothilone A, epothilone B, epothilone C, epothilone D, desoxyepothilone A, desoxyepothilone B, [lS-[ 1R*,3R*(E),7R*,10S*,11R*,12- R*,16S*]]-7,ll-dihydroxy- 8,8,10,12,16-pentamethyl-3-[l-methyl-2-(2-methyl~ 4-thiazolyl)ethenyl]-4-aza-17- oxabicyclo[14.1.0]hepta-decane-5,9-dione (disclosed in WO 99/02514), [lS-[ lR*,3R*(E),7R*,10S*,llR*,12R^*,16S*]]-3~ [2-[2-(aminomethyl)-4-thiazolyl]-l- methylethenyl]-7,ll-di-hydroxy-8,8,10,l- 2,16-pentamethyl-4,17-dioxabi-cyclo[14.1.0]- heptadecane-5,9-dione (disclosed in US Serial No. 09/506,481 filed on Feb. 17, 2000), and derivatives thereof; microtuble-disruptor agents; alkylating agents; anti-metabolites; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; and platinum coordination complexes. Other classes of suitable antineoplastic agents include the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, discodermolide, the pteridine family of drugs, diynenes, aromatase inhibitors, and the podophyllotoxins. Particularly suitable members of those classes not previously mentioned include, for example, epirubicin, doxorubicin, idarubicin, dactinomycin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloro-methotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytarabine, fludarabine, fludarabine phosphate, etoposide, etoposide phosphate, teniposide, melphalan, vinorelbine, vinblastine, vincristine, leurosidine, vindesine, leurosine, and the like. Other suitable antineoplastic agents include estramustine, cisplatin, carboplatin, oxaliplatin, cyclophosphamide, bleomycin, ifosfamide, melphalan, hexamethyl melamine, thiotepa, idatrexate, trimetrexate, dacarbazine, L-asparaginase, camptothecin, irinotecan and topotecan.

The present invention may also be used with radiotherapy treatments such as, but not limited to, x-rays or gamma rays which are delivered from either an externally applied source such as a beam or by implantation of small radioactive sources.

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non- limiting examples.

EXAMPLE 1

The purpose of this in vitro study was to determine:

(i) the effect of lovastatin dose and incubation time on epithelial cell proliferation, and (ii) the ability of lovastatin to afford chemoprotection to epithelial cells exposed to antineoplastic drugs. Definitions LOV - Lovastatin

CCM - Cell culture medium (DMEM medium containing 10 % foetal calf serum) CCL64 - Mink lung epithelial cell line DMEM - Dulbecco's modified Eagle medium MeCN - Methyl cyanide (acetonitrile) PBS - Phosphate buffered saline

Methods

STUDY (A) EFFECT OF LOVASTATIN DOSE AND INCUBATION TIME ON EPITHELIAL CELL PROLIFERATION

This study was conducted to examine lovastatin dose and incubation time on the inhibition of CCL64 cell growth.

The study was carried out by seeding CCL64 cells into the wells of four 12-well plates in a volume of 1.5 mL CCM, followed by incubation of the plate overnight at 37°C in a humidified atmosphere of 5 % CO₂ and 95 % air. 1.5 mL medium containing 0, 5, 10, 20 μM lovastatin was then added to wells in triplicate (final concentration lovastatin = 0, 2.5, 5, 10 μM respectively), and the plates again incubated as described above for 0, 24, 48, 72 h. At each time point, one plate was removed, the wells washed with PBS, the cells lifted using Pronase and counted in the presence of Trypan blue. The proliferation index was expressed as the multiplicity of the cell number after 24, 48 and 72 h, in relation to the cell number in the cultures at 0 time. The viability index represents the fraction of cells excluding Trypan blue at the time point analysed (Jacobisiak et al.,1991 PNAS 883628).

A cell cycle analysis was performed for cells treated with either 5 or 10 μM lovastatin. For each concentration of lovastatin, this was done by seeding cells in CCM into eight 10 cm culture dishes and incubating overnight as described above. The medium was removed from each of the plates, the cells rinsed with PBS and 10 mL CCM added to four plates (control) and 10 mL medium containing lovastatin added to the remaining four plates (lovastatin-treated). The dishes were then incubated for 0, 24, 48, 72 h. At each time point, the cells were harvested with Pronase as described above. The cells were then resuspended in 0.5 mL PBS and 5 mL ice cold 95 % ethanol slowly added whilst vortexing gently. Following incubation on ice for 15 minutes, the cells were spun down and washed in PBS. The cells were resuspended in 0.5 mL 40 mM sodium citrate pH 7.0 containing 10 μg/mL propidium iodide and 0.25 mg/mL RNase A, and incubated at 37°C in the dark for 30 minutes. Cell cycle parameters were analysed by flow cytometry (FACS Calibur, Becton Dickinson). The percentage of cells in each phase of the cell cycle was determined using Modfit LT software (Verity Software House, Topsham, ME) (Dimitroulakos et al. 2001, Clin. Cancer Res., 7 158).

STUDY (B) CHEMOPROTECTION STUDY

This study was conducted by seeding cells into three 96-well microtitre plates. The plates were incubated overnight and CCM containing lovastatin was then added to each of 30 wells in one half of each plate (final concentration = 2.5, 5 or 10 μM) and CCM only to 30 wells in the other half. The plate was then incubated for a further 24 h and the medium then removed from all the wells and rinsed once with CCM.

Medium containing one antineoplastic drug (either cytarabine, paclitaxel or cisplatin) was then added at five concentrations to each of the CCM wells in quintuplicate (see Figure 1). Also, medium containing lovastatin and one antineoplastic drug (either cytarabine, paclitaxel or cisplatin) was added at five concentrations to each of the lovastatin wells in quintuplicate. The remaining five lovastatin wells were incubated in CCM containing lovastatin (lovastatin control) and the remaining five CCM wells were incubated in CCM only (CCM control). The plates were incubated for a further 48 h.

Cell growth inhibition was determined using SRB staining (Skehan etal., 1990, JNCI, 82, 1107-1112). The results were presented as percentage values relative to the CCM control cells that received neither lovastatin nor antineoplastic drug. This analysis demonstrates the inhibition of cell growth induced by lovastatin and how this growth arrest translates into reduced cell growth inhibition following exposure to antineoplastic drugs. In a second analysis, data from the lovastatin treated and untreated wells was analysed separately, such that IC50 values can be calculated for each antineoplastic drug studied (McCormack et al, 1997 Biochem. Pharmacol. 1149-

Results

STUDY (A) EFFECT OF LOVASTATIN DOSE AND INCUBATION TIME ON

EPITHELIAL CELL PROLIFERATION

The results of two experiments are summarised in the Tables 1 and 2 below.

P = proliferation index = multiplicity of cells at different time points in relation to the cell number at 0 time V= viability index = fraction of cells excluding Trypan Blue at each time point

Table 1: Effect of lovastatin on cell proliferation and viability (Experiment 1)

Table 2: Effect of lovastatin on cell proliferation and viability (Experiment 2)

Proliferation of CCL64 cells was inhibited as a function of lovastatin dose and incubation time. Inhibition of cell proliferation was evident at 24 h following treatment with 5 μM or 10 μM lovastatin. At 72 h, cell proliferation was inhibited 4- and 8-fold by 5 μM and 10 μM lovastatin, respectively. While minimal cell death was observed in the 5 μM lovastatin samples after 72 h, a reduction in cell viability was observed at 10 μM lovastatin.

The percentage of cells in the different phases of the cell cycle after incubation with either 5 or 10 μM lovastatin are summarised in the Tables 3 and 4 below. Table 3: Percentage of cells in Gl, S and G2/M phase after treatment with 5 μM lovastatin

Table 4: Percentage of cells in Gl, S and G2/M phase after treatment with 10 μM lovastatin

Incubation of CCL64 cells with either 5 μM or 10 μM lovastatin reduced the percentage of cells in S-phase of the cell cycle at 24 h by 3- and 7- fold, respectively. This inhibition was associated with a corresponding increase in the percentage of cells in G0/G1 phase of the cell cycle. This S-phase inhibitory effect was maintained over the 72 h course of the experiment.

Incubation of cells with both 5 μM andlO μM lovastatin blocked cell cycle progression and inhibited cell proliferation with these effects more apparent at 10 μM lovastatin. A decrease in cell viability following incubation of cells at 10 μM lovastatin was not observed at 5 μM lovastatin.

STUDY (B) CHEMOPROTECTION STUDY

This study was performed at 2.5, 5 and 10 μM lovastatin. The results at 5 μM lovastatin are summarised in Figures 2, 3 and 4.

Cells in microtitre plates were incubated with 2.5, 5 and 10 μM lovastatin for 24 h followed by exposure to the antineoplastic drug for 48 h in medium containing lovastatin. Each point represents the mean percentage cell growth of five replicates from one experiment + standard error of the mean. Graph (A): All values are shown as a percentage of the cell growth in the CCM only control wells (ie. no lovastatin, no antineoplastic drug). Graph (B): The same percentage growth values from Graph (A) were replotted with the CCM and lovastatin-treated samples analysed separately and expressed as a percentage of their respective no-antineoplastic drug controls (McCormack etal., 1997 Biochem. Pharmacol. 1149-1159).

The results of the study demonstrate that preincubation of cells with 5 μM lovastatin for 24 h reduced the growth inhibitory activity of two anticancer drugs representing cell cycle dependent (paclitaxel and cytarabine) modes of action. Lovastatin protected cells from the growth inhibitory effects of each of these two antineoplastic drugs at each of the concentrations tested. The results observed with cisplatin (representing a cell cycle independent antineoplastic drug) revealed a protective effect at high cisplatin concentrations.

Preincubation of CCL64 cells with 5 μM LOV for 24 h effectively protects CCL64 cells from the growth inhibitory effects of each of the antineoplastic drugs. This chemoprotective effect was greater at 5 μM lovastatin than 2.5 μM lovastatin. The experiment was repeated at 5 μM lovastatin and the results confirmed the above findings.

Discussion

The results show that lovastatin provides significant chemoprotection to epithelial cells exposed to antineoplastic drugs indicating that HMG-CoA reductase inhibitors such as statins could be used as the basis of methods for preventing or reducing the severity of epithelial cell toxicity side-effects such as alopecia. Since it has been reported (Jakobisiak, M. etal, "Cell cycle-specific effects of lovastatin", Proc Natl Acad Sci USA 88:3628-3632, 1991) that the inhibition of epithelial cell proliferation by lovastatin is reversible, with the cell cycle resuming after a lag of about 6 hours following removal of the drug, it is also anticipated that such methods would permit the return of the epithelial cells to normal growth and function upon completion of the treatment (eg hair follicle cells ought to resume normal growth and function after treatment).

EXAMPLE 2

A limited clinical trial was conducted to assess the safety of lovastatin for use in the prevention or reduction of alopecia caused by chemotherapy.

Methods

Three adult patients due to receive chemotherapy were administered with lovastatin formulated as a topical solution containing 0.2 mg/ml lovastatin dissolved in ethanol 96% (80%w/ w) and caprylic/capric triglyceride (20%w/ w). 5mL of the formulation was dispensed into 15mL plastic dropper bottles for administration by topical application to the scalp of the patients. The dosing schedule for the three patients was as follows:

The patients applied the solution to the entire scalp every 6 hours, four times prior to chemotherapy and once after chemotherapy (total of five applications per cycle).

Table 5: Dosing schedule

0 hours = chemotherapy commences

The patients applied the solution directly to their hair (dry) and scalp using the dropper bottles. This was done by placing the tip of a bottle in light contact with the scalp and thereafter gently squeezing the bottle to allow a small amount of the solution to flow onto the scalp. The tip of the bottle was then moved and the process repeated until the entire contents was applied evenly to the entire scalp. Results

The patients were observed over three weeks after chemotherapy. The lovastatin solution was well tolerated by the patients with no observed side-effects.

EXAMPLE 3

The purpose of this in vitro study was to determine the ability of atorvastatin to afford chemoprotection to the growth inhibitory effects of epithelial cells exposed to antineoplastic drugs. Definitions ATOR- Atorvastatin CCM - Cell culture medium (DMEM medium containing 10 % foetal calf serum) CCL64 - Mink lung epithelial cell line LOV - Lovastatin

Methods

STUDY (A) CHEMOPROTECTION STUDY

This study was conducted by seeding cells into three 96-well microtitre plates. The plates were incubated overnight and CCM containing ATOR was then added to each of 15 wells in one half of each plate (final concentration = 5 μM) and CCM only to 15 wells in the other half. The plate was then incubated for a further 24 h and the medium then removed from all the wells and rinsed once with CCM.

Medium containing one antineoplastic drug (either cytarabine or paclitaxel; one drug per plate) was then added at two concentrations to each of the CCM wells in quintuplicate (see Figure 5). Also, medium containing ATOR and one antineoplastic drug (either cytarabine or paclitaxel) was added at two concentrations to each of the ATOR wells in quintuplicate. The remaining five ATOR wells were incubated in CCM containing ATOR and the remaining five CCM wells were incubated in CCM only. The plates were then incubated for a further 48 h.

Cell growth inhibition was determined using SRB staining (Skehan etal., 1990, supra). Briefly, the cells were fixed with 10% cold trichloroacetic acid for 1 h at 4°C and the plates then rinsed with distilled water, left to air dry and then stained with 0.4 % SRB (Aldrich) in 1% acetic acid (v/ v) for 30 min (binds to basic amino acids of cellular macromolecules). Unbound dye was removed by washing twice with distilled water and finally with 1% acetic acid. Protein-bound dye was then solubilised in 10 mM unbuffered Tris base and the absorbance was read at 550 nm using an automatic plate reader. The mean absorbance for each drug dose was expressed as a percentage of the relevant control, untreated well absorbance.

Results

The results of the study are summarised in Figures 6 and 7. In the figures, each point represents the mean percentage cell growth of five replicates from one experiment ± standard error of the mean. For figures 6(A) and 7(A), all values are shown as a percentage of the cell growth in the medium-only control wells (ie no ATOR, no antineoplastic drug). For figures 6(B) and 7(B), the same percentage growth values from the respective (A) figures were retabulated with the control and ATOR treated samples analysed separately and expressed as a percentage of the respective no-antineoplastic drug control (McCormack etal, "Transforming growth factor-beta3 protection of epithelial cells from cycle-selective chemotherapy in vitro", Biochem Pharmacol 53(8):1149-1159, 1997).

Discussion

Due to a study error, the dose of atorvastatin (5 μM) used was less than that which was preferred (ie 10 μM). Despite this, a trend towards a chemoprotective effect of atorvastatin was observed at relatively high doses of the antineoplastic drugs.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application. It will be appreciated by persons skilled in the art that numerous variations and /or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

1. A method of preventing or reducing the severity of epithelial cell toxicity side- effects of chemotherapy and /or radiotherapy treatment(s) of a subject, said method comprising administering to said subject an effective amount of a HMG- CoA reductase inhibitor optionally in combination with a pharmaceutically- acceptable carrier, excipient and /or diluent.

2. The method of claim 1, wherein said method is used to prevent or reduce the severity of alopecia, mucositis and /or plantar-palmar syndrome.

3. The method of claim 1, wherein said method is used to prevent or reduce the severity of alopecia.

4. The method of any one of claims 1 to 3, wherein the HMG-CoA reductase inhibitor is a statin, or derivative, analogue or prodrug thereof.

5. The method of claim 4, wherein the HMG-CoA reductase inhibitor is a statin selected from the group consisting of atorvastatin, cerivastatin, dalvastatin, fluvastatin, lovastatin, mevastatin, pravastatin, simvastatin, pitavastatin and rosuvastatin and mixtures thereof.

6. The method of claim 4, wherein the HMG-CoA reductase inhibitor is a statin selected from lovastatin, atorvastatin and pravastatin.

7. The method of any one of claims 1 to 6, wherein the administration of the HMG- CoA reductase inhibitor to the subject is, at least, partially prior to the chemotherapy and /or radiotherapy treatment(s).

8. The use of a HMG-CoA reductase inhibitor in the preparation of a medicament for preventing or reducing the severity of epithelial cell toxicity side-effects of chemotherapy and /or radiotherapy treatment(s).

9. The use of claim 8, wherein said use is for the prevention or reduction of the severity of alopecia, mucositis and /or plantar-palmar syndrome.

10. The use of claim 8, wherein said use is for the prevention or reduction of the severity of alopecia.

11. The use of any one of claims 8 to 10, wherein the HMG-CoA reductase inhibitor is a statin, or derivative, analogue or prodrug thereof.

12. The use of claim 11, wherein the HMG-CoA reductase inhibitor is a statin selected from the group consisting of atorvastatin, cerivastatin, dalvastatin, fluvastatin, lovastatin, mevastatin, pravastatin, simvastatin, pitavastatin and rosuvastatin and mixtures thereof.

13. The use of claim 11, wherein the HMG-CoA reductase inhibitor is a statin selected from lovastatin, atorvastatin and pravastatin.

14. A medicament for preventing or reducing the severity of epithelial cell toxicity side-effects of chemotherapy and /or radiotherapy treatment(s), said medicament comprising a HMG-CoA reductase inhibitor.

15. The medicament of claim 14, wherein said medicament is for the prevention or reduction of the severity of alopecia, mucositis and/ or plantar-palmar syndrome.

16. The medicament of claim 14, wherein said medicament is for the prevention or reduction of the severity of alopecia.

17. The medicament of any one of claims 14 to 16, wherein the HMG-CoA reductase inhibitor is a statin, or derivative, analogue or prodrug thereof.

18. The medicament of claim 17, wherein the HMG-CoA reductase inhibitor is a statin selected from the group consisting of atorvastatin, cerivastatin, dalvastatin, fluvastatin, lovastatin, mevastatin, pravastatin, simvastatin, pitavastatin and rosuvastatin and mixtures thereof.

19. The medicament of claim 17, wherein the HMG-CoA reductase inhibitor is a statin selected from lovastatin, atorvastatin and pravastatin.