WO1998040102A1

WO1998040102A1 - Cytoprotective agents comprising monoamine oxidase inhibitors

Info

Publication number: WO1998040102A1
Application number: PCT/IE1998/000023
Authority: WO
Inventors: Keith Tipton; Carmel Mothersill; Robert Mooney
Original assignee: FORBAIRT TRADING AS BIORESEARCH IRELAND; College of the Holy and Undivided Trinity of Queen Elizabeth near Dublin
Current assignee: FORBAIRT TRADING AS BIORESEARCH IRELAND; College of the Holy and Undivided Trinity of Queen Elizabeth near Dublin
Priority date: 1997-03-13
Filing date: 1998-03-13
Publication date: 1998-09-17
Anticipated expiration: 1999-09-13
Also published as: EP0975366A1; AU6417198A; CA2284001A1

Abstract

The present invention concerns the use of monoamine oxidase (MAO) inhibitors in the protection or rescue of normal cells from the toxic side effects caused by irradiation or the administration of chemotherapeutic agents such as certain anti-cancer agents. Deprenyl is a particularly preferred MAO inhibitor.

Description

CYTOPROTECTIVE AGENTS COMPRISING MONOAMINE OXIDASE INHIBITORS

5 This invention relates to cytoprotective agents. In particular, it relates to the use of monoamine oxidase (MAO) inhibitors in the protection or rescue of normal cells from the toxic side effects caused by irradiation or the administration of chemotherapeutic 10 agents.

The use of MAO inhibitors in the treatment of certain neurodegenerative conditions is known. However, it has now surprisingly been discovered that MAO

15 inhibitors can selectively protect or rescue normal cells from the toxic side effects caused by irradiation or the administration of chemotherapeutic agents. Furthermore, the MAO inhibitors may be effective at dose levels much lower than those at

20 which they are effective in the treatment of neurodegenerative conditions, such as, for example, Parkinson's disease.

According to the present invention, there is provided 25 the use of a monoamine oxidase inhibitor in the preparation of a medicament for protecting and/or rescuing normal cells from toxicity resulting from irradiation and/or the administration of a chemotherapeutic agent, in particular a chemotherapeutic agent which can disrupt DNA or RNA replication.

The invention also provides a pharmaceutical product for protecting and/or rescuing normal cells from toxicity resulting from the administration of a chemotherapeutic agent, the product comprising: (a) a monoamine oxidase inhibitor; and

(b) a therapeutically effective amount of the chemotherapeutic agent,

as a combined preparation for simultaneous, separate or sequential administration.

Preferred MAO inhibitors for use in the present invention are deprenyl (N-1-phenylisopropyl-N-methyl- 2-propynylamine) and structural analogues and derivatives thereof. Preferred MAO inhibitors are selected from the following:

(a) (-) -N-l-phenylisopropyl-N-methyl-2-propynylamine and pharmaceutically acceptable salts thereof;

(b) (-) -N-2, 4-dichlorophenoxypropyl-N-methyl-2- propynylamine and pharmaceutically acceptable salts thereof ;

(c) compounds of the formula I: Z₁ -0- Z₂ -0-C_nH2n-N ( CH₃ ) - CH₂ -C≡CH

wherein Z_ and Z₂ are each independently optionally substituted phenyl; and n is an integer from 2 to 4; and pharmaceutically acceptable salts thereof;

(d) compounds of the formula II: Z₃-X

wherein Z3 is indolyl substituted at the 1- position by hydrogen, methyl or ethyl, and at the 5- position by hydrogen, hydroxy or methoxy; and X, which may be present at the 2- or 3- position of the indolyl ring, is C_nH2_n-N(R.3) -R4, wherein R3 is a substituted ethylenic or acetylenic group; R4 is hydrogen, methyl or ethyl and n is an integer from 1 to 4; and pharmaceutically acceptable salts thereof;

(e) compounds of the formula III:

R-N(CH₃) -CH₂-C≡CH

wherein R is linear or branched aliphatic hydrocarbon; and pharmaceutically acceptable salts thereof ;

(f) compounds of the formula IV: Y- [N (R) ] _p - C_nH_2n-N ( CH₃ ) - CH₂ - C≡CH

wherein Y is optionally substituted indanoyl or indolyl; R, if present, is hydrogen or methyl; p is 0 or 1 ; and n is an integer from 1 to 4 ; and pharmaceutically acceptable salts thereof; and

(g) (R) -N-2-propynyl-l-indanamine (Rasagiline) and pharmaceutically acceptable salts thereof.

Possible substituents on the phenyl, ethylenic or acetylenic, or indanoyl/indolyl group in compounds of formula I, II or IV above include, but are not limited to, one or more of the following:

^cl-4 alkyl, C_]__4 alkoxy, C_]__4 thioalkyl, C]__4 alkyl- sulphinyl, Cτ__4 alkylsulphonyl, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, tri- fluoromethylsulphinyl, trifluoromethylsulphonyl, halogen (e.g. fluorine, chlorine or bromine) , cyano, nitro and amino.

As used herein, the term "Cτ__4 alkyl" refers to a saturated hydrocarbyl radical having a straight or branched chain of from 1 to 4 carbon atoms. Included within the scope of this term are methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl and tertiary butyl . As used herein, the terms "C1-4 alkoxy" , "C₁_₄ thioalkyl", "C_j^ alkylsulphinyl" and "Cτ__4 alkylsulphonyl" refer to the relevant radical, the Cχ_4 alkyl moiety of which is as defined above.

As used herein, the term "patient" refers to warmblooded animals or mammals including humans, who are in need of treatment to protect or rescue normal cells from the toxic side effects caused by irradiation or the administration of a chemotherapeutic agent.

The expression "pharmaceutically acceptable acid addition salt" encompasses both organic and inorganic acid addition salts including, for example, those prepared from acids such as hydrochloric, hydrobromic, sulphuric, sulphonic, tartaric, fumaric, citric, lactic, maleic, phosphoric, succinic, acetic, benzoic, ascorbic, p-toluenesulphonic, benzenesulphonic, propionic and the like. The hydrochloric acid addition salts are preferred.

Particularly preferred compounds for use in the present invention are ( -) -N-1-phenylisopropyl-N- methyl-2-propynylamine (known generically as selegiline or I-deprenyl) and (-)-N-2,4- dichlorophenoxypropyl-N-methyl -2 -propynylamine (known generically as clorgyline) . The hydrochloric acid addition salts of these compounds are particularly preferred for use in the present invention.

The MAO inhibitors used in the present invention can be prepared as described in Fowler, J.S., J.Org.Chem. 42, 2637 (1977); Cruces, M.A. , Eur. J. Med. Chem., 25:257 (1990); O'Brien, E.M. et al . , J. Neural Transm. [suppl.] 41, 295-305 (1994); and Yu, P.H. et al . , Adv. Exp. Med. Biol. 363: 17 (1994).

The MAO inhibitors used in the present invention act to protect and/or rescue normal or uninfected cells from the toxic side effects caused by the administration of chemotherapeutic agents, but have no effect on tumourigenic or infected cells. Certain chemotherapeutic agents can be useful in the treatment of diseases. However, these agents can also cause toxic side effects that can limit their use and/or effectiveness. Examples of such agents are DNA- intercalating agents (e.g. cisplatin) , pyrimidine bases (e.g. 5-fluorouracil) or DNA strand scission inducing agents (e.g. bleomycins) used in the treatment of cancers; and pyrimidine nucleosides (e.g. azidodeoxythymidine (AZT) ) used in the treatment of AIDS.

The MAO inhibitors may be administered simultaneously, separately or sequentially with chemotherapeutic agents of the above type. The MAO inhibitors are conveniently administered prior to the administration of the chemotherapeutic agent, for example, at least three hours prior thereto.

The MAO inhibitors also protect and/or rescue normal cells from toxicity resulting from irradiation, such as gamma-radiation, including radiation therapy used in the treatment of certain cancers. The MAO inhibitors are conveniently administered prior to irradiation, for example, at least three hours prior thereto, especially in the treatment of cancer.

The ability of MAO inhibitors to protect/rescue normal cells from the toxic side effects of irradiation or the administration of chemotherapeutic agents allows a more cytotoxic dose of radiation or chemotherapeutic agent to be administered while the surrounding normal cells remain biologically intact. Thus, MAO inhibitors are ideal agents for use in therapy requiring the use of radiation or chemotherapeutic agents, especially in the treatment of cancer.

The MAO inhibitors may be administered orally, for example with an inert diluent or with an assimilable edible carrier. They may also be enclosed in hard or soft shell gelatin capsules or they may be compressed into tablets. For oral administration, the MAO inhibitors may be incorporated with pharmaceutically acceptable excipients and used in the form of ingestible tablets, buccal tablets, capsules, suspensions, syrups and the like. Suitable excipients include solvents such as water, alcohol and propylene glycol, surface active agents, suspending agents, lubricants, flavouring agents, colourants, and the like. Such carriers and excipients are well known to those skilled in the art.

The MAO inhibitors may also be administered parenterally (e.g. intravenously). Injectable dosage forms of a solution or suspension of the MAO inhibitors can be prepared for example in a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Examples of oils which can be employed in these preparations are those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil and mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, ethanols and glycols (e.g. propylene glycol or polyethylene glycol) are preferred liquid carriers, particularly for injectable solutions.

As indicated above, the MAO inhibitors used in the present invention may be effective at very low dosage levels. For human use, an oral dosage could be as low as 70 ng (or even less) to 700 μg, preferably from 1 μg (or less) to 100 μg, per day for an average adult patient (70 kg) . Single dosages for parenteral (e.g. intravenous) administration could be as low as 10 ng (or even less) to 100 μg, preferably from 100 μg (or less) to 50 μg, for a 70 kg patient.

Variations on these dosages may occur depending on the weight and condition of the subject being treated, as will be determined by the medical practitioner. Transdermal administration of appropriate formulations would form the basis of continuous administration.

The following Examples illustrate the invention.

In the following Examples, the compound (-)-N-l- phenylisopropyl-N-methyl-2 -propynylamine hydrochloride is referred to as deprenyl, and the compound (-)-N- 2,4-dichlorophenoxypropyl-N-methyl-2 -propynylamine hydrochloride is referred to as clorgyline.

Unless otherwise stated, deprenyl and clorgyline are added to cell line cultures immediately after plating and to explant cultures immediately after attachment .

All irradiation studies were performed with cobalt-60 (gamma radiation) . Materials and Methods used in the Examples

Materials:

Collagenase type IV; clorgyline; 5-fluorouracil; hydrocortisone; insulin; hydrogen peroxide; and diaminobenzidene (DAB) were obtained from Sigma Chemical Company. Carbolfuchsin (Ziehl-Nielsen stain) and haematoxylin were obtained from British Drug Houses (BDH) .

All cell culture media were obtained from Gibco- BRL Life Technologies, as also were the following: Foetal calf serum (FCS) ; Horse serum (HS) ; HEPES Buffer; Versene (Trademark; 0.2g/l EDTA in isotonically buffered saline); Hank's balanced salt solution (0.4 g/1 KCl, 0.06 g/1 KH₂P0 , 8.0 g/1 NaCl ,

0.35 g/1 NaHC0₃, 0.048 g/1 Na₂HP0 , 1.00 g/1 D- glucose and 0.01 g/1 phenol red); trypsin (2.5% stock, containing 25 g/1 trypsin, 0.4 g/1 KCl, 2.2 g/1 NaHC0₃, 6.8 g/1 NaCl, 1.0 g/1 glucose, 0.005g/l phenol red) L-glutamine; and penicillin/streptomycin. Vectastain (Trademark) ABC kit was obtained from Vector Laboratories .

Glycergel was prepared by the inventors by dissolving 10 g gelatine (BDH) in 60 ml distilled water and adding 70 ml glycerol and 0.25 g phenol. The resulting product becomes solid and requires melting before use.

Isoton (Trademark) is a zwitterionic solution supplied by the Coulter Electronic Company and contains 7.9 g/1 NaCl, 1.9 g/1 Na₂HP0₄ , 0.4 g/1 EDTA disodium salt, 0.4 g/1 KCl.

Deprenyl was obtained from Semat Technical, St. Albans, England.

Cisplatin was obtained from David Bull Laboratories.

Bleomycin sulphate was obtained from Lundbeck, United Kingdom.

5-Fluorouracil was obtained from Roche.

All primary antibodies were obtained from DAKO. Phosphate buffered saline (PBS) was prepared by the inventors from 8 g/1 NaCl, 0.2 g/1 KCl, 1.44 g/1 Na₂HP0₄, 0.24 g/1 KH₂P0₄.

ethods

Cell Culture and Maintenance of Cell Lines

The HACAT cell line was derived from human skin keratinocytes spontaneously transformed in vi tro during long term incubation of a primary culture under selected culture conditions. This cell line was obtained as a gift from Dr. P. Boukamp (Heidelberg University, Germany) . The HPV-G (human papilloma virus-G) cell line was obtained as a gift from Dr. J. Dipaolo, NIH, (Bethesda, MD, USA) . This line was transformed from human neonatal foreskin transfected with the HPV-16 virus to cause immortalisation. The HPV-G cell line is also available from the European Collection of Cell Cultures (ECACC) . The CHO-K1 cell line was established from a subclone of the parent CHO cell line which was derived from the ovary of a Chinese hamster and was supplied by the ECACC. The PC3 cell line was established from a grade 4 prostatic adenocarcinoma from a sixty-two year old male Caucasian and this cell line was obtained from the ECACC.

The stock cells were grown in 260 ml nunc flasks in the appropriate growth medium for the particular cell line involved. To avoid rapid loss of differentiation, the cells were not passaged too often or split at too high a ratio. When the cells reached confluence, they were passaged in the following way: cells were rinsed for approximately one minute with 0.25% w/v trypsin solution (2.5% stock trypsin diluted 1:10 with Hank's balanced salt solution) and 1 mM Versene solution in a 1:1 mixture. This solution was then poured off and a fresh 0.25% w/v trypsin/lmM Versene solution (1:1) was used to trypsinise the cells at 37°C for approximately 12-15 minutes depending on the cell line. 10 ml of cell growth medium were then placed in the flask to stop the action of trypsin on the cells. This stock cell suspension was poured into a universal container and aspirated to ensure a single cell suspension resulted. Depending on the experimental work that was to be conducted, the cells were then counted, if appropriate, to determine the number of cells per ml in the stock cell suspension. The cells were counted using a Coulter Counter model Dn. Three background readings were taken using a cuvette containing 20 ml of Isoton only, three readings were then taken using a cuvette containing 20 ml of Isoton and 1 ml of stock cell suspension. The number of cells per ml was thus determined, so that it was possible to carry out various dilutions and plate known numbers of cells into the flasks.

Plating Efficiencies, % Surviving Fraction and Cell Survival Tests

At 9 or 10 days post irradiation when the irradiated cells produced colonies, the flasks were stained with carbolfuchsin diluted 1:15 with tap water. The colonies were then counted and the plating efficiencies determined. These were then expressed as a percentage of the control plating efficiency. The plating efficiency is the fraction of cells plated that grow to form colonies after significant time has elapsed i.e. "stainable groups of cells, surface attached, and large enough to be recognised by the eye" [Elkind, M.M., Whitmore, G.S., The Radiobiol ogy of Cultured Mammalian Cells (1967, New York, Gordon & Breach) ] . This definition is not adequate for cells that survive irradiation because these cells may form giant cells or may produce an abortive colony. Puck and Marcus [J. Exptl. Med. Vol.103, pp.653-666, 1956] have set a lower limit of 50 cells per colony to be representative of a surviving cell. This is the number per colony after about six divisions of the surviving cell.

Lethal Mutation Assay

As heritable defects may take several generations to be expressed, the plating efficiency was also determined per dose point at 18-20 days (i.e. the time taken for at least 15 cell doublings) post irradiation. Appropriate cell numbers were seeded into six flasks per dose point. These cells were allowed to attach to the surface of the flask and were then irradiated after 6 hours. When the cells produced macroscopic colonies (9-10 days) , three of the original six flasks were stained with carbol- fuschin, and the colonies were counted. The medium was then removed from the remaining three flasks and a fresh 5 ml of culture medium was placed into each flask. Upon reaching confluence (9-10 days) , the cells were trypsinised and the cell suspension counted to determine the number of cells per ml. A plating efficiency experiment was then set up for each flask. The cells in these flasks were grown to macroscopic colonies and were then stained using carbolfuchsin. By taking into account the surviving fraction of the progeny of the initially irradiated cells and that of the initial surviving fraction (S.F.) it was possible to determine the residual surviving fraction (R.S.F.) from the equation :

% Residual surviving fraction = Initial % S.F. x Pmσpnγ % S.F.

100

The residual percentage surviving fraction is a measure of the level of survival at times distant, i.e. 18-20 days, from the initially irradiated cells.

Tissue Culture Assay

All connective tissue was removed from the urether or bladder biopsy using a scalpel. The tissue was then cut into 2-3 mm² pieces using a scalpel. This tissue was then placed in 50 mg collagenase type IV/5 ml 0.25% w/v trypsin solution (2.5% stock trypsin diluted 1:10 with Hank's balanced salt solution) for 30 minutes at 37°C. The digested explants were then placed singly in 25 cm² nunc flasks with 2 ml of growth medium. When attachment occurred after approximately 3 days, the medium was removed and replaced with serum free medium to inhibit the growth of fibroblasts. Deprenyl or clorgyline was also added at this stage. Cultures were grown for 14 days before irradiation and fixation for immunocytochemical analysis .

Immunocytochemical Assay

Immunostaining was performed using a Vectastain ABC kit which employs a standard immunoperoxidase method. The cell culture was washed in phosphate buffered saline (PBS) and the endogenous peroxidase activity blocked with 3% H2O2 in PBS for 5 minutes. The culture was then washed in buffer for 20 minutes. All excess serum was washed from the sections and the slides were incubated with primary antibody diluted (1:20) in PBS for 60 minutes. The slides were washed again in buffer for 10 minutes. The sections were incubated for 30 minutes with diluted biotinylated antibody solution from the Vectastain ABC kit and then washed for 10 minutes in PBS buffer. Sections were then incubated for 45 minutes with Vectastain ABC reagent and washed in PBS buffer for a further 10 minutes. Sections were incubated in diaminobenzidine (DAB) prepared as follows : -1000 μl of DAB, 900 μl of PBS and 10 μl of H₂0₂ until staining became apparent. The slides were then washed in tap water and counter- stained with haematoxylin for 1 minute. The sections were then washed in hot water and mounted in glycergel. A positive reaction was indicated by a brown staining.

Cell culture media used

HACAT and HPV-G Cell Medium

Dulbecco's Modified Eagle's Medium (DMEM) /F: 12 plus

7.5% FCS

5 ml L-Glutamine (200mM)

5 ml Penicillin/streptomycin (5000 IU/ml) 12.5 ml 1M Hepes buffer

1 μg/ml Hydrocortisone

PC3 and CHO-K1 cell medium Hams F-12 plus 10% FCS 5 ml L-Glutamine (200mM)

5 ml Penicillin/streptomycin (5000 IU/ml) 12.5 ml 1M Hepes buffer

Urether and Bladder Explant culture medium RPMI-1640 plus 7.5% FCS and 10% HS 5 ml L-Glutamine (200mM)

5 ml penicillin/streptomycin (5000 IU/ml) 1 μg/ml Hydrocortisone (final concentration) 0.05 IU/ml Insulin (final concentration) Addition of Monoamine Oxidase Inhibitors to Cell Cultures

The monoamine oxidase (MAO) inhibitors, deprenyl and clorgyline, were made up in distilled water to the required molarities and were added to the cell - culture flasks at least 3 hours before irradiation to allow these MAO inhibitors to take effect prior to irradiation. The standard volume of inhibitor which was added to the required flask was always 100 μl . In the control flasks 100 μl of distilled water were added to keep the volume of cell culture medium constant throughout the experiment . The same protocol was employed when conducting the chemotherapy studies and the hydrogen peroxide induced cell death studies.

Addition of Chemotherapeutic Agents to Cell Cultures

The chemotherapeutic agent, cisplatin, consisted of a 1.0 mg/ml solution of cisplatin made up in a sodium chloride/ mannitol solution. In order to achieve the correct concentration of cisplatin, this stock solution was diluted with distilled water. 5- Fluorouracil was supplied in a solid form and was dissolved and made up to the desired concentration with distilled water. Bleomycin sulphate was dissolved in distilled water to give a 20 mg/ml stock solution which was diluted in medium as required. All additions of chemotherapeutic agents were made 6 hours after the cells had attached to the flasks and were incubated with the cells for 1 hour. In order to terminate the effect of these cytotoxic compounds, the- medium was poured off and the cells in monolayer culture were washed with Hank's balanced salt solution for 1 minute approximately. This solution was then poured off and the flask was re-filled with 5 ml of the appropriate cell culture medium.

Addition of Hydrogen Peroxide to Cell Cultures

Hydrogen peroxide was supplied in a 30% w/w solution. The desired concentration of hydrogen peroxide was obtained by diluting appropriately with distilled water. All additions of hydrogen peroxide to cell culture flasks were made 6 hours after the cells were plated initially.

Example 1

The effects of deprenyl and clorgyline on the responses of primary tissue from human patients to radiation

Normal and tumour human explants were exposed to the monoamine oxidase-B inhibitor, deprenyl, or to the monoamine oxidase-A inhibitor, clorgyline, at concentrations of 10^"9M and 10^"5M. These were then irradiated 72 hours after attachment at 0.5 and 5.0 Gy with Cobalt-60 gamma radiation before the cell numbers were determined and the percentage survival calculated with respect to control cells that had not been treated with monoamine oxidase inhibitors or exposed to radiation. The results shown in Tables la and lb indicate that 10"⁹M deprenyl has a significant radioprotective effect on normal tissue, but has no such effect on malignant tissue.

L-Lactate, a known radio-sensitiser, was also used to investigate whether deprenyl could overcome such chemical-induced radio-sensitisation. The results shown in Table la indicate a pronounced protective effect.

Example 2

The effects of deprenyl and clorgyline on the responses of cell lines to radiation

(a) Human skin keratinoσytes : The cell lines used were HACAT cells which are a non-tumourigenic but immortalized cell line and human papilloma virus (HPV) transfected keratinocytes which are tumourigenic.

Deprenyl and clorgyline were added immediately after plating and the cells were irradiated 6 hours after plating at the dosage indicated with cobalt-60 gamma radiation. The results in Tables 2a, 2b and 2c show that both deprenyl and clorgyline give rise to radio- protection and enhanced cloning efficiency in the normal, but not the tumourigenic, cell line. The results in Table 2d show that significant protection is obtained in HaCaT cells at 10^" and 10^" M deprenyl over a range of radiation doses from 2.5 Gy (therapeutic) to 10 Gy.

(b) Human tumour cell lines: A malignant human prostate cell line (PC3) was also tested. The results in Table 3a show no significant effect of deprenyl or clorgyline on the radiation response of malignant cells. These results are confirmed in Table 3b where the effect of InM deprenyl on PC3 and HaCaT cells is compared. These data show that deprenyl protects a normal human epithelial call line (HaCaT cells) but does not protect a human epithelial tumour cell line against the effects of radiation.

(c) Investigations using a "generic" cell line:

Radiation studies were also performed with the immortalized, tumourigenic CHO Kl cells in culture. As shown in Table 4a, clorgyline exerted no significant radio-protective effect and responses to deprenyl were small compared to those seen in the normal cell lines and tissue explants.

(d) Effect of different concentrations of deprenyl:

Radiation studies were performed on the HaCaT cell line using concentrations of deprenyl ranging from zero to 10^" M to establish the concentrations of deprenyl giving maximum radioprotection. Deprenyl was added immediately after plating and the cells were irradiated 6 hours after plating at 5 Gy with cobalt- 60 gamma radiation. The results given in Table 4b show an effective concentration range of deprenyl between 10^"5M and 10^"10M.

Example 3

Cisplatin, bleomycin and 5-fluorouracil induced cell death

To investigate whether deprenyl could rescue or prevent cells from undergoing cell death in response to treatment with cisplatin, bleomycin sulphate and 5-fluorouracil, two cell lines were used, one of which was non-tumourigenic (a human keratinocyte cell line (HACAT) ) and the other of which was tumourigenic (a Chinese hamster ovary cell line (CH0-K1) ) . The results shown in Tables 5a - 5e indicate that deprenyl protects the non-tumourigenic cell line from cell death induced by the chemotherapeutic agent but that it has no such protective effect on the tumourigenic cell line (see Table 5b) . Example 4

(a) Apoptosis gene expression: Cultures exposed to deprenyl and clorgyline were fixed and stained 1 hour after irradiation for expression of the product of the gene bcl-2 involved in damage control and repair. The results in Tables 6a- 6f show very strong induction of bcl-2 in the deprenyl and clorgyline treated cultures whether exposed to radiation or not. Controls induced bcl-2 expression following irradiation but this induction took at least one hour.

(b) Oxidative stress induced cell death: To investigate whether deprenyl could rescue or prevent cells from undergoing cell death in response to another noxious stimulus apart from gamma radiation, oxidative stress induced cell death of HaCaT cells incubated with increasing concentrations of the oxidant hydrogen peroxide was studied. The results in

Table 7 show that deprenyl at a concentration of 10^"

⁹M has a large protective effect on cells exposed to a hydrogen peroxide concentration of lOOμM. As the hydrogen peroxide concentration rises in the cell culture medium to 200μM, deprenyl at the above concentration (10^~9M) fails to protect cells to the same extent. However, it does have a small protective effect under these conditions . It was also found that at hydrogen peroxide concentrations above 200μM up to 400μM, deprenyl has no protective effect at all. Mechanistically these results are very important since it is well documented that hydrogen peroxide at a concentration of lOOμM leads to programmed cell death or apoptosis and as the cell experiences concentrations above this, death by necrosis or general cell damage ensues. Since deprenyl was found to found to have its peak protective action at a concentration of lOOμM hydrogen peroxide this would suggest that it is acting as an anti-apoptotic compound .

In the following Tables : n = not significant

* = P < 0.05

** = P < 0.01

*** = P < 0.001

Table la.

The effects of Deprenyl on the survival of normal human urothelial explant cultures after irradiation in the presence and absence of L-lactate. Explants were irradiated 72 hours after attachment .

Table lb.

The effects of Deprenyl and Clorgyline on the survival of human bladder tumour explants after irradiation . Explants were irradiated 72 hours after attachment .

Table 2a.

The effects of Deprenyl and Clorgyline on the survival of normal Human keratinocytes (HaCaT cells) irradiated at 6 hours post plating.

Table 2b.

The effects of Deprenyl and Clorgyline on the survival of HPV-G cells (Human keratinocyte cell line, HPV 16 transfected, immortalised, tumourigenic) irradiated at 6 hours post plating.

Table 2c.

The effects of Deprenyl and Clorgyline on the late damage (LD) and induction of lethal genetic instability of HPV-G cells (Human keratinocyte cell line, HPV 16 transfected, immortalised, tumourigenic ) exposed to radiation. The cells were irradiated under the same conditions as the experiments in Table 2b but were then allowed to go through at least 15 cell doublings prior to assay.

Table 2d

Effect of different combinations of radiation and L-Deprenyl on HaCaT cells. Irradiation occurred at 6 hours post plating.

Table 3a.

The effects of Deprenyl and Clorgyline on the survival of PC3 cells (human prostate carcinoma cells) after exposure to radiation 6 hours post plating.

Table 3b.

Effect of InM L-Deprenyl on the radiation response of PC 3 and HaCaT cells. Irradiation occurred at 6 hours post plating.

Table 4a.

The effects of Deprenyl and Clorgyline on clonogenic cell survival post-irradiation for CHO K-l cells (Chinese hamster ovary established cell line, immortalised, tumourigenic) irradiated at 6 hours post plating.

Table 4b.

The effects of different concentrations of deprenyl on the survival of HaCaT cells after exposure to radiation (5 Gy) 6 hours post plating. (Significance values for both columns are against the control with no deprenyl).

Table 5a.

Clonogenic assay results for HaCaT cell line (human keratinocyte non-tumourigenic cell line) treated with 2,4,6 and 8 microgrammes per ml of cisplatin (DNA intercalating agent used in cancer therapy) for 1 hour 6 hours post plating and treated with Deprenyl at a concentration of 1 nanomolar where indicated immediately after plating cells.

Table 5b.

Clonogenic assay results for CHO-Kl cell line (Chinese Hamster Ovary tumourigenic cell line) treated with 4, 6, 8 and 10 microgrammes per ml of cisplatin (DNA intercalating agent used in cancer therapy) for 1 hour 6 hours post plating and treated with Deprenyl at a concentration of 1 nanomolar where indicated immediately after plating.

Table 5c.

Clonogenic assay results for the HaCaT cell line treated with cisplatin for 1 hour at 6 hours post plating and treated with InM Deprenyl where indicated immediately after plating cells.

Table 5d.

Clonogenic assay results for the HaCaT cell line treated with bleomycin sulphate for 1 hour at 6 hours post plating and treated with InM Deprenyl where indicated immediately after plating cells.

Table 5e.

Clonogenic assay results for the HaCaT cell line treated with 5-fluorouracil for 1 hour at 6 hours post plating and treated with InM Deprenyl where indicated immediately after plating cells.

Table 6a.

Effect of Deprenyl on normal human urothelial explant cultures after irradiation in the presence and absence of L-lactate. Explants were irradiated 72 hours after attachment.

In Tables 6a-6f, pos=positive

Table 6b.

Effect of Deprenyl and Clorgyline on bladder tumour explants after irradiation. Explants were irradiated 72 hours after attachment.

Table 6c.

Effect of Deprenyl and Clorgyline on HaCaT cells (normal human keratinocytes) irradiated at 6 hours post plating.

Table 6d.

Effect of Deprenyl and Clorgyline on PC3 cells (human prostate carcinoma) irradiated at 6 hours post plating.

Table 6e.

Effects of Deprenyl and Clorgyline on HPV-G cells (Human tumourigenic keratinocytes) irradiated at 6 hours post plating.

Table 6f.

Effect of Deprenyl and Clorgyline on CHO-Kl cells (Chinese Hamster ovary established cell line, immortalised, tumourigenic) irradiated at 6 hours post plating.

Table 7.

Clonogenic assay results for HaCaT cell line (human keratinocyte non- tumourigenic cell line) treated with 100 to 400 micromolar solutions of hydrogen peroxide to promote oxidative stress induced cell death 6 hours after plating and treated with Deprenyl at a concentration of 1 nanomolar where indicated immediately after the cells were plated.

It is clear from the above results that the MAO

-10 inhibitors at concentrations as low as 10 M have a relatively large radioprotective effect on normal cells and tissues but do not protect tumour cells to any great extent .

The MAO inhibitors also have a protective effect on non-tumourigenic cells treated with chemotherapeutic agents, but have no such protective effect on tumourigenic cells treated with said agents. Thus, a more cytotoxic dose of radiation or chemotherapy can be delivered to a tumour while the surrounding normal cells remain protected.

The MAO inhibitors can also rescue damaged cells making them fit to deal with damage rather than improve survival of already fit cells. This is dramatically illustrated by the behaviour of the HACAT cell line plated at low cell numbers. As shown in Table 2a, the plating efficiency of this cell line is always relatively low and under these conditions plating efficiencies in the range <0.002 - 0.08% were obtained in the absence of deprenyl or clorgyline. Plating under exactly the same conditions but in the presence of 10~⁹M deprenyl or clorgyline had the effect that plating efficiencies increased to approximately 1.9 and 4.0% respectively. These survival figures were not greatly affected by radiation at either 0.5 or 5.0 Gy (plating efficiencies 2.6 and 2.2, respectively, in the presence of 10 ^~9M deprenyl) .

The mechanism of action of the MAO inhibitors is at present unknown. However, the results in Table 6c showing very strong induction of the bcl-2 gene after irradiation of normal cells implicate the activation of anti-apoptosis pathways. Tumour cells generally have bcl-2 already induced thus increasing their resistance to damage. Thus, one action of the MAO inhibitors could be to induce this anti-apoptotic pathway in the, relatively radiosensitive, normal cells, thereby increasing their survival to a level comparable to the more radio-resistant tumour cells, Another theory is that the MAO inhibitors could down regulate the expression of another protein known as BAX, in normal cells. BAX and bcl-2 act as a rheostat in the apoptotic process since when bcl-2 is in excess within the cell, apoptosis is inhibited. However, when BAX is in excess apoptosis proceeds. These two proteins cooperate and bind to each other via their BH1 and BH2 protein domains and the effect that BAX has on the apoptotic process is to act somewhat like a mopping device for bcl-2, to drive the cell in the direction of apoptosis since when in complex with bcl-2 it renders this protein inactive, thereby preventing it from exerting its apoptotic repressive effects. If the MAO inhibitors down regulated BAX expression in normal cells, more bcl-2 would be available and hence one would see a higher degree of apoptotic inhibition. In effect, this is what is seen from the present results.

Apoptosis or programmed cell death is a major problem in many diseases such as acquired immunodeficiency syndrome (AIDS) . AIDS is characterized by profound immunosuppression with diverse clinical features, including opportunistic infections, malignancies and central nervous system (CNS) degeneration. AIDS is one of a group of clinical syndromes caused by a retrovirus called human immunodeficiency virus (HIV) .

HIV primarily infects CD4 expressing T cells, including helper T cells and macrophages . The massive immunosu- pression is a result of virus induced apoptosis of subsets of T cells. Certain autoimmune diseases are also largely dependent on the apopototic response. Cytotoxic T lymphocytes which do not recognise other cells within the body as "self" excrete a toxin which may be lymphotoxin or a related molecule. The toxin kills cells by activating target cell enzymes that cleave DNA in the target cell nucleus. Once the nuclear DNA is fragmented, target cell nuclei also undergo fragmentation, i.e. apoptosis. Apoptosis is also a major cause of cell loss in biotechnological processes involving mass cell culture. It is a major cause of cell death in critical tissues such as blood, gut and liver following exposure to certain classes of toxic pollutants, e.g. cadmium or nickel, and also as a result of ischaemia. The anti-apoptosis function of MAO inhibitors makes them of potential use in all of these areas .

Claims

C AIMS ;

1. Use of a monoamine oxidase inhibitor for the preparation of a medicament for protecting and/or rescuing normal cells from toxicity resulting from irradiation and/or the administration of a chemotherapeutic agent.

2. Use of a combination of a monoamine oxidase inhibitor with a chemotherapeutic agent which can disrupt DNA or RNA replication for the preparation of a medicament for the treatment of a neoplastic disease state or AIDS.

3. Use according to claim 1 or 2 , wherein the medicament is adapted for oral, parenteral or transdermal administration.

A pharmaceutical product for protecting and/or rescuing normal cells from toxicity resulting from the administration of a chemotherapeutic agent, the product comprising :

(a) a monoamine oxidase inhibitor; and

(b) a therapeutically effective amount of the chemotherapeutic agent, as a combined preparation for simultaneous, separate or sequential administration.

5. A pharmaceutical composition for oral administration in unit dosage form for protecting and/or rescuing normal cells from toxicity resulting from irradiation and/or the administration of a chemotherapeutic agent, comprising from 70 ng (or less) to 700 μg, preferably from 1 μg (or less) to 100 μg, of an MAO inhibitor or an equivalent amount of a pharmaceutically acceptable salt thereof, per unit dose .

6. A pharmaceutical composition for parenteral administration in unit dosage form for protecting and/or rescuing normal cells from toxicity resulting from irradiation and/or the administration of a chemotherapeutic agent, comprising from 10 ng (or less) to 100 μg, preferably from 100 ng (or less) to 50 μg, of an MAO inhibitor or an equivalent amount of a pharmaceutically acceptable salt thereof, per unit dose.

A pharmaceutical composition according to claim 5 or 6 additionally comprising a chemotherapeutic agent which can disrupt DNA or RNA replication.

8. The invention according to any of claims 1, 4 or 8, wherein the chemotherapeutic agent is an antineoplastic agent, such as cisplatin, 5- fluorouracil or a bleomycin.

9. The invention according to any of claims 1, 4 or 8, wherein the chemotherapeutic agent is an anti -AIDS agent, such as azidodeoxythymidine (AZT) .

10. The invention according to any preceding claim, wherein the monoamine oxidase inhibitor is deprenyl (N-1-phenylisopropyl -N-methyl-2 -propynylamine) or a structural analogue or derivative thereof, or a pharmaceutically acceptable salt thereof.

11. The invention according to any preceding claim, wherein the monoamine oxidase inhibitor is selected from any of the following :

(a) (-) -N-l-phenylisopropyl-N-methyl-2- propynylamine and pharmaceutically acceptable salts thereof ;

(b) (-) -N-2,4-dichlorophenoxypropyl-N-methyl-2- propynylamine and pharmaceutically acceptable salts thereof ;

(c) compounds of the formula I: Z₁-0-Z₂-0-C_nH_2n-N(CH₃) -CH₂-C≡CH

wherein Z_λ and Z₂ are each independently optionally substituted phenyl; and n is an integer from 2 to 4; and pharmaceutically acceptable salts thereof;

(d) compounds of the formula II:

Z₃-X

wherein Z₃ is indolyl substituted at the 1- position by hydrogen, methyl or ethyl, and at the 5- position by hydrogen, hydroxy or methoxy; and X, which may be present at the 2- or 3- position of the indolyl ring, is C_πH_2n-N (R₃) -R₄, wherein R₃ is a substituted ethylenic or acetylenic group; R₄ is hydrogen, methyl or ethyl and n is an integer from 1 to 4; and pharmaceutically acceptable salts thereof;

(e) compounds of the formula III:

R-N(CH₃) -CH₂-C≡CH

(f) compounds of the formula IV: Y- [N (R) ] _pC_nH_2n-N ( CH₃) - CH₂ - C≡CH

(g) (R) -N-2-propynyl-1-indanamine (Rasagiline) and pharmaceutically acceptable salts thereof .

12. The invention according to claim 11, wherein the monoamine oxidase inhibitor is (-)-N-l- phenylisopropyl-N-methyl-2-propynylamine or a pharmaceutically acceptable salt thereof, in particular the hydrochloric acid addition salt thereof .

13. The invention according to claim 11, wherein the monoamine oxidase inhibitor is (-)-N-2,4- dichlorophenoxypropyl-N-methyl-2-propynylamine or a pharmaceutically acceptable salt thereof, in particular the hydrochloric acid addition salt thereof .