AU1761500A - Methotrexate derivatives - Google Patents

Description

WO 00/34281 PCT/AU99/01073 1 Methotrexate derivatives TECHNICAL FIELD The present invention is concerned with derivatives of methotrexate (MTX), 5 compositions containing the derivative and to methods of treatment involving use of these MTX derivatives. BACKGROUND MTX can bind to active sites on the enzyme dihydrofolate reductase (DHFR) and 10 through competitive inhibition block the formation of tetrahydrofolates (THF) needed in the biosynthesis of DNA and RNA. MTX also inhibits other folate dependent enzymes. The ability of MTX to inhibit nucleic acid synthesis has been used to treat abnormal cell growth. Since actively proliferating cells are more sensitive to MTX, it can be used to selectively impair cancerous cell growth. MTX is a widely used anti-cancer 15 agent used to treat neoplastic diseases. MTX also acts as an antiproliferative and immunoregulator and can be employed in the treatment of psoriasis and rheumatoid arthritis. Oral MTX is currently used for the treatment of moderate or severe arthritis and recalcitrant psoriasis. The weekly dose is 2.5 to 25 mg. Use of this drug is restricted by 20 its toxicity with the major long term side effect being liver damage. Attempts to reduce toxicity and improve efficacy in psoriasis treatment by applying MTX topically have met with little or no success, possibly due to either poor dermal penetration or residence time of the drug in the skin. 25 DISCLOSURE OF THE INVENTION In a first aspect, the present invention provides A I M-[Y]n-NH-C-CX 2 -O-R I B in which M is methotrexate or an analogue thereof, A=H, CX 2

-O-R

2 or halogen; 30 B=H, CX 2

-O-R

3 or halogen; X is independently H or halogen; n is 0 or greater than or equal to 1; Y is a linker group, and where n is greater than 1, each Y is the same or different; WO 00/34281 PCT/AU99/01073 2

R

1

,R

2 , and R3 are the same or different and are either hydrogen, substituted or unsubstituted methyl, ethyl, a saturated or unsaturated fatty acyl group, with the proviso that when n is 0 or 1, and A is CH 2

-O-R

2 and/or B is CH 2

-O-R

3 , then RI, R 2 and R3 are not selected from a fatty acyl group; 5 with the proviso that when n is greater than or equal to 2, and A is CH 2

-O-R

2 and/or B is CH 2

-O-R

3 , and at least one of RI, R 2 and R3 is a fatty acyl group, then -[Y],,- is other than -(AA),- or -Y-(AA).-i-, where AA is an amino acid. Examples of analogues of methotrexate are described in the prior art. See, for 10 example, .A.L Jackman ed (1999). "Antifolate Drugs in Cancer Therapy" Humana Press, Totowa, New Jersey; Matsuoka, H and Mihara, M (1998). " The synthesis and biological evaluation of new methotrexate derivatives in rheumatoid arthritis. Drugs of the Future 1998. 23(9);1015-1022; and Degraw, J et al. (1995) "New analogs of methotrexate in cancer and arthritis". Current Medicinal Chemistry 2:630-653, the 15 disclosures of which are incorporated herein by reference. Preferably M is methotrexate. Examples of linker groups that are useful in the present invention include: a) a linker with an amino group to M and a carboxyl group to the

NHC(A)(B)CX

2 ORI group, for example, an amino acid or antibiotic. 20 b) a linker with an amino group to M and a sulphonic acid group to the

NHC(A)(B)CX

2 OR, group, for example, 2-aminoethanesulphonic acid (taurine). c) a linker with an hydroxyl group to M and a carboxyl group to the

NHC(A)(B)CX

2 OR, group. The linker may be an hydroxy acid. The hydroxy acid may be analogue of an amino acid in which the amino group of the amino acid is replaced by an 25 hydroxy group. Examples of hydroxy acids include glycolic acid (HOCH 2 COOH); lactic acid (HOCH(CH 3 )COOH; 4-hydroxy-butyric acid HOCH 2

CH

2

CH

2 COOH; 6-hydroxy caproic acid; 10-hydroxy-decanoic acid; 2-hydroxy-caproic acid; 2-hydroxybutyric acid; 3-hydroxy-butyric acid, etc. The use of these acids allows linkage of the Tris, or Tris conjugates to the drug via a labile ester bond rather that via the more stable amide bond. 30 d) a linker with an hydroxyl group to M and a sulphonic acid group to the

NHC(A)(B)CX

2 OR, group, for example, 2-hydroxyethanesulphonic acid (isethonic acid). e) a linker with an hydroxyl group to M and a reactive halide group to the

NHC(A)(B)CX

2 OR, group, for example, 2-chloroethanol. f) a linker having an hydroxy group and an aldehyde group, for example, p 35 hydroxybenzaldehyde, WO 00/34281 PCT/AU99/01073 3 g) a linker having a halide group and an carboxyl group, for example, 2 chloroacetic acid; h) a linker having two reactive halide groups, for example, 1,2-dibromoethane; and 5 i) an alkylene oxide, for example, ethyleneoxide. The NHC(A)(B)CX 2

OR

1 group may be attached to the c or y carboxyl group of the methotrexate compound. Most preferably, the -NHC(A)(B)CX 2 OR group is attached to the y carboxyl of the methotrexate compound, optionally by the linker. Where Y is an amino acid it may be any suitable amino acid including, natural 10 and non-natural amino acids and analogues thereof Preferably the amino acid is glycine. Preferably R 1 , R 2 and R 3 are each H. Preferably n is in the range 0-5. In a preferred embodiment of the invention, M is methotrexate and -[Y],- is -[AA], -, where AA is an amino acid. Preferably AA is glycine and n is 1. Where n >2, 15 each AA need not be the same amino acid. A preferred methotrexate derivative is methotrexate-y-glycine-TRIS (MTX-y GT). In a second aspect, the present invention provides a pharmaceutical composition incorporating a compound of the formula I as defined above and a pharmaceutically 20 acceptable carrier or diluent. In a preferred form of the composition of the invention, M is methotrexate. Y is glycine and n is 1. One preferred methotrexate derivative is methotrexate derivative is methotrexate-y-glycine-Tris. 25 Compositions suitable for oral administration include liquid solutions, capsules, suspensions, emulsion etc. The composition of the invention may be in tablet form incorporating the usual tablet formulation ingredients. Composition suitable for parenteral administration include aqueous and non 30 aqueous injectable solutions or emulsions. Formulation suitable for topical administration may be in the form of a cream, gel, paste, salve, poultice, nasal spray, pulmonary aerosol or foam incorporating the compound of the present invention. The composition of the invention may be in the form of suppositories, pessaries, 35 tampons, creams, gels, pastes, lotions, implants, patches or foams. The composition of the invention may be in the form of an implant.

WO 00/34281 PCT/AU99/01073 4 The composition of the invention may be in the form of a liquid aerosol or powder for administration to the lung. The dosage level of the MTX derivative of the present invention in the pharmaceutical composition may be sufficient to effect a prophylactic or therapeutic 5 response in the selected time frame. The present invention also provides a method of treatment of cancer or a disease with an autoimmune component in a subject, the method including administration of a prophylactically or therapeutically effective amount of a compound of the formula I given above. 10 Accordingly, in a third aspect, the present invention provides a method of treatment of a disease with an autoimmune component or a cancer in a subject, the method including administration to the subject of an effective amount of a compound in accordance with of formula II: D I M-[Y]n-NH-C-CX 2

-O-PR

6 I E 15 wherein: D=H or CX 2

-O-R

4 or halogen; E=H or CX 2

-O-R

5 or halogen; X is as defined above; 20 n is as defined above; Y as defined above; and

R

4 , R 5 and R 6 are the same or different and either hydrogen, substituted or unsubstituted methyl, ethyl or fatty acyl groups with either saturated or unsaturated bonds. 25 The disease with an autoimmune component may be any condition where immunosuppression is called for. For example, the disease with an autoimmune component may be psoriasis or rheumatoid arthritis. The disease with an autoimmune component may be inflammatory bowel disease or Crohn's disease in a subject. 30 Preferably the compound used in the method of the third aspect is a compound of formula I. The method of treatment of the present invention may be achieved by any suitable route, for example, oral, parenteral, implant. and topical.

WO 00/34281 PCT/AU99/01073 5 The dosage level of the MTX derivative of the invention depends on the nature and severity of the condition being treated but generally the administered amount is sufficient to produce a prophylactic or therapeutic effect. Pharmaceutical compositions in accordance with the invention may contain the MTX derivative in an amount in the 5 range of about 0.001 to about 90% of the composition. A composition in an oral form, for example, a tablet, may contain up to 85% of the MTX derivative, preferably about 2 to 60% of the oral composition. The concentrations here set forth are provided by way of description only and not by way of limitation. In order that the invention may be more readily understood, the following non 10 limiting embodiments are provided. BRIEF DESCRIPTION OF DRAWINGS: Figure 1 is a boxplot demonstrating the effect of oral MTX-y-GT, MTX-y-GTP 1 and MTX-y-GTP3 in the rat assay of delayed type hypersensitivity footpad 15 oedema. MTX-y-GT and MTX-y-GTP1 are both active in this assay as is MTX. MTX-y-GTP1 is statistically more effective than MTX (p=0.021). Figure 2 shows the effects of MTX and MTX-y-GT in the rat adjuvant-induced arthritis model. The results indicate that MTX-y-GT is, at the two lower doses, equipotent to MTX in delaying the onset ofjoint inflammation in this model. 20 Figure 3 shows the suppressing effect of MTX-y-GTP3 on epidermal DNA synthesis following UVB irradiation. After UVB irradiation mice respond with an initial suppression of DNA synthesis followed 48h later by a marked increase in DNA synthesis. MTX-y-GTP3 is at least as active as MTX in suppressing this pulse of synthesis. 25 Figure 4 shows the suppressing effect of MTX and MTX-Tris conjugates on mitotic activity in epidermal cells following UVB irradiation. Figure 5 shows Filaggrin expression from the SPA proof-of-principle trial. The figure shows total filaggrin expression following topical MTX-y-GTP3 or MTX treatment versus the response with betamethasone. Patients in the upper right 30 hand quadrant responded positively to both betamethasone and 0.5% MTX-y GTP3 (indicated by *; patients 1, 2, 4, 6 & 8). Patients 5, 9 and 10 responded to betamethasone but failed to respond to 0.5% MTX-y-GTP3. MTX (circles) and 1.0% MTX-y-GTP3 (squares) also showed positive patients in a similar manner. The correlation between response to betamethasone and MTX-y 35 GTP3 suggests that there are two main groups in the trial: responders and non responders.

WO 00/34281 PCT/AU99/01073 6 Figure 6 is a box plot showing the pooled results for filaggrin expression in the stratum granulosum of SPA patients treated with oral MTX-y-GTP3 or MTX compared to betamethasone treatment. There is a statistically significant effect of the positive control betamethasone versus its vehicle. The comparison of 5 0.5% MTX-y-GTP3 with its vehicle gives a borderline significance of 0.08. Figure 7 shows granularity:parakeratosis ratios for patients treated with 1.0%, 0.5% MTX-y-GTP3 solution, 0.5% MTX and 0.05% betamethasone valerate cream. 1.0% MTX-y-GTP3 is showing positive results with this early indicator of plaque resolution. 10 MODES FOR CARRYING OUT THE INVENTION Abbreviations: AICAR phosphoribosylaminoimidazolecarboxamide 15 APA 4-amino-4-deoxy-N 10-methylpteroic acid DAHMP 2,4-Diamino-6-hydroxymethylpteridine DCC 1,3-dicyclohexylcarbodiimide DCM dichloromethane DECP diethyl cyanophosphonate 20 DMA N,N-dimethylacetamide DMAP 4-dimethylaminopyridine DMF N,N-dimethyl formamide Et 2 0 diethylether EtN'Pr2 di-isopropyl ethyl amine 25 EtOAc ethyl acetate GT glycine-Tris GTP 1 glycine-Tris monopalmitate GTP2 glycine-Tris dipalmitate GTP3 glycine-Tris tripalmitate 30 HOAc acetic acid HOSu N-hydroxysuccinimide MABA N-methyl aminobenzoic acid MTX methotrexate MTX-y-GT methotrexate-y-glycine-Tris 35 MTX-y-GTP 1 methotrexate-y-glycine-Tris monopalmitate MTX-y-GTP2 methotrexate-y-glycine-Tris dipalmitate WO 00/34281 PCT/AU99/01073 7 MTX-y-GTP3 methotrexate-y-glycine-Tris tripalmitate OMe methyl ester

P

2 0 5 phosphorus pentoxide PmCI palmitoyl chloride 5 PPh3 triphenyl phosphine RM reaction mixture RT room temperature tBu tert Butyl ester tBuOAc tert Butyl acetate 10 TFA trifluoroacetic acid TPPO triphenylphosphine oxide Z-GT benzyloxycarbonyl glycine-Tris Z-GTP 1 benzyloxycarbonyl glycine-Tris monopalmitate Z-GTP2 benzyloxycarbonyl glycine-Tris dipalmitate 15 Z-GTP3 benzyloxycarbonyl glycine-Tris tripalmitate Preparation of methotrexate-y-glycine-Tris (MTX-y-GT) Glycine-Tris (GT) 0 H 2 N -N O H NH( OH H OH 20

C

6

H

14

N

2 0 4 Mol. Wt. 178.19 N-(benzyloxycarbonyl)-glycine-Tris (Z-GT; USP 5,952,499) (1.70 g, 5.44 mmol) 25 was dissolved in ethanol (60 ml) with warming and stirring. After cooling to room temperature by immersion in a cool water bath, palladium-on-carbon (10%, 0.14 g) was added and the system evacuated and filled with hydrogen gas three times. The mixture was stirred vigorously under a hydrogen atmosphere at room temperature for 19 h then filtered through two glass-fibre (GF/A) filter papers (rinsing with fresh ethanol (approx. 30 10 ml)). The filtrate was evaporated leaving GT (0.97 g, 100%) as a white solid 1 H n.m.r. (d 6 -DMSO) 6 2.1, br s, NH 2 ; 3,1,s,NCH 2 CO; 3.55, s, 3xCH 2 0; 5.0, br s, 3xOH; 7.95, br s, NH.

WO 00/34281 PCT/AU99/01073 8 MTX-a-tBu-y-GT 0+ 0 0 HO NH-) N N OH NH 0H OH N 0 NH2N OH NH /N N MTX-a-tBu-y-G T C 30 H 42 N 100 8 Mol. Wt.: 670.72 Methotrexate-a-tert-butyl ester (MTX-c-tBu; USP 5,952,499) (0.438 g, 0.86 5 mmol) was dissolved with stirring, in DMF (4ml). HOSu (0.12 g. 1.03 mmol) was added and stirred until dissolution occurred. DCC (0.27 g. 1.29 mmol) was added and the resulting mixture stirred at room temperature under nitrogen for 16 h. More DCC (100 mg) was added and the mixture stirred for a further hour. GT (0.23 g., 1.29 mmol) in DMF (1.5 ml) was added and the resulting mixture stirred for 54 h. The mixture was 10 filtered through glass wool and the filtrate evaporated. The residue was radially chromatographed on silica gel. Elution with 15% methanol in DCM afforded the title compound (0.33 g. 57%) as a yellow solid. 'H n.m.r. (d 6 -DMSO) 5 1.39, s, C(CH 3

)

3 ; 1.96, m, glu-P1-CH 2 ; 2.24, m, glu-y-CH 2 ; 3.21, s, Nme; 3.52, d, J 6 Hz, 3xCH 2 OH; 3.70, d, J 6 Hz, gly NCH 2 CO; 4.22, m, glu-a-H; 4.69, t, J 6 Hz, 3xOH; 4.79, s, ArCH 2 n 6.62, 15 br s, NH 2 ; 6.82, 7.72 AA'BB', J 9Hz, ArH; 7.14, s, tris NH; 7.46, br s, NH 2 ; 8.10, t, J 6Hz, gly NH; 8.27, d, J 7Hz, glu NH; 8.56, s, H-7, Mass spectrum [electrospray ionization, positive ion mode (ES(+))] m/z 671 (M+1) WO 00/34281 PCT/AU99/01073 9 MTX-y-GT 0 CO 2 H 0 HH OH N H 2 H H 0 H N 0 N N OH NH 2 N N MTX-y- GT

C

26 H 34 N 1008 Mol. Wt.: 614.62 5 MTX-c-t-Bu-y-GT (0.459 g, 0.68 mmol) was dissolved, with stirring, in TFA (3 ml). The solution was stirred at room temperature for 1.5 h and the solvent evaporated under vacuum. The residue was dissolved in water and the solution pH (approx. 2) was adjusted to 6 with aqueous sodium bicarbonate solution (lg in 75 ml). Aqueous HOAc (10%) was added to pH 4. The solution was freeze-dried and the residue purified by 10 flash chromatography then radial chromatography (twice) on silica gel. Elution with 1 3% water in 3:5 methanol:chloroform afforded the title compound (0.086 g, 21%) as a yellow solid. 1H n.m.r. (d 6 -DMSO) 5 1.96, m, glu-3-CH 2 ; 2.14, m, glu-y-CH 2 ; 3.19, s, Nine; 3.5-3.7, m, 3xCH 2 OH + 3xCH 2 OH; 3.95, m, gly NCH 2 CO; 4.41, m, glu-ot-H; 4.76, s, ArCH 2 N; 5.37, br s, NH 2 ; 6.61, br s, NH 2 ; 6.84, 7.62 AA'BB', J 9Hz, ArH; 15 7.60, m, glu NH; 7.71, s, tris NH; 8.23, t, J 6Hz, gly NH; 8.57, s, H-7, ES(+) mass spectrum m/z 615 (M+1), 637 (M+Na), 659 (M+2Na). Preparation of methotrexate-y-glycine-Tris mono, di and tripalmitate (MTX-y GTP1, MTX-y-GTP2, MTX-y-GTP3) 20 The title compounds can be prepared as described in USP 5,952,499 or via the following procedures.

WO 00/34281 PCT/AU99/01073 10 Synthesis of 2,4-Diamino-6-hydroxymethylpteridine (DAIIMP)

NH

2

NH

2 N N N-'OH NH 4 0H H2N OH

H

2 N N N) .HCI H 2 N N" N MW 228.65 MW 192.19 5 Ref: Rosowsky el al, J. Med. Chem., 1985, Vol. 28, No. 5, 660-667. Reactants: DAHMP.hydrochloride (69.04g, 0.30mol) 10 HOAc (120ml) H20 (1200ml) Procedure: DAHMP.HCI was dissolved (with heating to 80oC) in aqueous acetic acid. The 15 solution was filtered through glass wool and cooled to 45oC. The pH was adjusted to 5.3-5.6 (monitored with pH paper) with concentrated ammonium hydroxide solution. RM cooled to RT and filtered. The yellow/caramel coloured solid collected was washed twice with water then dried in a vacuum oven over P205 at 80oC overnight x 2 to afford the desired DAHMP (free base) (52.90g, 92%) as a golden/tan coloured solid. 20 4-[[(2,4-Diamino-6-pteridinyl)methyln]methylamninof-benzoic acid, or 4-amnino-4 deoxy-N 1 0 -methylpteroic acid (APA) CO02H

NH

2

NH

2

CO

2 H H OH (1) PPh 3 /Br 2 N N

H

2 N (2) MABA/ H 2 N EtN'Pr2 MW 192.19 EtN'Pr 2 dihydate MW 361.37 25 Ref: Kralovec el al, J. Med. Chem., 1989, Vol. 32, No. 11, 2426-2431. Rosowsky et al, J. Med. Chem., 1985, Vol. 28, No. 5, 660-667.

WO 00/34281 PCT/AU99/01073 11 Reactants: DAHMP (15.00g, 78mmol) Br2 (3eqv, 12.lml, 37.53g, 0.23mol) PPh3 (3eqv, 61.44g, 0.23mol) 5 DMA (187ml) MABA (13.57g, 89mmol) EtNiPr2 (3eqv, 40.6ml, 30. lg, 0.23mol) Procedure: 10 Br2 was added slowly via syringe to an ice-cooled, stirred mixture of PPh3 in DMA under nitrogen. Solid DAHMP was added (nitrogen "breeze" over RM) and RM stirred at RT for 24h. The amine was added via syringe, followed by solid MABA (nitrogen "breeze"). RM was allowed to stir at RT for 67.5h. RM poured into aqueous sodium hydroxide solution (0.33M, 125ml), the resulting precipitate filtered off and the 15 filtrate adjusted to pH 5.5 with aqueous acetic acid (10% v/v). The precipitate was collected, washed sequentially with water, cold ethanol, and ether, and dried in a vacuum oven at 40 0 C for 4 days to afford a yellow solid (25.79g). To remove the TPPO and MABA contaminants, the crude product was stirred in 3:2 methanol:ethanol (50ml) at RT for 50h, filtered, washed with Et 2 0 (x2), air-dried and evacuated to give APA 20 (24.48g, 87%) as a yellow solid. L-Glutamic Acid- ca-tBu-r-Me ester CO 2 H (1) tBuOAc, HCIO 4

CO

2 tBu

H

2 N CO 2 Me (2) NaHCO 3

H

2 N" CO 2 Me MW 161.16 MW 217.26 25 Ref: Rosowsky et al, J. Med. Chem., 1981, Vol. 24, No. 12, 1450-1455. Reactants: L-Glutamic acid 5-Me ester (17.54g, 0.108mol) 30 tBuOAc (200ml) 70% HCIO4 (10.9ml, 0.12mmol) WO 00/34281 PCT/AU99/01073 12 Procedure: HCIO4 was added slowly to a suspension/partial solution of the substrate in tBuOAc (during acid addition, substrate started to dissolve). RM was stirred in loosely stoppered flask at RT for 40h. RM extracted with 0.5N HCI (2 x 200ml, 1 x 150ml). 5 The aqueous layer was cooled in ice/salt/dry ice bath (> -5oC) and neutralized with solid sodium hydrogen carbonate (until pH approx. 8 and no more NaHCO3 dissolves) with the temperature of the solution being maintained at approx. -4oC. The mixture was extracted with ether (RT, 4 x 200ml), the combined organic layers washed with brine (100ml), dried (MgSO4) and evaporated (rotary evaporator, then oil pump, without 10 heating) to afford the desired product (10.77g, 45%) as an almost colourless, viscous oil which partially solidified on storage at -20'C. MTX- a-tBu- y-Me diester

NH

2 0 CO 2 H N N N -I

H

2 N N N Dihydrate MW 361.37 APA (1) (EtO) 2 POCN (2) CO 2 tBu + iPr 2 NEt

H

2 N CO 2 Me O CO 2 tBu

NH

2 N CO 2 Me N"N N H 1 MW 524.59 15H 2 N NN Ref: (1) Rosowsky et al, J. Med. Chem., 1981, Vol. 24, No. 12, 1450-1455. (2) Kralovec et al, J. Med. Chem., 1989, Vol. 32, No. 11, 2426-2431. 20 Reactants: APA (8.56g, 23.7mmol) EtN'Pr2 (9.0ml, 51.7mmol) DMF (460ml) WO 00/34281 PCT/AU99/01073 13 DECP (7.20g, 44.1rmmol) L-Glutamic Acid a-tBu-y-Me ester (7.26g, 33.4mmol) EtNiPr2 (8.5ml, 48.8mmol) 5 Procedure: APA (granulated) was added portionwise over 10 minutes to a solution of DECP and EtN'Pr2 (first portion) in DMF (400ml). The solution was immersed in an oil bath (pre-heated to 80 0 C) for 2 minutes (internal temp. to 34 0 C). The flask was removed from the bath and stirred for 30 mins (internal temp. 22'C). The second portion of 10 EtN'Pr2 was added followed by glutamate in DMF (50+10ml rinse). RM was stirred at room temperature under nitrogen for 14 h (overnight). A solution of NaHCO3 (1.80g, 21mmol) in water (60ml) was added, the mixture stirred for 30 mins and then evaporated (rotary/oil pump, 45'C). The residue was dissolved in chloroform (approx. 400ml) and the solution washed with water (300ml) and 5% aqueous sodium 15 bicarbonate solution (200ml). The organic layer was washed with brine (30ml), dried (MgSO4) and evaporated. The residue (brown, viscous gum) was filtered through a small silica column with 5% methanol in DCM to remove polar impurities and the resulting product flash chromatographed on silica gel, eluting with 7-12% methanol in DCM to afford the product in three fractions: 1 (trace impurity) (2.17g, 18%); 2 (high 20 purity) (4.40g, 36%); and 3 (trace impurity) (2.77g, 22%) as bright yellow solids. MTX-a-tBu ester (This is the same intermediate as described in USP 5,952,499 - Compound IV) 0 C0O 2 tBu

NH

2 N CO 2 Me N H2N N MW 524.59

H

2 N Ba(OH) 2 .8H 2 0 0 CO 2 tBu

NH

2 N H C02 H N I MW 510.56 25

H

2 N N N WO 00/34281 PCT/AU99/01073 14 Ref: Rosowsky el al, J. Med. Chem., 1981, Vol. 24, No. 12, 1450-1455. Reactants: 5 MTX-c-tBu-y-Me diester (5.00g, 9.5mmol) Ba(OH)2.8H20 (1.82g, 5.8mmol) Ethanol (95ml) Water (110ml) Na2SO4.10H20 (1.93g, 6.0mmol) in water (10ml) 10 Procedure: The first three reactants were mixed and stirred at RT in a loosely-stoppered flask for 19 h. Sodium sulfate in water was added and the mixture stirred for a few minutes (BaSO4 precipitate seems to form immediately) then filtered through 2xGF/A 15 filter papers on a No. 3 or 4 glass sinter. The filtrate was adjusted to pH 5 with 10% HOAc in water. The resultant suspension (gelatinous precipitate) was refrigerated for 30m and then the solid collected by filtration using a No. 1 filter paper on a glass sinter. The collected solid was air-dried overnight (still on the filter paper in the funnel) then evacuated (oil pump) to afford the desired product (4.41g, 91%) as a bright 20 orange/yellow solid.

WO 00/34281 PCT/AU99/01073 15 Z-GTP3 (+ Z-GTP2 + Z-GTPl) 0 H ON OH 0O O ZGT0O H PmCl(2eqv) Et 3 N DMF/CH 2C12 (1:4) O o H OPm H,, OPm 0 NOPm + H' OPm 0~ OPm - 0 OH ZGTP3 ZGTP2 Ok HN OPm N OH OOH +O ZGTP1 Reactants: 5 Z-GT (50.00g, 0.16mol) PmCl (108ml, 97.85g, 0.356mol) Et3N (100ml, 72.4g, 0.72mol) DMF (450ml) DCM (1.60 + 0.20L) 10 Procedure: Z-GT was dissolved in DMF in a 5L, three necked round-bottomed flask. DCM (1.6L) was added and the stirred solution cooled with an ice-salt bath for lh. A solution of PmCl in DCM (100 ml) was added dropwise over 2h and the bath temperature 15 maintained at -13 to -10 0 C The dropping funnel was rinsed with DCM (100 ml). The RM was stirred at the same temperature for a further 2h and then at 4oC over the weekend. The solvent was removed under vacuum (the precipitate that appeared during the evaporation was filtered after the removal of DCM in order to remove DMF efficiently). The residue was extracted with EtOAc (400 ml) then DCM (800 ml) 20 (EtOAc extract resulted in precipitation on washing the extract with brine or water). The combined organic extracts were washed with brine and dried (MgSO4). Solvent WO 00/34281 PCT/AU99/01073 16 removal under vacuum gave the crude product as a yellow wax (133.48g). HPLC analysis indicated a mixture of Z-GTP3, Z-GTP2 and Z-GTP1 in a ratio of 28.8%, 47.4% and 23.8% respectively. 5 MTX-a-tBu-y-GTP3 0 C0 2 tBu NH2 N C02 H NHH N N N H2NJl N NI MW 510.56

H

2 N N N

GTP

3 DCC/DMAP O CO 2 tBu

NH

2 -N

GTP

3 N NO H2NA[.. N NMW 1385.91 Reactants: 10 MTX-ca-tBu ester (1 1.00g, 21.5mmol) DCC (1.2eqv, 5.50g, 26.7mmol) GTP3 (1.1eqv, 21.32g, 23.9mmol) DMAP (0. leqv, 258mg, 2. lmmol) DMF (86ml) 15 DCM (340mi) Procedure: MTX-oc-tBu ester was dissolved, with stirring, in DMF. DCM (200ml) was added. DCC was added and the solution stirred for 20 mins. GTP3 in DCM (70ml) was 20 added dropwise over 10 mins. DMAP was added and the RM stirred at RT under nitrogen overnight (24 h). The RM was filtered to remove white solid, urea by-product from DCC and concentrated (rotary/oil pump, 50 0 C) to remove DMF. The residue was dissolved in DCM, refiltered and evaporated. The residue was dissolved in EtOAc/DCM (150/20ml), washed with brine, dried (MgSO4) and evaporated. The residue was 25 divided into halves and each was flash chromatographed on silica gel, eluting the first WO 00/34281 PCT/AU99/01073 17 half with 5% methanol in DCM and the second half with 3.7-4.4% methanol in DCM. Re-chromatography of the impure fractions afforded the product in two fractions (bright yellow solids), (1) (>95%purity) (17.18g, 58%) and (2) (90-95% purity) (4.83g, 16%). Further elution with 8-10% methanol afforded a third fraction containing a bright yellow 5 solid by-product. MIX- r-GTP3 0 CO 2 tBu

NH

2 NGTP 3 N N O H2I MW 1385.91

H

2 N NN TFA ~ O CO 2 H

NH

2 N GTP 3 N iH0 NN O HI( MW 1329.81

H

2 N N N 10 Reactants: MTX-o-tBu-y-GTP3 (0.86g, 0.62mmol) TFA (1.7ml, 22mmol) DCM (5ml) 15 Procedure: TFA was added dropwise to an ice-cooled, stirred solution of the substrate in DCM. The RM was allowed to warm to RT then stirred under nitrogen for 5.5 h. The RM was placed at 4oC overnight then diluted with DCM (25ml) and washed with 20 aqueous, sodium bicarbonate solution (5% w/v, 35ml) (check that pH>7). HOAc (glacial, 1.8ml) was added and mixture shaken (check that pH-4). The organic layer was washed with water (2x25ml) (the emulsion was broken by addition of a little NaCI & methanol & fresh solvents, swirling and warming), dried (MgSO4) and evaporated. The residue was flash chromatographed on silica gel, eluting with an 8-30% step gradient WO 00/34281 PCT/AU99/01073 18 with methanol in DCM (3X4%; 2X5% steps) to afford the product in three fractions: (1) (trace higher Rf by-product, 187mg), (2) (higher purity, 476mg) and (3) (trace lower Rf by-product, 79mg). All fractions were yellow solids and of quite high purity. Fraction (2) -> 97.25% purity (by HPLC). Yield = 742mg, 90%. 5 MTX-y-GTP 1 and MTX-y-GTP2 can be prepared in a similar manner from Z GTP I & 2 respectively. In vitro DHFR Inhibition Dihydrofolate reductase (DHFR) is a target enzyme of MTX. It catalyses the 10 following reaction: DHFR Dihydrofolate + NADPH + H+ -> Tetrahydrofolate + NADP + Its activity can be measured in vitro by following the rate of NADPH oxidation 15 at 340nm. Method: The inhibition of DHFR by MTX, MTX-y-GT and MTX-y-GTP I was measured as described by Imbert AM, Pignon T, Lena N (1983) "Enzymatic assay for 20 methotrexate with a centrifugal analyser (Cobas-Bio)" Clin Chem, 29(6):1317-1318. Results: MTX-y-GT inhibited DHFR in the same concentration range as MTX (Table 1). 25 Table 1. Inhibitory activity of MTX and MTX-y-GT on DHFR at 2.86 nM Compound % inhibition MTX 45.5 MTX-y-GT 36.4 The inhibitory activity of MTX-y-GTP1 was also tested. The concentration required to cause a 50% inhibition of DHFR was 76nM. This is 16X less active than 30 MTX which causes 50% inhibition of DHFR at 4.8nM. MTX-ct-GTP 1 did not inhibit DHFR under these conditions. MTX-y-GTP 2 and MTX-y-GTP 3 were not tested as their solubility characteristics are incompatible with the assay system.

WO 00/34281 PCT/AU99/01073 19 Investigation of MTX-y-GT, MTX-y-GTP1 and MTX-y-GTP3 using the delayed type hypersensitivity (DTH) response in the rat Delayed type hypersensitivity (DTH) is an immune response which can be used 5 to assess the action of various therapeutic agents. It can be demonstrated by sensitising an individual to a compound, and then on challenge with the same compound, observing the development of oedema at the site of challenge. MTX has an immunosuppressive effect which reduces oedema, and this reduction in DTH response has been used to assess one aspect of the efficacy of MTX-y-GT, MTX-y-GTP 1 and MTX-y-GTP3. 10 Methods: Copenhagen (COP) rats had their abdomens shaved and 100 pl of 0.5% dinitroflurobenzene (DNFB) in acetone was applied daily for 3 days. Concurrently, the rats were administered orally either, soy oil, MTX (0.5 mg) or equimolar amounts of 15 MTX-y-GT, MTX-y-GTP1 or MTX-y-GTP3 daily for 5 days. On day 7, rats were anaesthetised with methoxyflurane and the footpad volume measured immediately prior to the addition of 25 pl of 0.2% DNFB in acetone to the left footpad. Under anaesthesia, footpad volume was measured at 23h. Plethysmography (the measurement of the volume of an object by observed changes in volume after immersion), using a 20 laboratory fabricated oncometer, was used to determine the volume of the footpad. The nett oedema was calculated by subtracting the volume of the foot at time 0 from time 23h. Drugs: 25 MTX (lmg/ml) in Soya oil MTX-y-GT - (1.4 mgs/ml) in soya oil - (Batch QY5-64MXS) MTX-y-GTP1 - (1.88 mg/ml) in soya oil - (Batch GB4-99 NB00077-p50) MTX-y-GTP3 - (3 mg/ml) in soya oil - (Batch QY 4-41 FP) Control - soya oil 30 Results: The nett footpad oedema data is presented in Figure 1. The mean nett footpad oedema was significantly less in those rats treated with MTX, MTX-y-GTP 1 and MTX-y-GT, but not MTX-y-GTP3 when compared with 35 control rats (Table 2). As well, the nett footpad oedema from the MTX-y-GTPl -treated rats was significantly less (p = 0.021) than that in the MTX-treated rats WO 00/34281 PCT/AU99/01073 20 Table 2: Mean, SD and p values of data Treatment n Mean SD p value when oedema compared to (ml) _Controls Control 38 0.363 0.254 MTX 8 0.216 0.150 0.044* MTX-y-GTP3 22 0.327 0.325 0.59 MTX-y-GT 18 0.192 0.237 0.0195* MTX-y-GTP1 11 0.055 0.050 <0.0001* * statistically significant 5 Discussion: DTH is an in vivo T cell-dependent immune response manifested as an inflammatory reaction after antigenic challenge. Thus these results indicate the immunosuppressive effects of MTX-y-GT and MTX-y-GTP1 are at least equal to or perhaps more effective than MTX. 10 In vitro Cytotoxicity The ability of MTX and its Tris-conjugates to inhibit proliferation of cultured cells were assessed. 15 Methods: The effect of MTX and MTX-Tris-conjugates on cell proliferation was studied using routine cell culture procedures. The following cell lines were tested: JURKAT (human acute T-cell leukaemia) Swiss mouse fibroblast (3T3) cells 20 CCRF-CEM (CEM) human T-cell leukaemia JURKAT cells were cultured in the presence of drug for 64 h whilst 3T3 and CEM cells were cultured for 48h and 96h respectively in the presence of compound. All studies were performed in 96-well plates and control wells contained no drug. Cell proliferation was then measured using the MTS or MTT detection: (D Marks, L Belov, 25 MW Davey, RA Davey, A Kidman. Leukemia Research 16, 1165-1173. "The MTT cell viability assay for cytotoxicity testing in multidrug-resistant human leukemic cells." WO 00/34281 PCT/AU99/01073 21 Results: All conjugates inhibited proliferation but were less toxic to all cell types tested than the parent compound, MTX. Results are summarised in the Table 3. 5 Table 3. Effect of MTX and MTX-Tris conjugates on the proliferation of various cell types. ICso = concentration of compound at which cell growth is inhibited by 50% of control growth. Compomund ICo 0 jiM 3T3 JURKAT CEM MTX 0.0525 8.2 0.019 MTX-a-GT - - 35.9 (1889) MTX-y-GT no effect* - 0.57 (30) MTX-y-GTP1 - - 0.21 (11) MTX-a-GTP2 - 10300 (1256) MTX-y-GTP2 0.41 (7.8) 1200 (146) 0.40 (21) - not tested; * no effect observed up to 250 nM; (n) Conjugate:MTX ratio. 10 Reduced activity is likely to be due to the modification of MTX on the aX- or y COOH. In vivo MTX is polyglutamylated (addition of (glu)n) on the y-COOH and this is an important aspect of MTX toxicity. It increases intracellular retention and consequently results in a sustained block in tetrahydrofolate synthesis via DHFR. MTX y-(glu)n, also have greater inhibitory effects on other enzymes involved in DNA and RNA 15 synthesis including thymidylate synthetase and AICAR transformylase. Polyglutamate formation and accumulation may have unwanted effects in vivo as they form in significant amounts in some organs (kidney and liver) where they may give rise to unwanted toxicity due to prolonged intracellular retention. Some of our conjugates (ie MTX-y-GT, MTX-y-GTP1 and MTX-y-GTP3) are 20 modified on the y-COOH and are unlikely to be polyglutamylated thus explaining their reduced toxicity. The y-COOH is also involved in cellular uptake and modification may reduce uptake. In our study, the various MTX-y-conjugates display differences in their effect on cell growth. MTX-y-GT is less inhibitory than MTX-y-GTP1 or 2. It could be 25 postulated that whilst addition of GT impedes the drug uptake (and therefore toxicity) via the folate transport system, this effect may, in part, be over ridden by the addition of palmitate groups. It is likely that palmitate-conjugates enter the cell by endocytosis. Whilst MTX-y-GT is less toxic to cells than the palmitoylated conjugates, it is more WO 00/34281 PCT/AU99/01073 22 inhibitory to DHFR in a biochemical assay (see above). This suggests that it is the transport mechanism rather than direct enzyme inhibition that differentiates between MTX-y-GT and MTX-y-GTP1 or 2. It is widely reported that modification to the oc-COOH reduces cell uptake to a 5 greater extent than modification to the -y-COOH. Our results are consistent with this. Both MTX-a-GT and MTX-GTP2 are much less toxic than MTX-y-GT and MTX-y GTP2, respectively. Overall our results are consistent with literature reports. Some relevant references are listed: 10 Rosowsky et al.; J. Med. Chem. (1984) 27, 600-604 Rosowsky et al. J. Med. Chem. (1984) 27, 605-609 Rosowsky et al., J. Med. Chem. (1981) 24, 1450-1455 Fan et al. (1991) Biochem. 30, 4573-4580. 15 Testing of MTX and MTX-y-GT in the rat adjuvant-induced arthritis model. Doses of 40, 15 and 5tmol/kg of MTX have been shown by Baggott and co workers (Arthritis Rheum 1998, 41:1407-10) to give dose-dependent inhibition of adjuvant induced arthritis in Lewis rats. A similar model using Dark Agouti (DA) rats has been used to test the effect of 20 MTX-y-GT. Method: DA rats were injected subcutaneously with Freund's Complete Adjuvant into the tail base. Arthritis developed 10-12 days later in untreated rats. Rats were treated with 25 PBS (control) or molar equivalent doses of MTX or MTX-y-GT in PBS orally (by gavage) once at day 6; six rats per treatment group. The effect of treatment on arthritis was monitored daily by assessing body weight and evidence of arthritis, which was quantified using an established scoring system. This involves giving a score from 0 to 4 for each paw (0= no evidence of arthritis or inflammation, 4= global swelling of the 30 paw). The theoretical maximum score was 16 but rats were euthanased if a score greater than 12 was observed. In treated rats, in which mild arthritis occurred, the disease was followed beyond the treatment period to determine whether arthritis was delayed rather than suppressed or prevented. 35 WO 00/34281 PCT/AU99/01073 23 Results: The results of a single dose of MTX and MTX-y-GT at day 6 are shown in Figures 1: A to F 5 Conclusions: MTX and MTX-y-GT were effective in delaying the onset of inflammation by 2 4 days. At the doses tested and within experimental error the effects of the two compounds were indistinguishable. From the DTH rat footpad results it would be expected that MTX-y-GTP 1 might also delay the onset of inflammation in this model. 10 MTX-y-GTP3 was negative in the DTH assay but shows activity in the human SPA trial in the treatment of psoriasis (see below). Its effect on adjuvant-induced arthritis in rats remains to be determined. Toxicity of MTX-Tris Conjugates 15 Whilst no experiment has been performed that specifically examines toxicity, observations have been made during experiments examining biological activity which indicate that MTX-TRIS conjugates have reduced systemic toxicity compared to MTX. The following observations have been noted: 20 1. Experiments examining the effect of daily topical applications over three consecutive days of vehicle, MTX, MTX-y-GTP2 or MTX-y-GTP3 on UVB-induced erythema in female Skh-1 mice have been performed. During this study mice were assessed for toxic signs (dehydration, diarrhoea and anorexia) for 6 days 25 followed by assessment at necropsy for gross signs of GIT toxicity (enlarged fluid-filled intestines). MTX-y-GTP2 and MTX-y-GTP3 appeared much less toxic than MTX and results are shown in the following table.

WO 00/34281 PCT/AU99/01073 24 Toxicity results from topical application to Skh-1 hairless mice TREATMENT SIGNS OF DEATH GROSS GIT TOXICITY vehicle 0/24 0/24 MTX 4 mg/kg 1 5/24 8/24 MTX-y-GTP2 9.6 mg/kg' 0/24 0/24 MTX-y-GTP3 11.7 mg/kg' 0/14 0/14 5 2Equimolar amounts of MTX, MTX-y-GTP2 and MTX-y-GTP3 were used. They were formulated in oil/water cream:ethyl oleate (4:1) and 0. 1ml was applied to each mouse. GIT = gastrointestinal tract. In many mice treated with MTX, clinical signs of toxicity and at necropsy, 10 enlarged fluid-filled intestines were prevalent. Histological examination of these intestines showed marked microvilli damage and necrotic mucosal cells. In comparison, neither GIT damage nor mortality were observed in the mice receiving MTX-y-GTP2 or MTX-y-GTP3. In all mice, the liver and kidneys appeared normal. 15 2. In Delayed Type Hypersensitivity assays studies, Copenhagen rats were administered orally either soy oil, MTX (0.5 mg) or equimolar amounts of MTX-y-GT, MTX-y-GTP 1 or MTX-y-GTP3 daily for 5 days, whilst being concurrently sensitised by topical application of DNFB. Following challenge with DNFB and necropsy 24h later, the intestines from a representative group of rats were examined for gross changes and 20 samples collected for histology. All MTX-treated rats (n=7) had an enlarged stomach and intestines filled with milky fluid. Histology of these GIT samples revealed villi with thickened, inflammed cores, separation and sloughing of the epithelium and mucosal necrosis. This is indicative of mild to severe damage. Rats receiving MTX-y-GT (n=9) had a normal GIT 25 with the exception of one animal which had an enlarged fluid-filled large intestine. Macroscopic examination of the GIT from MTX-y-GTP 1 (n

=

14) and MTX-y-GTP3 WO 00/34281 PCT/AU99/01073 25 (n=14) treatment groups revealed no gross abnormalities. Reduced histopathology was displayed in the GIT from rats receiving MTX-Tris conjugates. 3. In studies examining the effects of MTX and MTX-y-GT on adjuvant-induced 5 arthritis in rats, the following observations were noted: a) Following a single oral dose of MTX or MTX-y-GT (40 umole/kg), diarrhoea was observed 1-2 days later in the MTX group. This was not evident in the MTX-y-GT and vehicle only groups. b) In a second experiment, rats were given 3 oral doses, 3 days apart. Toxic side effects 10 (ruffled fur and ataxia) were evident in rats treated with MTX (5 umole/kg). Consequently, these animals were euthanased earlier than originally planned. Post mortem showed marked changes in the GIT, particularly the small intestine which appeared bloated and contracted. Histology showed marked crypt hyperplasia and loss of villous structure in the mucosa of the jejunum and the ileum. In some areas 15 there was loss of both crypt and villous epithelium. The mesenteric lymph nodes of these rats were also enlarged. None of these effects was seen in the vehicle- and MTX-y-GT-treated groups. In addition, peripheral blood samples from these MTX treated rats showed profound leucopaenia (approx. 2 x 10 9 /L), due largely to neutropaenia. Leucocyte counts in the vehicle control and MTX-y-GT groups were 20 not different (approx. 30 x 10 9 /L). Conclusions: All MTX-Tris conjugates have reduced toxicity compared with MTX. Similar results were observed with cytotoxicity experiments (see above). 25 Distribution of MTX-Tris Conjugates in Mice Introduction: MTX is an effective oral treatment for a disease with an autoimmune component such as psoriasis and rheumatoid arthritis. However its long-term use requires ongoing 30 liver assessment due to the risk of hepatic atrophy, necrosis and cirrhosis. MTX also induces gastrointestinal irritation, renal toxicity and abortion. We have demonstrated that MTX-Tris conjugates are biologically active but are less toxic in vitro and in vivo (see above). In addition, the distribution of radiolabelled MTX-Tris conjugates as well as MTX, administered topically, orally (p/o) and intravenously (i/v) have been examined 35 in mice using liquid scintillation and whole body autoradiography. This was to determine if MTX-Tris conjugates (or their breakdown products) accumulate in organs of concern WO 00/34281 PCT/AU99/01073 26 for MTX toxicity and if their excretion patterns varied from those of MTX. Pertinent results are summarised below. Results: 5 1. Mice given [ 1 4 C]-MTX-y-GT, both i/v and orally, demonstrated a higher level of radioactivity excreted in the faeces, but similar amounts in the urine compared with animals given [14C]-MTX. The tissue distribution of [1 4 C] was similar, but at lower levels in mice given [14C]-MTX-y-GT. These findings suggest that MTX-y-GT has greater hepatic clearance than MTX 10 2. Following topical application, radiolabel from MTX-y-GTP3 was detected in the skin epidermis for longer periods than MTX treated mice. This suggests that MTX y-GTP3 is retained in the skin to a greater extent than MTX. In addition, radioactivity is present in the skin from mice given oral [1 4 C]-MTX-y-GTP 1 and

[

3 H]-MTX-y-GTP3. These findings may have important implications regarding their 15 potential use as anti-psoriatic treatments. 3. Following oral administration, both radiolabelled MTX-y-GTP 1 and MTX-y-GTP3 show significantly reduced levels of counts in organs of concern for MTX toxicity. This suggests either decreased absorption or increased hepatic excretion (ie enterohepatic circulation). However once these conjugates enter the circulation (ie 20 following i/v administration) they appear to accumulate in the liver, spleen and lung. This offers the potential to targeting drugs to specific organs following their conjugation to fatty acyl groups via Tris. 4. Radiolabel from [14C]MTX is mainly excreted in the urine and to a lesser extent in the faeces. In contrast, when [1 4 C]MTX-y-GTP 1 and [ 1 4 C]MTX-y-GTP3 are 25 delivered i/v, radiolabel is mainly eliminated in the faeces via hepatic excretion, with very low levels detected in the urine. Discussion: MTX-Tris conjugates, when given orally, accumulate to a lesser extent than 30 MTX in organs of concern for MTX toxicity because they either have greater hepatic clearance (MTX-y-GT) or low levels of absorption in the formulations tested (MTX-y GTP 1 and MTX-y-GTP3). The distribution patterns may partially explain the reduced toxic side effects seen with the conjugates (see Toxicity section above). Reduced cellular uptake and polyglutamylation is also likely to contribute (see Cytotoxicity 35 Section above). Whilst MTX-y-GTP 1 and MTX-y-GTP3 may accumulate in the liver, lung and spleen when administered i/v, we have not assessed toxic side effects. However WO 00/34281 PCT/AU99/01073 27 it is unlikely that these conjugates are polyglutamylated, which is the mechanism widely attributed to MTX toxicity. Both MTX-y-GTP 1 and MTX-y-GTP3 were found in the skin following oral delivery. This may have implications for their potential use as a therapeutic for psoriasis. 5 Clinical trial of MTX-y-GTP3 as a topical treatment for psoriasis Psoriasis is a skin condition that affects 1-2% of people of European origin. It is an inflammatory and epidermal hyperproliferative disorder of unknown aetiology with a poor prognosis. 10 MTX, when taken orally, is an effective treatment for moderate to severe psoriasis. However its use is associated with immediate gastrointestinal problems and long-term liver toxicity. Approximately 6 weeks of treatment are needed before a clinical response. It has been postulated that topically-applied MTX would provide drug at the 15 affected site and have fewer side-effects. This has been trialed (Weinstein et al., 1989) but overall the results were not promising, perhaps due to poor skin retention. From our observations it was considered likely that MTX-y-GTP3 would have increased skin retention as well as the biological activities of MTX. 20 Preclinical studies Preclinical studies indicated that topically-applied MTX-y-GTP3 had biological activity that may make it suitable for the treatment of psoriasis. It had equivalent or better activity to MTX in reducing UVB-induced epidermal hyperproliferation in hairless mice (Figure 3) and suppressed UVB-induced epidermal DNA synthesis (Figure 4) as 25 determined by the method of Molek et al.(Brit J Derm(1983) 108:25-31). Intradermal injection of MTX-y-GTP2 was more effective than MTX in reducing the growth of B 16 melanomas (USP 5,952,499). Distribution studies (above) show that topically-applied MTX-y-GTP3 is retained in the epidermis of hairless mice for longer periods than MTX. MTX-y-GTP3 administered topically or orally is less toxic to hairless mice than MTX 30 (See above). These positive pre-clinical results and the lack of a good animal model for psoriasis led to a proof-of-concept trial of topical MTX-y-GTP3 in humans. Clinical study design 35 The Small Plaque Assay (SPA) assesses the anti-psoriatic potential of compounds over a two week time course. The assay simultaneously tests 6 compounds WO 00/34281 PCT/AU99/01073 28 on a single plaque under occlusion. The position of the test compounds on each plaque was randomised to eliminate evaluation bias and any drug interaction effects. Eleven patients were studied. 5 Treatments: 0.5% MTX-y-GTP3 solution (test formulation) 1.0% MTX-y-GTP3 solution (test formulation) MTX-y-GTP3 vehicle (negative control) 0.5% MTX solution (reference formulation) 10 MTX vehicle (negative control) 0.05% betamethasone valerate (positive control) (All concentrations of MTX-y-GTP3 solutions are with respect to their MTX content). Schedule Day Tratment Assessment .. ...... ...... ....................... ....... ...................................................... I ............. t.. ............................................................. 0 2% salicylic acid clinical & histology 1 drugs applied 3 drugs applied 6 drugs applied 8 drugs applied clinical 10 drugs applied 13 drugs applied 15 clinical & histology 29 clinical 15 Parameters Approximately 6 weeks of oral MTX treatment are needed before a clinical response is evident. Therefore it was unlikely that a clinical result would be seen with topical MTX-y-GTP3 in the two week duration of the SPA (restricted for ethical 20 reasons). However, early histological changes consistent with psoriasis resolution would be expected with an active agent. Therefore, as well as clinical assessment, a range of histopathological and immunohistological markers were assessed as follows: WO 00/34281 PCT/AU99/01073 29 * Clinical Infiltration and Erythema on days 0, 8, 15 and 29. * Histopathology 5 Parakeratosis Inflammatory infiltrate Granularity Acanthosis Vascular changes Epidermal thickness Papillomatosis 10 * Immiunohistology Involucrin ICAM- 1 Filaggrin Ki-67' keratinocytes CD4 and CD8+ T-cells CD4 and CD8 T-cells WO 00/34281 PCT/AU99/01073 30 Patient Selection Inclusion Criteria Exclusion Criteria Male of any ethnic group, 18 to 70 years of Pustular psoriasis, erythrodermic psoriasis, age. Females of non-reproductive status inverse psoriasis and/or active psoriasis also allowed, arthritis. Serious intercurrent illness or active Chronic plaque type psoriasis vulgaris for at infections requiring treatment, including least 6 months. hepatitis or AIDS. Known to be HIV positive. Capable of understanding the purposes and Known hypersensitivity to trial medication risks of the trial, giving the impression that components. she/he is willing to partake in all History of and/or present systemic assessments for the whole duration and has malignancy. given written informed consent. Simultaneous participation in any other investigational drug trial or receiving Required plaque characteristics: investigational drug 4 weeks prior to start SOn trunk or extremities; not in a flexure of trial. area. Received any systemic anti-psoriatic SArea at least 5.5 x 8.3 cm. therapy 4 weeks prior to start of trial. SHomogeneous appearance (erythema Received any topical anti-psoriatic therapy and infiltration), for the plaque tested two weeks prior to SSevere erythema & infiltration start of trial. Laboratory values that contra-indicate participation in the trial. Any form of substance abuse, psychiatric disorder or condition which may invalidate communication with the investigator. Known to have cirrhosis of the liver or other liver disease. Patients taking medications which may interact with MTX. Patients who have a calculated creatinine clearance < 30 ml/min.

WO 00/34281 PCT/AU99/01073 31 Results: Clinical The positive control, betamethasone, significantly improved the clinical appearance of test areas indicating that the trial methodology was preforming correctly. 5 Little or no change was observed in the MTX-y-GTP3 or MTX test spot areas over the two week period. This was not unexpected as the time course required for anti-psoriatic activity of oral MTX is 6 weeks. No serious side effects or local irritation were observed with any test compounds. 10 Histological Statistically, betamethasone demonstrated anti-psoriatic activity in all parameters except hyperkeratosis and involucrin. MTX-y-GTP3 treatment had positive effects on filaggrin expression (a marker of terminal epidermal differentiation), granularity (granular layer is absent or incomplete in 15 psoriasis) and parakeratosis (imperfect formation of epidermal horn cells) in five out of eleven patients. * Filaggrin. Of all the immunohistological markers examined, filaggrin proved to be the most powerful indicator of the action of the test drugs. Examination of filaggrin results 20 within patients showed a correlation between response to betamethasone and response to MTX-y-GTP3 solution (Figure 5). MTX-y-GTP3 solution showed the best filaggrin response at a concentration of 0.5% while 1% MTX-y-GTP3 and 0.5% MTX gave approximately equivalent results. As a group, there were differences in filaggrin expression in the stratum granulosum with 0.5% MTX-y 25 GTP3 solution compared to vehicle. There was a similar response to MTX (Figure 6). * Granularity:Parakeratosis Ratio. The granularity to parakeratosis ratio was calculated to give an index of therapy - an increase in the ratio indicates a trend towards psoriasis resolution. Histopathological 30 changes in granularity and parakeratosis consistent with early resolution of psoriasis were evident with 0.05% betamethasone valerate cream (10 out of 11 patients) and 1.0% MTX-y-GTP3 solution (5 out of 11 patients) - Figure 7 a) and b). Treatment with 0.5% MTX-y-GTP3 and MTX resulted in little response in granularity:parakeratosis ratio (1 & 2 patients respectively). 35 WO 00/34281 PCT/AU99/01073 32 Clinical study conclusions MTX-y-GTP3 demonstrated anti-psoriatic effects in a number of patients in comparison to its vehicle as judged by histopathology (granularity and parakeratosis) 5 and the keratinocyte maturation marker, filaggrin. This, coupled with its relatively low toxicity compared to MTX makes it a potential therapeutic for the topical treatment of recalcitrant psoriasis. 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 10 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 (20)

1. A compound having the formula I: A I M-[Y]n-NH-C-CX2-O-R 1 B 5 in which M is methotrexate or an analogue thereof; A=H, CX 2 -O-R 2 or halogen; B=H, CX 2 -O-R 3 or halogen; X is independently H or halogen; n is 0 or greater than or equal to 1; 10 Y is a linker group, and where n is greater than 1, each Y is the same or different; R 1 ,R 2 , and R 3 are the same or different and are either hydrogen, substituted or unsubstituted methyl, ethyl, a saturated or unsaturated fatty acyl group, with the proviso that when n is 0 or 1, and A is CH 2 -O-R 2 and/or B is CH 2 -O-R 3 , then R 1 , R 2 and R 3 are not selected from a fatty acyl group; 15 with the proviso that when n is greater than or equal to 2, and A is CH 2 -O-R 2 and/or B is CH 2 -O-R 3 , and at least one of RI, R 2 and R 3 is a fatty acyl group, then -[Y],,- is other than -(AA)n- or -Y-(AA)n. 1 -, where AA is an amino acid.

2. A compound according to claim 1 wherein M is attached via its y carboxyl group. 20

3. A compound according to claim 1 wherein Y is selected from the group consisting of a) a compound having a carboxyl group and an amino group; b) a compound having an amino group and a sulphonic acid group; 25 c) a compound having an hydroxyl group and a carboxyl group; d) a compound having an hydroxyl group and a sulfonic acid group; e) a compound having an hydroxyl group and a reactive halide group; f) a compound having a halide group and a carboxyl group; g) a compound having two reactive halide groups; 30 h) a compound having an hydroxyl group and an aldehyde group; and i) an alkylene oxide. WO 00/34281 PCT/AU99/01073 34

4. A compound as claimed in any one of the preceding claims wherein -[Y]n- is [AA],n-, where the or each AA is an amino acid.

5. A compound according to any one of claim 4 wherein n is 1 and AA is glycine. 5

6 A compound according to claim 4 wherein n is 2 and AA is glycine.

7. A compound according to any one of the preceding claims wherein A and B are CH 2 OH and R 1 is H. 10

8. Methotrexate-y-glycine Tris.

9. Methotrexate-y-glycine glycine Tris. 15

10. A pharmaceutical composition including a compound according to any one of the preceding claims and a pharmaceutically acceptable carrier or diluent.

11. A pharmaceutical composition according to claim 10 wherein the composition is in a form suitable oral administration. 20

12. A pharmaceutical composition according to claim 10 wherein the composition is in a form suitable for parenteral administration.

13. A method of treatment of a disease with an autoimmune component or a cancer 25 in a subject, the method including administration to the subject of an effective amount of a compound in accordance with of formula 1: A I M-[Y]n-NH-C-CX2-O-RI I B in which M is methotrexate or an analogue thereof; 30 A=H, CX 2 -O-R 2 or halogen; B=H, CX 2 -O-R 3 or halogen; X is independently H or halogen; n is 0 or greater than or equal to 1; WO 00/34281 PCT/AU99/01073 35 Y is a linker group, and where n is greater than 1, each Y is the same or different; RI,R 2 , and R3 are the same or different and are either hydrogen, substituted or unsubstituted methyl, ethyl, a saturated or unsaturated fatty acyl group, with the proviso that when n is 0 or 1, and A is CH 2 -O-R 2 and/or B is CH 2 -O-R 3 , 5 then RI, R2 and R3 are not selected from a fatty acyl group; with the proviso that when n is greater than or equal to 2, and A is CH 2 -O-R 2 and/or B is CH 2 -O-R 3 , and at least one of R 1 , R2 and R 3 is a fatty acyl group, then -[Y]n- is other than -(AA)n- or -Y-(AA)n- 1 -, where AA is an amino acid. 10

14. A method according to claim 13 wherein M is attached via its y carboxyl group.

15. A method according to claim 13 or claim 14 wherein the treatment is of a disease with an autoinunune component. 15

16. A method according to any one of claims 13 to 15 wherein the disease with an autoinumune component is selected from the group consisting of psoriasis, rheumatoid arthritis, inflammatory bowel disease and Crohn's disease.

17. A method according to claim 16 wherein the disease is psoriasis. 20

18. A method according to claim 16 wherein the disease is rheumatoid arthritis.

19. A method according to any one of claims 13 to 18 wherein the compound of formula I is administered by oral or parenteral administration. 25

20. A method according to any one of claims 13 to 19 wherein the compound of formula I is methotrexate-y-glycine-Tris.