CA2353839A1 - Methotrexate derivatives - Google Patents

Abstract

Methotrexate conjugates of formula (I) are disclosed as well as their use in the treatment of a disease with an autoimmune component and cancers. A
particular example of a compound of formula (I) is methotrexate-.gamma.-glycine Tris, in which M is methotrexate or an analogue thereof; A=H, CX2-O-R2 or halogen; B=H, CX2-O-R3 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; R1, R2, 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 CH2-O-R2 and/or B is CH2-O-R3, then R1, 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 CH2-O-R2 and/or B is CH2-O-R3, and at least one of R1, R2 and R3 is a fatty acyl group, then -[Y]n- is other than -(AA)n- or -Y-(AA)n-1-, where AA is an amino acid.

Description

WO 00/34281 PC'T/AU99/OI073 Methotrexate derivatives TECHNICAL FIELD
The present invention is concerned with derivatives of methotrexate (MTX);
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 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 25 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 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.
DISCLOSURE OF THE INVENTION
In a first aspect, the present invention provides A
M-[Y]ri NH-C-CX2-O-R~
B
in which M is methotrexate or an analogue thereof;
A H, CXz-O-R2 or halogen;
B=H, CXz-O-R3 or halogen;
X is independently H or halogen;
n is 0 or greater than or equal to l;
Y is a linker group, and where n is greater than i, each Y is the same or different;

R1,R2, and R3 are the same ar 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 l, and A is CH2-O-R2 and/or B is CH2-O-R3, then R,, RZ and Rz are not selected from a fatty acyl group;
with the proviso that when n is greater than or equal to 2, and A is CH2-O-R2 and/or B is CH2-O-Rz, and at least one of R,, R2 and R3 is a fatty acyl group, then -[Y)" is other than -(AA)" or -Y-(AA)"_x-, where AA is an amino acid.
Examples of analogues of methotrexate are described in the prior art. See, for 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 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)CXZOR, group, for example, an amino acid or antibiotic.
b) a linker with an amino group to M and a sulphonic acid group to the NHC(A){B)CXZOR, 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)CXzOR, 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 hydroxy group. Examples of hydroxy acids include glycolic acid (HOCH2COOH);
lactic acid (HOCH(CH3)COOH; 4-hydroxy-butyric acid HOCHZCH2CHZCOOH; 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.
d) a linker with an hydroxyl group to M and a sulphonic acid group to the NHC(A){B)CXzOR, 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)CXZOR, group, for example, 2~chloroethanol.
~ a linker having an hydroxy group and an aidehyde group, for example, p-hydroxybenzaldehyde, g} a linker having a halide group and an carboxyl group, for example, 2-chloraacetic acid;
h) a linker having two reactive halide groups, for example, 1,2-dibromoethane;
and i) an alkylene oxide, for example, ethyleneoxide.
The NHC(A}(B}CX20R1 group may be attached to the a or y carboxyl group of the methotrexate compound. Most preferably, the -NHC(A)(B)CX20RI 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 and non-natural amino acids and analogues thereof. Preferably the amino acid is glycine.
Preferably Rl, R2 and R3 are each H.
Preferably n is in the range 0-S.
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, 25 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 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.
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-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, tampons, creams, gels, pastes, lotions, implants, patches or foams.
The composition of the invention may be in the form of an implant.

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 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.
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
M-~ri ~-C~C~-O-Rs E
wherein:
D=H or CXZ-O-R4 or halogen;
E=H or CX2-O-RS or halogen;
X is as defined above;
n is as defined above;
Y as defined above; and R4, RS and R~ are the same or. different and either hydrogen, substituted or unsubstituted methyl, ethyl or fatty acyl groups with either saturated or unsaturated bonds.
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.
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, parenterai, implant. and topical.

WO 001342$1 PCT/AU99/01073 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 range of about 0.00 i 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-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 oedema. MTX-'y-GT and MTX-y-GTP1 are both active in this assay as is MTX. MTX-'y-GTP 1 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 of joint inflammation in this model.
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.
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 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-GTP3 suggests that there are two main groups in the trial: responders and non-responders.

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 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.
MODES FOR CARRYING OUT THE INVENTION
Abbreviations:
AICAR phosphoribosylaminoimidazolecarboxamide APA 4-amino-4-deoxy-N 10-methyipteroic acid DAHMP 2,4-Diamino-6-hydroxymethylpteridine DCC 1,3-dicyclohexylcarbodiimide DCM dichloromethane DECP diethyl cyanophosphonate DMA N,N-dimethyiacetamide DMAP 4-dimethylaminopyridine DMF N,N-dimethyl formamide Et20 diethylether EtN'Pr2 di-isopropyl ethyl amine EtOAc ethyl acetate GT glycine-Tris GTP 1 glycine-Tris monopalmitate GTP2 glycine-Tris dipahnitate GTP3 glycine-Tris tripalmitate HOAc acetic acid HOSu N-hydroxysuccinimide MABA N-methyl aminobenzoic acid MTX methotrexate MTX-y-GT methotrexate-y-giycine-Tris MTX-y-GTP 1 methotrexate-y-glycine-Tris monopalmitate MTX-y-GTP2 methotrexate-y-glycine-Tris dipalmitate MTX-y-GTP3 methotrexate-y-glycine-Tris tripalmitate OMe methyl ester P205 phosphorus pentoxide PmCI palmitoyl chloride PPh3 triphenyl phosphine ItM reaction mixture RT room temperature tBu tert Butyl ester tBuOAc tert Butyl acetate TFA trifluoroacetic acid TPPO triphenylphosphine oxide Z-GT benzyloxycarbonyl glycine-Tris Z-GTP1 benzyloxycarbonyl glycine-Tris monopalmitate Z-GTP2 benzyloxycarbonyl glycine-Tris dipalmitate Z-GTP3 benzyloxycarbonyl glycine-Tris tripalmitate Preparation of methotrexate-y-gtycine-Tris (MTX-y-GT) Glycine-Tris (GT) O
H2N~N OOH
H _ OH
CGH14N2~4 Mol. Wt. 178.19 N-(benzyloxycarbonyl}-glycine-Tris (Z-GT; USP 5,952,499} (1.70 g, 5.44 mmol) 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 f ltered through two glass-fibre (GFIA) filter papers (rinsing with fresh ethanol (approx.
10 ml)}. The filtrate was evaporated leaving GT (0.97 g, 100%) as a white solid 1H
n.m.r. (dG-DMSO) 8 2.1, br s, NH2; 3, l,s,NCHzCO; 3.55, s, 3xCH20; 5.0, br s, 3xOH;
7.95, br s, NH.

MTX a tBu-y-GT
O
O
H
N OH
N
H OH

N OH

MTX-a-tB u- y G T
~30~42~1008 M 01. W t.: 670.72 Methotrexate-a-tent-butyl ester (MTX-a-tBu; USP 5,952,499) (0.438 g, 0.86 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 haur. 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 filtered through glass wool and the filtrate evaporated. The residue was radially chrornatographed an silica gel. Elution with 15% methanol in DCM afforded the title compound {0.33 g. S7%) as a yellow solid. 1H n.m.r. (d~-DMSO) 8 1.39, s, C(CH3)3;
1.96, m, glu-~3-CHz; 2.24, m, glu-y-CH2; 3.21, s, Nme; 3.52, d, J 6 Hz, 3xCH20H; 3.70, d, J 6 Hz, gly NCH2C0; 4.22, m, glu-a-H; 4.69, t, 3 6 Hz, 3xOH; 4.79, s, ArCH2n 6.62, br s, NH2; 6.82, 7.72 AA'BB', J 9Hz, ArH; 7.14, s, tris NH; 7.46, br s, NHZ;
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(+))] rrrlz 671 {M+1) WO 00/34281 PCT/AI~99/01073 MT.Y y~GT
0 COzH 0 N
OH
N HZ ~ N N
H H ON
N ~ N~ N ~ 0 OH
~ -' ~ MTX-y- G T
/ 'N N
NHZ

MoI.Wt.:614.62 MTX-a-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 flash chromatography then radial chromatography (twice) on silica gel. Elution with 1-3% water in 3:5 methanol:chloroform ai~orded the title compound (0.086 g, 21%) as a yellow solid. 1H n.m.r. (ds-DMSO) 8 1.96, m, glu-(3-CH2; 2. i4, m, glu-y-CH2;
3.19, s, Nme; 3.5-3.7, m, 3xCH20H + 3xCHZOH; 3.95, rn, giy NCH2C0; 4.41, rn, glu-a-H;

4.76, s, ArCH2N; 5.37, br s, NH2; 6.61, br s, NH2; 6.84, 7.62 AA'BB', J 9Hz, ArH;
7.60, m, glu NH; 7.71, s, tris NH; 8.23, t, 3 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-slvcine-Tris mono, di and triualmitate ~MTX-y-GTPls, MTX-y-GTP2, MTX-y-GTP3) The title compounds can be prepared as described in USP 5,952,499 or via the following procedures.

Synthesis of 2,4 Diamino-~rhydroxynzetl:ylpteridine (DAHMP) NH2 NHz N ''~ N~ OH NH40H N w ~ OH
I ~ ~ I
H2N~N N .HCI H2N~N N
MW 228.65 MW 192.19 Ref~. Rosowsky et al, J. Med. Chem., 1985, Vol. 28, No. 5, 660-667.
Reactants:
DAHMP.hydrochloride (69.04g, 0.30mo1) 10 H(~Ac ( 120m1) H20 ( 1200m1) Procedure:
DAHMI'.HCI was dissolved {with heating to 80°C) in aqueous acetic acid. The solution was filtered through glass wool and cooled to 45°C. 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 80°C
overnight x 2 to afford the desired DAHMP (free base) (52.94g, 92%) as a golden/tan coloured solid.
4 jj(2,4-Dinmino-6 rteridinyl)ntethylJnzethylan:inoJ-benzoic acid, or 4-nn:ino-rleoxy NI ~-methylhteroic acid (APA) CO ZH

N ~ N~ OH (1 ) PPh3iBr~ N ~ ~ N
I / I
H2N'~ N N (2) MABAI H2N N N
EtN Pr2 MW 192.19 dihydate MW 361.37 Ref: Kralovec et al, J. Med. Chem., 1989, Vol: 32, No. 11, 2426-2431.
Rosowsky et al, J. Med. Chem., 1985, Vol. 28, No. 5, 660-667.

Reactants:
DAHMP {15.008; 78mmo1) Br2 (3eqv, l2.Iml, 37.538, 0.23mo1) PPh3 (3eqv, 61.448, 0.23mo1) DMA ( I 87m1) MABA (13.578, 89mmol) EtN~Pr2 (3eqv, 40.6m1, 30.18, 0.23mo1) Procedure:
1U Br2 was added slowly via syringe to an ice-cooled, stirred mixture of PPh3 in DMA under nitrogen. Solid DAHIVa' 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.Sh. RM poured into aqueous sodium hydroxide solution (0.33M, 125m1), the resulting precipitate filtered off and the 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°C for 4 days to afford a yellow solid {25.798). To remove the TPPO
and MABA contaminants, the crude product was stirred in 3:2 methanol:ethanol (SOmI) at RT for 50h, filtered, washed with Et20 (x2), air-dried and evacuated to give APA
{24.488, 87%) as a yellow solid.
L-Glutarnic Acirl a tBu-y Me ester C02H (1}tBuOAc, HCl04 _C02tBu H2N ~ CO ZMe {2) NaHCO ~ H2N'~ CO 2Me MW 161.16 MW 217.26 Ref: Rosowsky et al, J. Med. Chem., 1981, Vol. 24, No. 12, 1450-1455.
Reactants:
L-Glutamic acid 5-Me ester (17.548, O.I08mol) 3U tBuOAc (200m1) 70% HC104 ( 10.9m1, 0. I2mmo1) Procedure:
HCI04 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 200m1, 1 x 150m1).
The aqueous layer was cooled in ice/salt/dry ice bath (> -5°C) and neutralized with solid sodium hydrogen carbonate (until pH approx. 8 and no more NaHC03 dissolves) with the temperature of the solution being maintained at approx. -4°C. The mixture was extracted with ether (RT, 4 x 200m1), the combined organic Layers washed with brine ( 100m1), dried (MgS04) and evaporated (rotary evaporator, then oil pump, without 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 tl3it-yLMe ~liester NH2 , CO~H
N ~ ~ N ~ I
I ~ I
H2N~ N N pih drate MW 361.37 Y
APA
(1) (Et0)2POCN (2) ~02tBu + iPr2NEt H2N ~' COZMe r O C02tBu NH2 / I H~'/'' CO2Me N ~ N~ N
i MW 524.59 H2N ~ N N
Ref: (1) Rosowsky et. al, J. Med. Chem., 1981, Voi. 24, No. 12, 1450-1455.
(2) Kralovec et al, J. Med. Chem., 1989, Vol. 32, No. 11, 2426-2431.
2o Reactants:
APA (8.56g, 23.7mmo1) EtN'Prz (9.Oml, 51.7mmo1) DMF (460m1) DECD (7.20g, 44.1 mmol) L-Glutamic Acid a-tBu-y-Me ester (7.26g, 33.4mmol) EtNiPr2 (8.Sml, 48.8mmo1) Procedure:
APA (granulated) was added portionwise over 10 minutes to a solution of DECP
and EtN'Pr2 (first portion) in DMF (400m1). The solution was immersed in an oil bath {pre-heated to 80°C) far 2 minutes {internal temp. to 34°C). The flask was removed from the bath and stirred for 30 rains {internal temp. 22°C). The second portion of EtN'Pr2 was added followed by glutamate in DMF (50+lOml rinse). RM was stirred at room temperature under nitrogen for 14 h (overnight). A solution of NaHC03 {1.808, 2lmmol) in water (60m1) was added, the mixture stirred for 30 rains and then evaporated {rotary/oil pump, 45°C). The residue was dissolved in chloroform {approx.
400m1) and the solution washed with water (300m1) and 5% aqueous sodium bicarbonate solution {200m1). The organic layer was washed with brine (30m1), dried {MgS04) 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 purity) (4.408, 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) O CO ztBu NH2 ~ I H~C02Me N ~ N~ N
I ~ I MW 524.59 H2N J~ N N i Ba(OH)2.8H20 r O CO ~tBu NH2 ~ I H~COZH
N W N~ N w H N ~ N N~ I MW 510.56 Ref: Rosowsky et al, J. Med. Chem., 1981, Vol. 24, No. 12, 1450-1455.
Reactants:
MTX-a-tBu-y-Me diester (5.00g, 9.Smmo1) Ba(OH)2.8H20 (1.828, 5.8mmo1) Ethanol (95m1) Water (110m1) Na2S04.1OH20 {1.93g, 6.0mmo1) in water (l0ml) 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 (BaS04 precipitate seems to form immediately) then filtered through 2xGF/A
filter papers on a No. 3 or 4 glass sinter. The f Itrate 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 orange/yellow solid.

Z GTP3 (+ Z GTP2 + Z GTPI) O~ N N OH
I H 'I OH
ZGT O OH
PmCI(2eqv) Et3N
DMF/CH 2C12 (1:4) r OPm ~ N OPm O H~ -OPm + ~ ' O H~ OPm O OPm ~ O OH

O~ N N OPm + ~ H~ OH
/ O OH

Reactants:
Z-GT (SO.OOg, 0.16mo1) PmCI (108m1, 97.85g, 0.356mo1) Et3N ( 100m1, 72.4g, 0.72mo1) DMF (450m1) DCM { 1.60 + 0.20L) Procedure:
Z-GT was dissolved in DMF in a SL, 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 PmCI in DCM { 100 ml) was added dropwise over 2h and the bath temperature maintained at -13 to -10°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 4°C
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) (EtOAc extract resulted in precipitation on washing the extract with brine or water).
The combined organic extracts were washed with brine and dried (MgS04).
Solvent 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-GTPl in a ratio of 28.8%, 47.4% and 23.8% respectively.
MTX a tliu- y GTP3 O CO 2tBu NHz ~ ~ H~ CO2H
N ~ N.. N
MW 510.56 H2N ~ N N

O C02tBu NH2 N w ~ ' H
N ~ ~N O
H N~ N~ N~ ~ MW 1385.91 Reactlnts:
MTX-a-tBu ester (11.00g, 2l.Smmol) DCC (l.2eqv, S.SOg, 26.7mmol) GTP3 ( 1. I eqv, 2I .32g, 23.9mmol) DMAP (0.1 edv, 258mg, 2.1 mmol) DMF (8bm1) DCM (340m1) Procedure:
MTX-oc-tBu ester was dissolved, with stirring, in DMF. DCM (200m1) was added. DCC was added and the solution stirred for 20 mins. GTP3 in DCM (70m1) was added dropwise over 10 rains. 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°C) to remove DMF. The residue was dissolved in DCM, refiltered and evaporated. The residue was dissolved in EtOAc/DCM
( I 50/20m1}, washed with brine, dried (MgS04) and evaporated. The residue was divided into halves and each was flash chromatographed on silica gel, eluting the first 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) (4.838, 16%).
Further elution with 8-10% methanol afforded a third fraction containing a bright yellow solid by-product.
MT'X y~-GTP3 O C02tBu NH2 -'', ~ H
N ~ N~ N ~ O
H N~ N~ I MW 1385.91 TFA

NH2 N ~ ~ ' H
N ~ ~N O
H N~ N N I MW 1329.81 Reactants:
MTX-oc-tBu-y-GTP3 (0.86g, 0.62mmo1) TFA ( 1.7m1, 22mmo1) DCM (5ml) 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 4°C overnight then diluted with DCM (25m1) and washed with aqueous, sodium bicarbonate solution (5% w/v, 35m1) (check that pH>7). HOAc (glacial, 1.8m1) was added and mixture shaken (check that pH~4). The organic layer was washed with water (2x25m1) (the emulsion was broken by addition of a little NaC1 &
methanol & fresh solvents, swirling and warming), dried (MgS04) and evaporated. The residue was flash chromatographed on silica gel, eluting with an 8-30% step gradient 1$
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%.
MTX-y-GTP 1 and MTX-y-GTP2 can be prepared in a similar manner from Z-GTP 1 & 2 respectively.
In vitro DHFR Inhibition Dihydrofolate reductase (DHFR) is a target enzyme of MTX. It catalyses the following reaction:
DHFR
Dihydrofolate + NADPH + H+ -~ Tetrahydrofoiate + NADp+
Its activity can be measured ifi vitro by following the rate of NADPH
oxidation ~5 at 340nm.
Method:
The inhibition of DHFR by MTX, MTX-y-GT and MTX-y-GTPi was measured as described by Imbert AM, Pignon T, Lena N (1983) "Enzymatic assay for 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).
Table 1. Inhibitory activity of MTX and MTX-y-GT on DHFR at 2.86 nM
Cam ound % inhibition MTX 45.5 MTX-y-GT 36.4 The inhibitory activity of MTX-y-GTP 1 was also tested. The concentration required to cause a 50% inhibition of DHFR was 76nM. This is 16X less active than MTX which causes 50% inhibition of DHFR at 4.8nM.
MTX-a-GTP1 did not inhibit DHFR under these conditions.
MTX-y-GTP2 and MTX-y-GTP3 were not tested as their solubility characteristics are incompatible witlx the assay system.

Investigation of MTX-y-GT, MTX-y-GTP1 and MTX-y-GTP3 using the delayed tyue hypersensitivity (DTH) response in the rat Delayed type hypersensitivity {DTH) is an immune response which can be used 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 .
Methods:
Copenhagen {COP) rats had their abdomens shaved and 100 ~,l 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 MTX-y-GT, MTX-y-GTP 1 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 p.l of 0.2% DNFB in acetone to the left footpad. Under anaesthesia, footpad volume was measured at 23h. Piethysmography (the measurement of the volume of an object by observed changes in volume after immersion), using a 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:
MTX (1mg/mI) in Soya oil MTX-y-GT - (1.4 mgslml) in soya oil - (Batch QY5-64MXS) MTX-y-GTP1 - (1.88 mglml) 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 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 control rats (Table 2). As well, the nett footpad oedema from the MTX-y-GTP 1-treated rats was significantly less (p = 0.02 i ) than that in the MTX-treated rats Table 2: Mean, SD and values of data Treatment n Mean SD p value when oedema compared to .....__~__...._ _...~.............___._.._._-_._.~Inl~._ .~......~..~._..._..__ Controls Control 38 0.363 0.254 -MTX 8 0.216 0.1 SO 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 lI 0.055 0.050 <0.0001*
* statistically significant 5 Discussion:
DTH is an irr 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-GTP 1 are at least equal to or perhaps more effective than MTX.
In vitro Cytotoxicity The ability of MTX and its Tris-conjugates to inhibit proliferation of cultured cells were assessed.
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 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, 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."

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.
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.
Com ound ICsa M

MTX 0.0525 8.2 0.019 MTX-a-GT - - 35.9 1889 MTX-y-GT no effect* - 0.57 30 MTX-y-GTP I - - 0.21 11 MTX-a-GTP2 - 10300 1256 -MTX-y-GTP2 __ ___ 0.40 (21) _ 1200 (146) ~ 0.41 {7.8) ~

- not tested; * no effect observed up to 250 nM; (n) Conjugate:MTX ratio.
Reduced activity is likely to be due to the modification of MTX on the a- or Y-COOH. Itr vivv MTX is polyglutamylated (addition of (glu)") on the y-COOH and this is an important aspect of MTX toxicity. It increases intracellular retention and conseduently results in a sustained block in tetrahydrofolate synthesis via DHFR. MTX-y-(glu)" also have greater inhibitory effects on other enzymes involved in DNA
and RNA
synthesis including thymidylate synthetase and AICAR transformylase.
Polyglutamate formation and accumulation may have unwanted effects ifT vivo as they fozrn 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 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-GTP 1 or 2. It could be 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 mare 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-GTP 1 or 2.
It is widely reported that modification to the a-COOH reduces cell uptake to a 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:
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.
Testing of MTX and MTX-w-GT in the rat adjuvant-induced arthritis model.
Doses of 40, I 5 and 5 p.mollkg 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 MTX-y-GT.
Method:
DA rats were injected subcutaneously with Freund's Complete Adjuvant into the tail base. Arthritis developed 10-I2 days later in untreated rats. Rats were treated with PBS (control) or molar equivalent doses ofMTX 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 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.

Results:
The results of a single dose of MTX and MTX-y-GT at day 6 are shown in Figures 1: A to F
Conctusio~~s:
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 efl'ects 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.
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 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:
za 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 z5 followed by assessment at necropsy for gross signs of GIT toxicity (enlarged fluid-filled intestines). MTX-y-GTPZ and MTX-y-GTP3 appeared much less toxic than MTX and results are shown in the following table.

Toxicity results from topical application to Skh-1 hairless mice TREATMENT SIGNS OF Dg,ATH
GROSS GIT
TOXICITY
vehicle 0/24 0/24 MTX 4 mg/kgi 5/24 8/24 MTX-y-GTP2 9.6 mg/kg1 0/24 0/24 MTX-y-GTP3 11.7 mg/kgs 0/14 0/14 IEquimolar amounts of MTX, MTX-y-GTP2 and MTX-y-GTP3 were used. They were formulated in oil/water cream:ethyl oleate (4:1) and O.lml was applied to each mouse. GIT = gastrointestinal tract.
In many mice treated with MTX, clinical signs of toxicity and at necropsy, 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.
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 samples collected for histology.
All MTX-treated rats (rt=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
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 (n=14) treatment groups revealed no gross abnormalities. Reduced histopathology was displayed in the GIT from rats receiving MTX-Tris conjugates.
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 20 (ruffled fur and ataxia) were evident in rats treated with MTX (5 umolelkg).
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 10s/L), due largely to neutropaenia. Leucocyte counts in the vehicle control and MTX-y-GT groups were 20 not different (approx. 30 x 109/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 Coniiug~tes 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 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 ira 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 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 zs for MTX toxicity and if their excretion patterns varied from those of MTX.
Pertinent results are summarised below.
Results:
1. Mice given [14C]-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 ['4C]-MTX. The tissue distribution of [14C] 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.
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-GTP'~ is retained in the skin to a greater extent than MTX. In addition, radioactivity is present in the skin from mice given oral ['4C]-MTX-y-GTP 1 and ['H]-MTX-y-GTP3. These findings may have important implications regarding their potential use as anti-psoriatic treatments.
3. Following oral administration, both radiolabelled MTX-y-GTP 1 and MTX-y-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 2o 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 ['aC]MTX is mainly excreted in the urine and to a lesser extent in the faeces. In contrast, when ['4C]MTX-y-GTP 1 and [14C]MTX-y-GTP3 are 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 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 Section above). Whilst MTX-y-GTPI and MTX-y-GTP3 may accumulate in the liver, lung and spleen when administered ilv, we have not assessed toxic side effects. However WO 001342$1 PCTIAU99/01073 it is unlikely that these conjugates are polyglutamylated, which is the mechanism widely attributed to MTX toxicity.
Both MTX-y-GTP1 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.
Clinical trial of MTX-y-GTP3 as a tolvical treatment for usoriasis Psoriasis is a skin condition that affects 1-2% of people ofEuropean origin.
It is an inflammatory and epidermal hyperproliferative disorder of unknown aetiology with a poor prognosis.
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 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.
Pneclir~ical slz~dies 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 determined by the method of Molek et al.(Brit J Derm(1983) 108:25-31).
Intradermal injection of MTX-y-GTP2 was mare effective than MTX in reducing the growth of 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
(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 sta~dy design The Small Plaque Assay (SPA) assesses the anti-psoriatic potential of compounds over a two week time course. The assay simultaneously tests 6 compounds 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.
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) MTX vehicle (negative control) 0.05% betamethasone valerate (positive control) (AIl concentrations of MTX-y-GTP3 solutions are with respect to their MTX
content}.
Schedule Da Treatment Assessment .......... ..................-.._.................................................................._...._....
................_.....~...................._-._....................................._.......................
..~'..........__.

0 2% salicylic acid clinical & histology I drugs applied 3 drugs applied 6 drugs applied 8 drugs applied clinical I0 drugs applied 13 drugs applied I 5 clinical & histology 29 clinical 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 ZO 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:

~ Clinical Infiltration and Erythema on days 0, 8, 1S and 29.
~ Histopatlaology Parakeratosis Inflammatory inf Itrate Granularity Acanthosis Vascular changes Epidermal thickness Papillomatosis ~ Imnzunohistology Involucrin ICAM-1 Filaggrin Ki-67+ keratinocytes CD4 and CD8+ T-cells Patient Selection Inclusion Criteria Exclusion Criteria E Male of any ethnic group, I8 to 70 years of Pustular psoriasis, erythrodermic psoriasis, age. Females of non-reproductive status inverse psoriasis and/or active psoriasis f also allowed. arthritis.
Serious intercurrent illness or active Chronic plaque type psoriasis vulgaris for at infections requiring treatment, including f 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 E ~ On trunk or extremities; not in a flexure of trial.
area. Received any systemic anti-psoriatic Area at least 5. 5 x 8.3 cm. therapy 4 weeks prior to start of trial.
F ~ Homogeneous appearance (erythema Received any topical anti-psoriatic therapy and infiltration). for the plaque tested two weeks prior to E ~ Severe 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 i other liver disease.
Patients taking medications which may interact with MTX.
i v Patients who have a calculated creatinine clearance < 30 mUmin.

Results:
Clinical The positive control, betamethasone, significantly improved the clinical appearance of test areas indicating that the trial methodology was preforming correctly.
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.
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 psoriasis) and parakeratosis (imperfect formation of epidermal horn cells) in five out of eleven patients.
~ F~~grin.
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 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-GTP3 solution compared to vehicle. There was a similar response to MTX (Figure s).
~ 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 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).

WO 00/342$1 PCT/AU99/01073 Clinical study conclusions MTX-y-GTP3 demonstrated anti-psoriatic etFects in a number of patients in comparison to its vehicle as judged by histopathology {granularity and parakeratosis) 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 andlor modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims (20)

1. A compound having the formula I:
in which M is methotrexate or an analogue thereof;
A=H, CX2-O-R2 or halogen;
B=H, CX2-O-R3 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;
R1,R2, 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 CH2-O-R2 and/or B is CH2-O-R3, then R1, 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 CH2-O-R3 and/or B is CH2-O-R3, and at least one of R1, R2 and R3 is a fatty acyl group, then -[Y]n- 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 .gamma.
carboxyl group.

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;
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;
h) a compound having an hydroxyl group and an aldehyde group; and i) an alkylene oxide.

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.

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 CH20H and R1 is H.

8. Methotrexate-.gamma.-glycine Tris.

9. Methotrexate-.gamma.-glycine glycine Tris.

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.

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 in a subject, the method including administration to the subject of an effective amount of a compound in accordance with of formula I:
in which M is methotrexate or an analogue thereof;
A=H, CX2-O-R2 or halogen;
B=H, CX2-O-R3 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;
R1, R2, 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 CH2-O-R2 and/or B is CH2-O-R3, then R1, 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 CH2-O-R2 and/or B is CH2-O-R3, and at least one of R1, R2 and R3 is a fatty acyl group, then -[Y]m- is other than -(AA)n- or -Y-(AA)n-1-, where AA is an amino acid.

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

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

16. A method according to any one of claims 13 to 15 wherein the disease with an autoimmune 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.

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.

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