Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D475/00—Heterocyclic compounds containing pteridine ring systems
  • C07D475/06—Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4
  • C07D475/08—Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4 with a nitrogen atom directly attached in position 2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators

Definitions

• 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.
• MTX methotrexate
• 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.
• DHFR dihydrofolate reductase
• THF tetrahydrofolates
• 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 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.
• the present invention provides
• 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 ;
• Y is a linker group, and where n is greater than 1, each Y is the same or different; R ⁇ ,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 Ri, R 2 and R 3 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 Ri, R 2 and R is a fatty acyl group, then -[Y] shadow- is other than -(AA) n - or -Y-(AA) n-1 -, where AA is an amino acid.
• M is methotrexate.
• NHC(A)(B)CX 2 OR ⁇ group for example, 2-aminoethanesulphonic acid (taurine).
• 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.
• hydroxy acids examples 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.
• NHC(A)(B)CX 2 OR ⁇ group for example, 2-hydroxyethanesulphonic acid (isethonic acid).
• a linker having an hydroxy group and an aldehyde group for example, p- hydroxybenzaldehyde
• a linker having two reactive halide groups for example, 1,2-dibromoethane
• an alkylene oxide for example, ethyleneoxide
• the NHC(A)(B)CX 2 OR ⁇ group may be attached to the ⁇ or ⁇ carboxyl group of the methotrexate compound. Most preferably, the -NHC(A)(B)CX 2 OR ⁇ group is attached to the ⁇ carboxyl of the methotrexate compound, optionally by the linker.
• Y is an amino acid it may be any suitable amino acid including, natural and non-natural amino acids and analogues thereof.
• the amino acid is glycine.
• Ri, R 2 and R 3 are each H.
• n is in the range 0-5.
• M is methotrexate and -[Y] n - is -[AA] n -, where AA is an amino acid.
• AA is glycine and n is 1. Where n >2, each AA need not be the same amino acid.
• a preferred methotrexate derivative is methotrexate- ⁇ -glycine-TRIS (MTX- ⁇ - GT).
• the present invention provides a pharmaceutical composition incorporating a compound of the formula I as defined above and a pharmaceutically acceptable carrier or diluent.
• M is methotrexate.
• Y is glycine and n is 1.
• methotrexate derivative is methotrexate- ⁇ -glycine-Tris.
• Compositions suitable for oral administration include liquid solutions, capsules, suspensions, emulsion etc.
• 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.
• composition of the invention may be in the form of suppositories, pessaries, tampons, creams, gels, pastes, lotions, implants, patches or foams.
• composition of the invention may be in the form of an implant.
• 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:
• R-t, R5 and Re 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.
• 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.
• 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.
• 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 0.001 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.
• concentrations here set forth are provided by way of description only and not by way of limitation.
• Figure 1 is a boxplot demonstrating the effect of oral MTX- ⁇ -GT, MTX- ⁇ -GTPl and MTX- ⁇ -GTP3 in the rat assay of delayed type hypersensitivity footpad oedema.
• MTX- ⁇ -GT and MTX- ⁇ -GTPl are both active in this assay as is
• FIG. 1 shows the effects of MTX and MTX- ⁇ -GT in the rat adjuvant-induced arthritis model. The results indicate that MTX- ⁇ -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- ⁇ -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- ⁇ -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- ⁇ -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- ⁇ -
• FIG. 6 is a box plot showing the pooled results for filaggrin expression in the stratum granulosum of SPA patients treated with oral MTX- ⁇ -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- ⁇ -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- ⁇ -GTP3 solution, 0.5% MTX and 0.05% betamethasone valerate cream. 1.0% MTX- ⁇ -GTP3 is showing positive results with this early indicator of plaque resolution.
• AICAR phosphoribosylaminoimidazolecarboxarnide APA 4-amino-4-deoxy-N 1 ⁇ -methylpteroic acid
• 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 filtered through two glass-fibre (GF/A) 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.
• Methotrexate- ⁇ -tert-butyl ester (MTX- -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 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 filtered through glass wool and the filtrate evaporated.
• MTX- ⁇ -t-Bu- ⁇ -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 HO Ac (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-
• DAHMP.HCl 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
• 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 HC1 (2 x 200ml, 1 x 150ml).
• 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 NaHCO3 dissolves) with the temperature of the solution being maintained at approx. -4°C.
• 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 U C) for 2 minutes (internal temp, to 34°C). The flask was removed from the bath and stirred for 30 mins (internal temp. 22°C). The second portion of EtN'Pr2 was added followed by glutamate in DMF (50+10ml rinse). RM was stirred at room temperature under nitrogen for 14 h (overnight).
• N-1HCO3 (1.80g, 21 mmol) 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 bicarbonate solution (200ml). The organic layer was washed with brine (30ml), dried
• 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 filter papers on a No. 3 or 4 glass sinter.
• the filtrate was adjusted to pH 5 with 10%
• MTX- ⁇ -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 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°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.
• DHFR Dihydrofolate reductase
• MTX- ⁇ -GT inhibited DHFR in the same concentration range as MTX (Table 1).
• the inhibitory activity of MTX- ⁇ -GTPl 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- ⁇ -GTPi did not inhibit DHFR under these conditions.
• MTX- ⁇ -GTP 2 and MTX- ⁇ -GTP were not tested as their solubility characteristics are incompatible with the assay system.
• Delayed type hypersensitivity 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- ⁇ -GT, MTX- ⁇ -GTPl and MTX- ⁇ -GTP3.
• 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- ⁇ -GT, MTX- ⁇ -GTPl or MTX- ⁇ -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 ⁇ l of 0.2% DNFB in acetone to the left footpad. Under anaesthesia, footpad volume was measured at 23 h.
• DNFB dinitroflurobenzene
• Plethysmography (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.
• the mean nett footpad oedema was significantly less in those rats treated with
• DTH is an in vivo T cell-dependent immune response manifested as an inflammatory reaction after antigenic challenge.
• MTX and its Tris-conjugates were assessed.
• JURKAT human acute 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:
• IC 50 concentration of compound at which cell growth is inhibited by
• Reduced activity is likely to be due to the modification of MTX on the ⁇ - or ⁇ -
• MTX is polyglutamylated (addition of (glu) n ) on the ⁇ -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- ⁇ -(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 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.
• MTX- ⁇ -GT is less inhibitory than MTX- ⁇ -GTPl 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- ⁇ -GT is less toxic to cells than the palmitoylated conjugates, it is more 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- ⁇ -GT and MTX- ⁇ -GTPl or 2.
• Both MTX- ⁇ -GT and MTX-GTP2 are much less toxic than MTX- ⁇ -GT and MTX- ⁇ - GTP2, respectively.
• MTX and MTX- ⁇ -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- ⁇ -GTPl might also delay the onset of inflammation in this model.
• MTX- ⁇ -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.
• mice were assessed for toxic signs (dehydration, diarrhoea and anorexia) for 6 days followed by assessment at necropsy for gross signs of GIT toxicity (enlarged fluid-filled intestines).
• toxic signs dehydration, diarrhoea and anorexia
• GIT toxicity enlarged fluid-filled intestines
• mice treated with MTX 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- ⁇ -GTP2 or MTX- ⁇ -GTP3. In all mice, the liver and kidneys appeared normal.
• MTX is an effective oral treatment for a disease with an autoimmune component such as psoriasis and rheumatoid arthritis.
• an autoimmune component such as psoriasis and rheumatoid arthritis.
• MTX also induces gastrointestinal irritation, renal toxicity and abortion.
• MTX-Tris conjugates are biologically active but are less toxic in vitro and in vivo (see above).
• 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 for MTX toxicity and if their excretion patterns varied from those of MTX. Pertinent results are summarised below.
• radiolabel from MTX- ⁇ -GTP3 was detected in the skin epidermis for longer periods than MTX treated mice. This suggests that MTX- ⁇ -GTP3 is retained in the skin to a greater extent than MTX.
• radioactivity is present in the skin from mice given oral [ 1 C] -MTX- ⁇ -GTPl and [ 3 H]-MTX- ⁇ -GTP3.
• both radiolabelled MTX- ⁇ -GTPl and MTX- ⁇ -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 following i/v administration) they appear to accumulate in the liver, spleen and lung.
• Radiolabel from [ 14 C]MTX is mainly excreted in the urine and to a lesser extent in the faeces.
• radiolabel is mainly eliminated in the faeces via hepatic excretion, with very low levels detected in the urine.
• 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- ⁇ -GT) or low levels of absorption in the formulations tested (MTX- ⁇ - GTPl and MTX- ⁇ -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- ⁇ -GTPl and MTX- ⁇ -GTP3 may accumulate in the liver, lung and spleen when administered i/v, we have not assessed toxic side effects. However it is unlikely that these conjugates are polyglutamylated, which is the mechanism widely attributed to MTX toxicity.
• 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. 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.
• MTX- ⁇ -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- ⁇ -GTP2 was more effective than MTX in reducing the growth of B16 melanomas (USP 5,952,499).
• MTX- ⁇ -GTP3 is retained in the epidermis of hairless mice for longer periods than MTX.
• MTX- ⁇ -GTP3 administered topically or orally is less toxic to hairless mice than MTX (See above).
• SPA Small Plaque Assay
• betamethasone valerate 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- ⁇ -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.
• betamethasone demonstrated anti-psoriatic activity in all parameters except hyperkeratosis and involucrin.
• MTX- ⁇ -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.
• 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- ⁇ -GTP3 solution (Figure 5). MTX- ⁇ -GTP3 solution showed the best filaggrin response at a concentration of 0.5% while 1% MTX- ⁇ -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- ⁇ - GTP3 solution compared to vehicle. There was a similar response to MTX ( Figure
• 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- ⁇ -GTP3 solution (5 out of 11 patients) - Figure 7 a) and b). Treatment with 0.5% MTX- ⁇ -GTP3 and MTX resulted in little response in granularity:parakeratosis ratio (1 & 2 patients respectively). Clinical study conclusions
• MTX- ⁇ -GTP3 demonstrated anti-psoriatic effects 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.

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