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Topical methotrexate: percutaneous penetration and clinical efficacy Wallace, Sylvia Mary Gloria 1972

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TOPICAL METHOTREXATE Percutaneous Penetration and C l i n i c a l E f f i c a c y by SYLVIA MARY GLORIA WALLACE B.S.P., University of B r i t i s h Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Faculty of Pharmaceutical Sciences D i v i s i o n of Pharmaceutics We accept th i s thesis as conforming to the required standard UNIVERSITY OF BRITISH COLUMBIA September 1972 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Pharmaceutics  Faculty of Pharmaceutical Sciences The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada October 6,1972 ABSTRACT Because of the effectiveness of systemically administered methotrexate (MTX) i n p s o r i a s i s , t o p i c a l application of the drug has been suggested as a possible therapeutic regimen for the disease. However, controversy reigns over the c l i n i c a l e f f i c a c y of such t o p i c a l therapy and l i t t l e i s known of the absorption k i n e t i c s of t o p i -c a l l y applied MTX. In the present work percutaneous penetration of MTX was investigated i n v i t r o and i n vivo using h a i r l e s s mouse and human skin. C l i n i c a l e f f i c a c y of t o p i c a l l y applied and i n t r a l e s i o n a l l y injected MTX was tested i n p s o r i a t i c patients. An unsuccessful attempt was made to i s o l a t e and obtain k i n e t i c data for dihydro-folate reductase, the proposed target enzyme for MTX i n h i b i t i o n , i n human skin. However, estimates of k i n e t i c parameters for p a r t i a l l y p u r i f i e d dihydrofolate reductase from chicken l i v e r acetone powder are reported and are i n agreement with l i t e r a t u r e values. In vivo t o p i c a l l y applied MTX was ra p i d l y absorbed through h a i r l e s s mouse skin but not through the skin of p s o r i a t i c patients. In mice, t o p i c a l l y applied MTX was detected by fluorometric analysis i n the plasma and l i v e r i n amounts comparable to those obtained a f t e r i n t r a p e r i t o n e a l administration. • Skin levels were also analyzed. Following t o p i c a l application i n p s o r i a t i c patients, MTX could be detected i n neither plasma nor skin. In_ v i t r o , t r i t i a t e d MTX penetrated through excised samples of h a i r l e s s mouse and human autopsy skin at com-parable rates when analyzed using l i q u i d s c i n t i l l a t i o n counting techniques. Vehicle pH affected steady state penetration rates. As pH of the suspension vehicle was increased from 3.49 to 8.15, and thus i o n i z a t i o n of the weakly a c i d i c MTX, steady state penetration rates decreased. Changes i n penetration rates were correlated with changes i n s o l u b i l i t y and octanol-water p a r t i t i o n c o e f f i c i e n t as determined spectrophotometrically. Increas-ing the concentration of MTX i n cream and suspension vehicles increased the penetration rates but not the con-centration of MTX within the skin. Levels of MTX reached in v i t r o i n h a i r l e s s mouse skin were within the same range as those reached in_ vivo. In 17 out of 18 p s o r i a t i c patients t o p i c a l l y applied (0.2% cream or 5 mg MTX deposited on the skin from an alcohol suspension) or i n t r a l e s i o n a l l y injected (0.5 mg) MTX was c l i n i c a l l y i n e f f e c t i v e . Any changes in the treated areas which suggested c l i n i c a l improvement i n the p s o r i a t i c lesions were p a r a l l e l e d by s i m i l a r changes i n control areas. Apparently, neither lack of percutaneous penetra-t i o n nor low serum folate i s the determining factor for a c t i v i t y of t o p i c a l MTX. Evidence suggests that the s i t e of action of MTX i n p s o r i a s i s involves tissues other than the affected skin. i v . TABLE OF CONTENTS PAGE LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS x i INTRODUCTION 1 LITERATURE SURVEY 3 A. METHOTREXATE IN PSORIASIS 3 1. Biochemical mechanism of MTX 5 2. Side e f f e c t s of systemic MTX 11 3. Pharmacokinetics of systemic MTX 14 4. Increasing the therapeutic index of MTX 17 a) "Leucovorin rescue" 17 b) Modified dosage schedule 18 c) Alternative routes of administration 19 B. PERCUTANEOUS PENETRATION 2 2 1. Skin structure and d i f f u s i o n pathways 22 2. The "b a r r i e r " 2 5 3. Mathematical models for percutaneous penetration 2 5 4. Factors a f f e c t i n g percutaneous absorption 29 a) Skin v a r i a t i o n 29 b) Cha r a c t e r i s t i c s of the penetrant 31 c) Vehicle e f f e c t s 33 STATEMENT OF PROBLEM 37 EXPERIMENTAL 3 9 PAGE 1. METHODOLOGY 39 A. ANALYTICAL METHODS FOR MTX 39 1. Fluorometry 39 2. Spectrophotometry 42 3. Liquid S c i n t i l l a t i o n Counting 45 B. ANALYSIS OF DHFR ACTIVITY 48 1. ^Spectrophotometry 4 8 2. Fluorometry 5 3 I I . EXPERIMENTS AND RESULTS 5 5 A. ACTIVITY OF DHFR AND ITS INHIBITION BY MTX 5 5 B. IN VIVO DISTRIBUTION OF MTX 62 1. Intraperitoneal absorption i n h a i r l e s s mice 62 2. Oral absorption i n p s o r i a t i c patients 65 3. Percutaneous absorption i n h a i r l e s s mice and p s o r i a t i c patients 68 C. CLINICAL EFFICACY OF LOCAL MTX IN PSORIASIS 74 D. DETERMINATION OF SOLUBILITY AND PARTITION COEFFICIENT OF MTX 78 1. S o l u b i l i t y 78 2. P a r t i t i o n C o e f f i c i e n t 79 E. IN VITRO PERCUTANEOUS PENETRATION 8 5 1. Of t r i t i a t e d water 85 2. Of MTX 88 v i . P A G E D I S C U S S I O N 1 0 3 A . A C T I V I T Y O F D H F R A N D I T S I N H I B I T I O N B Y M T X 1 0 3 B . C O R R E L A T I O N O F D H E F F E C T S O N I N V I T R O P E R C U T A N E O U S P E N E T R A T I O N O F M T X W I T H P A R T I T I O N C O E F F I C I E N T A N D I O N I Z A T I O N 1 0 4 C . I N V I V O D I S T R I B U T I O N O F M T X 1 1 2 D . C L I N I C A L E F F I C A C Y O F L O C A L M T X I N P S O R I A S I S 1 1 5 E . P E R C U T A N E O U S P E N E T R A T I O N A S R E L A T E D T O T H E I N E F F E C T I V E N E S S O F T O P I C A L M T X 1 1 6 F . S I T E O F A C T I O N O F M T X I N P S O R I A S I S 1 1 7 S U M M A R Y A N D C O N C L U S I O N S 1 2 0 A P P E N D I C E S I . M A T E R I A L S 1 2 4 I I . A P P A R A T U S 1 3 0 I I I . C A L C U L A T I O N O F T H E F R A C T I O N O F U N I O N I Z E D M T X 1 3 3 R E F E R E N C E S 1 3 5 LIST OF TABLES Kin e t i c constants for chicken l i v e r dihydro-fo l a t e reductase at pK 7.4 determined with and without the presence of MTX Ef f e c t of pH on s o l u b i l i t y of MTX Ef f e c t of pH on octanol/water p a r t i t i o n c o e f f i c i e n t of MTX Comparison of permeability constants f o r d i f f u s i o n of t r i t i a t e d water through h a i r l e s s mouse skin i n v i t r o from buffers of varying pH Comparison of d i f f u s i o n of MTX through h a i r l e s s mouse and human autopsy skin i n v i t r o from a 0.2 5% solution at pH 8.15 Ef f e c t of vehicle pH in v i t r o percutaneous penetration of MTX through h a i r l e s s mouse skin E f f e c t of vehicle composition and concen-t r a t i o n on i n v i t r o percutaneous penetra-t i o n of MTX through h a i r l e s s mouse skin Steady state d i f f u s i o n rate, s o l u b i l i t y , p a r t i t i o n c o e f f i c i e n t and f r a c t i o n of unionized MTX at varying pH V l l l . LIST OF FIGURES PAGE 1. Chemical structures of f o l i c acid and methotrexate 6 2. Interconversions and metabolic roles of tetrahydrofolate cbfactors 7 3. Metabolic pathways for synthesis of thymidylate 8 4. Enzymes catalyzing the interconversions of reduced folates 10 5. Diagrammatic representation of human skin structure 2 3 6. a) Concentration p r o f i l e through a membrane b a r r i e r under membrane flux control (Higuchi, 1960) 30 b) Concentration p r o f i l e through a membrane-d i f f u s i o n layer b a r r i e r under d i f f u s i o n layer flux control (Flynn et a l . , 1972) 30 7. Fluorescence spectra of MTX (0.4 ug/ml) 40 8. Fluorometric standard curve for MTX 41 9. UV absorption spectra for MTX at pH 4.3 and 8.9 43 10. E f f e c t of pH on abs o r p t i v i t y of MTX at three d i f f e r e n t wavelengths 44 11. Spectrophotometric standard curve f o r MTX from UV absorbance at three d i f f e r e n t pK values 46 12. Quench correction curve f o r t r i t i u m with an 13 7 external Cs standard i n the Picker Nuclear Liquimat 4 7 13. Quench correction curve for t r i t i u m using an external standard (l-^Ba) r a t i o method i n the Nuclear Chicago Isocap 300. 49 14. Reactions involved i n the spectrophotometric assay for dihydrofolate reductase a c t i v i t y 50 i x . PAGE 15. a) Spectrophotometric standard curves for DHF and THF at 340 nm 51 b) Spectrophotometric standard curves f o r NADP at 340 nm 52 16. Lineweaver-Burk plot of chicken l i v e r dihydro-folate reductase a c t i v i t y with and without 9.17 x I O - 9 M MTX 59 17. Eadie plot of chicken l i v e r dihydrofolate reductase a c t i v i t y with and without 9.17 x 10~ 9 M MTX 60 18. Hofstee plot of chicken l i v e r dihydrofolate reductase a c t i v i t y with and without 9.17 x 10" y M MTX 61 19. Dixon plot of MTX i n h i b i t i o n of chicken l i v e r dihydrofolate reductase with 3.1 x 10~ 6 M and 9.3 x I O - 6 M DHF 63 20. Ackermann-Potter plot of chicken l i v e r dihydro-f o l a t e reductase a c t i v i t y at 9.3 x 10~ 6 M DHF and i n h i b i t e d by 9.17 x 10~ 9 M MTX 64 21. Plasma leve l s of MTX i n h a i r l e s s mice following i n t r a p e r i t o n e a l i n j e c t i o n of a 1.25 mg/kg dose 66 22. Plasma l e v e l s of MTX i n three p s o r i a t i c patients following a 25 mg o r a l dose 67 23. Comparison of plasma, l i v e r and skin l e v e l s of MTX i n h a i r l e s s mice following i n t r a p e r i t o n e a l i n j e c t i o n (1.25 mg/kg dose) and t o p i c a l application (20 mg of a 5% cream) 69 24. a) Control autoradiograph of h a i r l e s s mouse skin 71 b) Autoradiograph of hairless-mouse skin one hour a f t e r a p p l i c a t i o n of H-MTX 72 c) Autoradiograph of h a i r l e s s mouse skin two and a ha l f hours a f t e r a p p l i c a t i o n of ^ H - M T X 73 25. E f f e c t of P H on s o l u b i l i t v of MTX 81 X . PAGE 26. E f f e c t of pK on octanol/water p a r t i t i o n c o e f f i c i e n t of MTX 84 27. Log-log plot of octanol/water p a r t i t i o n c o e f f i c i e n t (K°) as a function of f r a c t i o n of MTX unionized at varying pH 86 28. Cumulative d i f f u s i o n of t r i t i a t e d water through h a i r l e s s mouse skin i n v i t r o at pH 7.90 and 2.47 90 29. Representative percutaneous penetration curve of MTX — cumulative d i f f u s i o n of MTX through h a i r l e s s mouse skin i n v i t r o from a 0.25% suspension i n pH 3.49 buffer 92 30. Cumulative d i f f u s i o n of MTX through h a i r l e s s mouse skin i n v i t r o from a 0.2 5% solution i n 0.5% bicarbonate (pH 8.15) 9 5 31. Cumulative d i f f u s i o n of MTX through human autopsy skin i n v i t r o from a 0.25% solution i n 0.5% bicarbonate (pH 8.15) 96 32. Cumulative d i f f u s i o n of MTX through human autopsy skin in v i t r o from an intracutaneous i n j e c t i o n of 0.5 mg MTX i n 0.5% bicarbonate soluti o n 102 33. a) Log-log plot of steady state f l u x (J ) as a function of f r a c t i o n of MTX unionized (o^) 108 b) Log-log plot of steady state flux (J ) as a function of concentration of dissolved, unionized drug ([MTX°]) 109 34. Steady state flux of MTX through h a i r l e s s mouse skin i n v i t r o at varying pH as a function of octanol/water p a r t i t i o n c o e f f i c i e n t 110 35. Flux of MTX through h a i r l e s s mouse and human leg skin from dirr.ethylacetamide solutions as a function of concentration of penetrant (Data from Mewbold and Stoughton , 19 72 ) 113 36. Diagrammatic representation of the d i f f u s i o n c e l l 132 x i . LIST OF ABBREVIATIONS AICAR ... aminoimidazolecarboxamide r i b o t i d e BSP ... bromosulphothalein DHF ... dihydrofolate DHFR ... dihydrofolate reductase DMA ... dimethylacetamide DMF ... dimethylformamide DMSO ... dimethylsulphoxide DNA ... deoxyribonucleic acid DUMP ... deoxyuridine monophosphate FAICAR ... formyl aminoimidazolecarboxamide r i b o t i d e FGAR ... formyl glycineamide r i b o t i d e FIGLU ... formimino glutamic acid GAR ... glycineamide r i b o t i d e 3H-MTX ... t r i t i a t e d methotrexate 3 . . . . H-UdR ... t r i t i a t e d deoxyuridine MTX ... methotrexate NADP ... nicotinamide adenine dinucleotide phosphate NADPH ... reduced nicotinamide adenine dinucleotide phosphate SGOT ... serum glutamate-oxalacetate transaminase SGPT ... serum glutamate-pyruvate transaminase THF ... • tetrahydrofolate TMP ... thymidine monophosphate UdR ... deoxyuridine ACKNOWLEDGEMENTS To Dr. J.O. Runikis, thesis supervisor. To Dr. Wm. D. Stewart for his advice and guidance in the clinical aspects of this thesis. To Drs. R. Ongley, R. Adams and D. Tsang for adminis-tration and clinical evaluation of metho-trexate treatments in psoriatic patients. To the Departments of Pathology and Surgery, Vancouver General Hospital for their co-operation in obtaining autopsy and surgical skin. To the Department of Pathology3 University of British Columbia for their advice and use of histological equipment and supplies. To the Medical Research Council of Canada, for its generous financial support. INTRODUCTION A safe and consistently e f f e c t i v e treatment for psoria s i s has been sought for decades. Although systemic administration of MTX has proven e f f e c t i v e i n a high per-centage of cases treated, suppressive e f f e c t s on a l l rapidl y p r o l i f e r a t i n g tissues and progressive hepatic damage l i m i t s i t s use in t h i s benign disease. To circum-vent the side effects of systemic dosage, other hopefully less t o x i c , modes of administration have been investigated. Therapeutic e f f e c t s of l o c a l a p p l i c a t i o n -- t o p i c a l and intradermal -- have been evaluated. However, d i s p a r i t y between the e f f i c a c y of l o c a l and systemic therapy has been notable. Although some investigators have obtained improvement i n ps o r i a s i s with t o p i c a l MTX, l o c a l treatment i s generally considered i n e f f e c t i v e . T r i a l s with t o p i c a l MTX, however, reveal one possible shortcoming -- a b r i e f duration of therapy, (one to two weeks). It should be noted that a lag period of up to three weeks may be required before c l i n i c a l improvement i s noted with systemic MTX. Since the introduction of MTX as a therapeutic agent, the pharmacokinetics of systemically administered MTX in cancer patients had been thoroughly investigated. However, l i t t l e i s known of the k i n e t i c s of t o p i c a l absorption of MTX. It i s the purpose of t h i s study to expand experience with t o p i c a l use of MTX in the treatment o p s o r i a s i s and to determine the k i n e t i c s of MTX absorp t i o n following t o p i c a l a p p l i c a t i o n . 3 . LITERATURE SURVEY A. METHOTREXATE IN PSORIASIS Psoriasis i s a chronic, inflammatory skin disease characterized by fa u l t y k e r a t i n i z a t i o n , hyperplasia and increased mitotic a c t i v i t y of the epidermis (Montgomery, 1967). Despite numerous biochemical and histochemical in v e s t i g a t i o n s , r e s u l t s are non-specific and o f f e r l i t t l e i n explanation of the etiology of the disease (Montgomery, 1967; Roe, 1968). Rather they r e f l e c t the increased r e p l i c a t i o n rate of p s o r i a t i c epidermal c e l l s with the consequent f a i l u r e of d i f f e r e n t i a t i o n and k e r a t i n i z a t i o n . Since there i s no known cause or therapy for the metabolic-mitotic stimulus, therapy attempts to control the most obvious symptom, cutaneous hyperplasia (Stankler,1970). For 90% of p s o r i a t i c patients, symptoms can be r e l i e v e d by routine therapy with coal t a r , d i t h r a n o l , t o p i c a l c o r t i -costeroids, u l t r a v i o l e t l i g h t or s u p e r f i c i a l i r r a d i a t i o n (Defeo et a l . , 1965). However, for those patients with generalized p s o r i a s i s , r e s i s t a n t to these more conventional treatments, MTX provides a therapeutic a l t e r n a t i v e . MTX i s acknowledged as an e f f e c t i v e agent for the treatment of severe, r e c a l c i t r a n t p s o r i a s i s (Edmundson and Guy, 1958 ; Hunter, 1962 , Van Scott et a l . , 1964; Auerbach, 1964; Callaway et a l . , 1966; Carpenter and J o l l y , 1967; 4 . Rees et a l . , 1967 ; Frank et a l . , 196 8 ; Schewach-Millet and Ziprowski, 1968 ; McDonald and Bertino, 1969 ; Roenigk et a l . , 1969; Grupper and Bourgeois-Spinasse, 1970). Therapy regimens for p s o r i a s i s vary. O r a l l y , MTX i s administered as 2.5 to 5.0 mg d a i l y doses with intervening rest periods or as a single 25-50 mg dose once weekly. By the parenteral route, intravenous or intramuscular i n j e c t i o n , 2 5-50 mg of MTX i s administered once weekly or biweekly. Which dosage regimen i s the most e f f e c t i v e and least toxic i s a matter of opinion. C l i n i c a l e f f i c a c y , incidence and nature of side e f f e c t s of the d i f f e r e n t treatment schedules are s i m i l a r (Weinstein and Frost, 1971). Good to excellent r e s u l t s (85-100% clearing of body surface area affected with p s o r i a s i s ) are achieved i n greater than 60% of patients treated. 1. BIOCHEMICAL MECHANISM OF MTX MTX, an antimetabolite synthesized i n 1948 (Seeger et a l . , 1948) as a f o l i c acid analogue, d i f f e r s from the vitamin by substitution of a 4-amino group for the 4-hydroxy and addition of an N"^-methyl group (Figure 1). This s l i g h t s t r u c t u r a l a l t e r a t i o n produces an i n h i b i t o r of DHFR which has 20,000 times the a f f i n i t y for the enzyme as has the natural substrate, DHF (Werkheiser, 1963). DHFR catalyzes the reduction of folate and DHF to THF. Binding of MTX to DHFR i n h i b i t s t h i s reduction and thus depletes the body's stores of reduced f o l a t e s . THF derivatives are e s s e n t i a l cofactors for the trans f e r of one-carbon units i n purine biosynthesis, methylation of deoxyuridylate, methionine biosynthesis, h i s t i d i n e degradation, serine and glycine metabolism, and possibly for i n i t i a t i n g formation of peptide chains for protein synthesis (Blakely, 1969; S i l b e r and Mansouri, 1971) (Figure 2). Thymidylate synthetase, catalyzing the transfer of the N^°-methyl group of N^-methylene THF to deoxyuridylate, occupies a unique position among the THF-dependent reactions. In the process of demethylation, THF i s oxidized to DHF (Figure 3). Since THF i s the rate l i m i t i n g factor for thymidylate synthesis (Neely, 1971) and i s depleted by t h i s c y c l i c process , the thymidylate pathway i s es p e c i a l l y vulnerable to MTX i n h i b i t i o n 6. H 2 N - r ^ F O L I C A C I D Pteroylglutamic ac id C O O H C O O H M E T H O T R E X A T E 10 4 - a m i n o - N -methyl pteroylglutamic acid FIGURE 1: Chemical structures of f o l i c acid and methotrexate 7. P u r i n e b i o s y n t h e s i s FIGURE 2: Interconversions and metabolic roles of tetrahydrofolate cofactors. 8. "Salvage pathway" 'de novo pathway" deoxyuridine thymidine dUMP 5,10-methylene THF V DHF J D N A M T X J. d i h y d r o f o l a ' " ""^ vOv r e d u c t a s e THF c a l c i u m l eucovor in FIGURE 3: Metabolic pathways for synthesis of thymidilate 9 . (Bertino, 1967). I n h i b i t i o n of DHFR ultimately leads to a depletion of the pool of thymidine required for DNA synthesis and c e l l r e p l i c a t i o n . . In v i t r o i n L - c e l l cultures thymidine and deoxyadenosine i n s u f f i c i e n t concentrations overcome MTX-induced i n h i b i t i o n of c e l l p r o l i f e r a t i o n (Borsa and Whitmore, 1959a). Borsa and Whitmore (1969b) have demonstrated that, in v i t r o , MTX also i n h i b i t s thymidylate synthetase d i r e c t l y . The greater magnitude of the constant f o r MTX i n h i b i t i o n of thymidylate synthetase than for i n h i b i t i o n of DHFR precludes consideration of thymidylate synthetase as a s i g n i f i c a n t s i t e of action (Harrap, 1971). I n h i b i t i o n of the purine biosynthetic pathway requires tenfold greater concentrations of MTX than the thymidylate biosynthetic pathway (Borsa and Whitmore, 1969a). Although binding with other enzymes of the fo l a t e pathway -- formate a c t i v a t i n g enzyme, serine hydroxy-methylase, and 5, 10-methylene THF dehydrogenase (Figure 4) -- has been postulated (Huennekens, 1969), i t i s generally agreed that the mechanism of action of MTX i n p r o l i f e r a t i v e diseases such as ps o r i a s i s i s rela t e d to i t s reaction with DHFR. I n h i b i t i o n of these fo l a t e enzymes can only be obtained i n v i t r o at le v e l s of MTX greater than those usually reached in vivo (10 ^ M) (Huennekens, 1969). FIGURE 4: Enzymes catalyzing interconversions of reduced f o l a t e s . 2. SIDE EFFECTS OF MTX Unfortunately, MTX i n h i b i t i o n of DKFR i s not tissue s p e c i f i c . The e f f e c t s observed c l i n i c a l l y are somewhat limited by the rate of d i v i s i o n and DNA require-ments of various c e l l types. Nonetheless, body tissues which normally p r o l i f e r a t e r a p i d l y are also subject to cy t o s t a t i c e f f e c t s . Stomatitis and g a s t r o i n t e s t i n a l bleeding have resulted from i n h i b i t e d regneration of sloughed gastro-i n t e s t i n a l e p i t h e l i a l c e l l s . Nausea and vomiting occur frequently although the exact causes are unknown. Bone marrow depression, r e s u l t i n g i n leukopenia, thrombocyto-penia and hemoglobinemia, i s the l i m i t i n g factor i n the use of MTX c l i n i c a l l y . Toxic e f f e c t s on the ha i r matrix a tis s u e with a regeneration time of 2^ hours, may produce temporary alopecia (McDonald and Bertino, 1969). Depression of the immune mechanism may leave patients susceptible to i n f e c t i o n . A l l i s o n (1968) has reported a generalized vaccinia reaction i n a patient receiving a smallpox vaccination while under therapy with MTX. Cases of pneumonia (Robertson, 1970) and active tuberculosis (Smith and Knox, 1971) have also been linked to MTX therapy. Chronic immunosuppression m a v f a c i l i t a t e tumour emergence, growth and metastases (Harris, 1971; Craig and Rosenberg, 1971; Schroter et a l . , 1971). Although l i v e r i s not a r a p i d l y r e p l i c a t i n g tissue and has large reserves of reduced f o l a t e s , the incidence of hepatoxicity linked with MTX administration i s notable. Increases have been detected i n serum transaminases (SGOT and SGPT) and BSP retention. H i s t o l o g i c examination of l i v e r biopsies reveals numerous pathological changes — f a t t y i n f i l t r a t i o n , p e r i - p o r t a l f i b r o s i s and post-necrotic c i r r h o s i s , but none s p e c i f i c f o r MTX damage (Weinstein et a l . , 1970 ; Roenigk e_t a l . , 1971). It has been suggested that an i n d i r e c t block of protein synthesis by MTX may lead to damage of organelles and plasma membranes of hepatic parenchymal c e l l s i n t e r f e r i n g with t h e i r function and allowing leakage of enzymes (Dubin, 1970). The evaluation of MTX-induced hepatotoxicity i s complicated by the frequency of h i s t o l o g i c l i v e r abnormalities which are known to occur i n p s o r i a t i c patients (Berge, 1970). In a recent study, h i s t o l o g i c examination of a control group of p s o r i a t i c patients who had never received MTX and who were c l a s s i f i e d as minimal to moderate drinkers revealed f o c a l necrosis, f a t t y metamorphosis or non-specific h e p a t i t i s i n four out of eight cases (Weinstein et a l . , 1970). Patho-l o g i c changes in l i v e r biopsies i n p s o r i a t i c s may be related to both the severity of the disease and prolonged t o p i c a l treatment with other therapeutic agents (Zachariae and Schidt, 1971). These side e f f e c t s delineate the contraindications to MTX administration; namely, kidney dysfunction, l i v e r abnormalities, hematopoeitic disturbances and active i n f e c t i o u s diseases. In addition, a b o r t i f a c i e n t and t e r a -togenic e f f e c t s of MTX (Baker, 1970) preclude i t s use during pregnancy, p a r t i c u l a r l y during the f i r s t trimester. Almeyda and his co-workers (1971) consider that at the present time i t i s not l o g i c a l to e n t i r e l y condemn the use of MTX i n p s o r i a s i s . However, they suggest that the surveillance of patients' status by conventional l i v e r function tests (SGOT, SGPT and BSP retention) should be augmented by annual l i v e r biopsy (Almeyda et_ a_l. , 1971) . Recent l e g i s l a t i o n by the United States Food and Drug Administration has o f f i c i a l l y approved the use of MTX i n p s o r i a s i s . Labelling precautions r e s t r i c t the use of MTX "to severe, disabling proven cases r e c a l c i t r a n t to more conservative treatment" (FDA Drug B u l l e t i n , 1971). 3. PHARMACOKINETICS The k i n e t i c s of MTX absorption, d i s t r i b u t i o n and elimination have been thoroughly investigated. Bischoff and his co-v;orkers (1971) developed a multicompartment a l mathematical model for computer simulation of MTX pharma-cokinetics at various dose l e v e l s i n several animal species. Oral absorption of MTX i s rapid and complete except for large doses (Freeman, 1958; Henderson et a l . , 1965; Halprin et a l . , 1971). The precise k i n e t i c s characterizing i n t e s t i n a l absorption of o r a l doses i n i n t a c t animals remains uncertain (Bischoff et a_l. , 1971). MTX may u t i l i z e the f o l i c acid active transport system for absorption across the i n t e s t i n a l mucosa ( B e r l i n et a_l. , 1963). U t i l i z a t i o n of a saturatable transport system could explain the incomplete absorption of large o r a l doses of the drug. In some anomalous patients, the so-called "slow absorbers" (Kettel et a l . , 1968), absorption and elimina-t i o n are prolonged. Peak plasma lev e l s of 0.74-1.55 yg/ml are obtained two hours a f t e r o r a l administration of 25 mg of MTX. 37-50% of the MTX c a r r i e d i n the blood i s r e v e r s i b l y bound to plasma protein (Freeman, 1958; Henderson et a l . , 1965; B e r l i n et a l . , 1963). The plasma h a l f - l i f e of such a dose has been estimated as 2-4 1/2 hours (Halprin et a l . , 1971). The plasma h a l f - l i f e of a given dose i s approximately proportional to the fourth root of the body weight (Dedrick et a l . , 1970). MTX i s rap i d l y d i s t r i b u t e d into tissue following intravenous administration (Su l l i v a n e_t a l . , 1966 ; Anderson et_ aJL., 1970). Highest l e v e l s are reached i n l i v e r and kidney with low or n e g l i g i b l e concentrations i n brain, lung, muscle and fat (Fountain et a l . , 1953; Anderson et' a l . , 1970). Peak skin lev e l s four hours a f t e r intravenous or o r a l administration average 450 to 850 ng/gm of tissue as estimated by microbiological assay a f t e r a 50 mg dose of MTX (Liguori et a l . , 1962) and 60 to 80 ng/gm a f t e r a 5 mg dose of r a d i o a c t i v e l y l a b e l l e d MTX (Sull i v a n et a l . , 1966; Anderson et a l . , 1970). The s i g n i f i c a n c e of MTX metabolism i n man i s uncertain. B e r l i n et a l . , (1963) reported there was no evidence of MTX metabolism. Although Henderson ejt a l . , (1965) report three urinary "metabolites" representing 10% 3 of the t o t a l r a d i o a c t i v i t y administered o r a l l y as H-MTX in one out of nine chromatograms, he feels that the "metabolites" may be due to i n v i t r o b a c t e r i a l or chemical conversion i n stored urine. However, Johns et a l . , (1964) have also detected traces of non-MTX r a d i o a c t i v i t y in chromatographed urine samples. Rothenberg (1969), studying resistance to MTX i n leukemic patients, has detected a change i n MTX by some leukemic c e l l s which a l t e r s i t s a f f i n i t y for an anion exchange r e s i n and DHFR. In mice and rats ( O l i v i e r o and Zaharko, 1971) i n t e s t i n a l bacteria meta-bolize MTX. However with human l i v e r enzyme only dichloro MTX oxidase a c t i v i t y has been demonstrated (Johns and Valerino, 1971). Most systemically administered MTX i s rapidl y excreted i n the urine. Estimates of the amounts excreted range from 54 to 88% i n 24 hours (Henderson et a l . , 1965) to 85 to 100% i n 12 hours (Freeman, 1958). Recently, L i e g l e r et a l . (1969) proposed that MTX u t i l i z e s a renal tubular secretory mechanism common to many organic acids. Co-administration of compounds such as s a l i c y l a t e s , aminohippurate and sulphonamides, which normally also u t i l i z e t h i s excretion pathway, i n h i b i t renal tubular sec-r e t i o n of MTX and thereby delay elimination. A small amount of MTX i s excreted i n the feces (Henderson et a l . 1965) through b i l i a r y excretion. Enterohepatic c i r c u l a t i o n i s a s i g n i f i c a n t consideration i n MTX pharmacokinetics ( O l i v e r i o and Zaharko, 1971). The r e s i d u a l MTX not r a p i d l y excreted i n the urine i s retained in animal tissues f o r long periods. MTX has been detected i n mouse l i v e r f or 3 weeks to 8 months (Fountain et_ a l . , 195 3) and in human l i v e r at autopsy as long as 116 days a f t e r cessation of MTX therapy (Charache et a l . , 1960). It has been suggested that binding of MTX by DHFR i s responsible for i t s prolonged retention i n the l i v e r (Werkheiser, 1963; Johns et a l . , 1964). 17. 4. I N C R E A S I N G T H E T H E R A P E U T I C I N D E X O F M T X Various approaches have been taken i n an attempt to increase the therapeutic index of M T X treatment of p s o r i a s i s . a) "Leucovorin rescue" The "leucovorin rescue" technique has been par-t i a l l y successful i n MTX cancer chemotherapy (Hyrniuk and Bertino, 1969). In p r i n c i p l e , co-adminstration of calcium leucovorin and MTX provides a s u f f i c i e n t supply of reduced folates to protect normal c e l l s without decreasing the response of the tumour to MTX. The increased therapeutic index i s attributed to differences in the k i n e t i c s of growth of normal and tumour c e l l s (Bertino, 19 71). In advanced cancer and leukemia, leucovorin rescue i s a well tolerated therapeutic regimen (Vogler and Jacobs, 1971). With leucovorin administered 24 to 36 hours a f t e r MTX, 2 patients t o l e r a t e up to 300 mg/m MTX i n divided doses over a 24 hour period at weekly i n t e r v a l s without undue t o x i c i t y . In p s o r i a s i s , r e s u l t s have been inconclusive. Ive and de Saram (197) and Roenigk e_t a l . (1969) have maintained p s o r i a s i s under control and decreased the incidence of side e f f e c t s using t h i s technique. However, Cipriano et a l • (1970) report f a i l u r e of leucovorin rescue with rapid exacerbation of p s o r i a t i c lesions previously controlled by MTX. b) Modified dosage schedule Current dosage regimens for o r a l and parenteral MTX i n p s o r i a s i s are l a r g e l y empirical. Weinstein's modified dosage schedule, based on the current knowledge of epidermal c e l l p r o l i f e r a t i o n k i n e t i c s i n p s o r i a s i s , i s an attempt to introduce a more r a t i o n a l MTX therapy regimen f o r p s o r i a s i s . 2.5 to 5.0 mg of MTX i s adminis-tered o r a l l y at 12 hour i n t e r v a l s f o r three doses each week. The epidermal c e l l s i n p s o r i a s i s complete a germina-t i v e c e l l cycle every 37 hours (Weinstein and Frost, 1968), whereas normal epidermal c e l l s complete a cycle every i+57 hours. A single 10 to 50 mg dose of MTX given system!-c a l l y i n h i b i t s DNA synthesis f o r 12 to 16 hours (Weinstein et a l . , 1971). Therefore, o r a l administration of MTX at 12 hour i n t e r v a l s f o r three doses each week should i n h i b i t DNA synthesis for 36 hours — the length of the p s o r i a t i c c e l l c ycle. This new dosage schedule lowers the t o t a l weekly dose of MTX from 2 0-2 5 mg per week to 7.5-15 mg per week; i . e . , three doses of 2.5-5.0 mg. Of the 26 patients with severe p s o r i a s i s treated with t h i s new three-dose regimen, 20 achieved 75-100% improvement with minimal side e f f e c t s (Weinstein and Frost, 1971). With a lower t o t a l dose per week and lower maximum blood l e v e l s , i t i s hoped that the c l i n i c a l effectiveness of t h i s schedule w i l l be better than and t o x i c i t y less than with established dosage schedules. Evaluation of the long term e f f e c t s of t h i s new dosage schedule must await further sudies. c) Alternative routes of administration Since p s o r i a s i s i s a skin disease, t o p i c a l medica-t i o n would seem a l o g i c a l a l t e r n a t i v e to systemic therapy. I d e a l l y , more l o c a l i z e d therapy applied d i r e c t l y to the diseased s i t e should decrease the systemic side e f f e c t s and concentrate maximal therapeutic e f f e c t in the skin. Although Fry and McMinn (19 67) claimed improvement i n p s o r i a t i c patches following t o p i c a l a p p l i c a t i o n of 0.2% MTX i n aqueous cream, t h e i r r e s u l t s have not been confirmed. The b e n e f i c i a l , e f f e c t s attributed to t o p i c a l MTX by Allenby (1966) are dubious because of a d d i t i o n a l intermittent applications of betamethasone-17-valerate. Other i n v e s t i -gators (Van Scott and Reinertson, 1959; Nurse, 1963; Frank et_ a_l. , 1968 ; Comaish and J u h l i n , 1969) have observed l i t t l e c l i n i c a l e f f e c t following t o p i c a l application of 0.1 to 10% MTX creams and solutions and also prolonged i n t r a l e s i o n a l infusions with MTX concentrations higher than peak plasma leve l s obtained a f t e r o r a l dosage. Various theories, most neither confirmed nor denied by experimental fact , have been proposed to explain the lack of effectiveness of l o c a l MTX. i ) A systematic s i t e of action Van Scott and Reinertson (1959) f i r s t suggested that pharmacological action of MTX i n pso r i a s i s might require i n i t i a t i o n at a distant systemic s i t e . MTX may act on skin c e l l s i n d i r e c t l y by. i t s e f f e c t on the l i v e r (Comaish, 196G). In h i b i t i o n of hepatic production of 5 N -methylTHF — one of the pool of metabolically active reduced folates -- may be more important for the c l i n i c a l effectiveness of MTX than i n h i b i t i o n of endogenous pro-duction of these compounds by the epidermal c e l l s (Cipriano ejt a l . , 1970). A generalized depletion of body stores of f o l i n i c acid may be necessary before MTX can act on the p s o r i a t i c epidermal c e l l s (Nurse, 1963). Malabsorption of dietary f o l a t e induced by MTX may also contribute to folate deficiency (Hepner, 1969). i i ) An active metabolite of MTX Evidence concerning i n vivo metabolism of MTX i n . man i s c o n f l i c t i n g ( B e r l i n et a_l. , 1963 ; Johns et_ a l . , 1964; Henderson et a l . , 1965). Comaish (1969) has suggested that conversion products, i f formed, may provide an explanation for the effectiveness of systemic over 3 t o p i c a l MTX. Recent demonstration of i n h i b i t i o n of H-UdR incorporation by l o c a l l y applied MTX i n explants of human skin (Marks et_ a l . , 1971) casts doubt on t h i s theory. i i i ) I n s t a b i l i t y of aqueous MTX preparations Fry and McMinn (1967) suggested that i n s t a b i l i t y of aqueous MTX preparations might play a part i n the negative r e s u l t s obtained by Van Scott and Reinerston (1959) and Nurse (1963) i n t h e i r t r i a l s with t o p i c a l MTX. However, Comaish and Juhlin (1969) refuted t h i s theory in t h e i r l a t e r study by using materials within a maximum of seven days a f t e r preparation and s t i l l obtaining negative r e s u l t s . iv) Lack of penetration Although lack of percutaneous penetration has often been considered as a possible mechanism for the ineffectiveness of t o p i c a l MTX, t h i s theory i s usually dismissed. Allenby (1966) i n f e r r e d that some MTX was absorbed a f t e r t o p i c a l a p p l i c a t i o n since two readings of serum folates were below the normal range. Condit (cited by Van Scott and Reinertson, 1959) has detected MTX i n the urine a f t e r t o p i c a l a p p l i c a t i o n . The only quantitative estimate of percutaneously absorbed MTX i n vivo reports 0.06 to 0.5% of the t o t a l t o p i c a l l y - a p p l i e d dose i n the urine a f t e r 72 hours i n 4 patients (Comaish and J u h l i n , 1969) v) High rate of t r a n s i t I n t r a l e s i o n a l MTX i s also i n e f f e c t i v e i n p s o r i a s i s . In t h i s case, too rapid d i s p e r s a l from the s i t e of i n j e c t i o n may account for the ineffectiveness of MTX. Comaish and Juh l i n (1969) also claim that rapid t r a n s i t rates through the epidermis may account for t h e i r i n a b i l i t y to demonstrate epidermal l a b e l l i n g by autoradiography af t e r t o p i c a l a p p l i c a t i o n . B. PERCUTANEOUS PENETRATION Although d e f i n i t i v e studies of the percutaneous penetration of MTX have not been reported, several excellent reviews on the percutaneous absorption of numerous other compounds have appeared i n the l i t e r a t u r e i n the past decade (Malkinson, 1964; Tregear, 1966; Barrett, 1969; Idson, 1971a, 19 71b; Scheuplein and Blank, 1971). 1. SKIN STRUCTURE AND DIFFUSION PATHWAYS The skin consists of a s u p e r f i c i a l e p i t h e l i a l layer, the epidermis, and a vascular bed of connective t i s s u e , the dermis. The epidermis i t s e l f i s composed of four layers or s t r a t a : the stratum germinativum or basal layer, the stratum spinosum, stratum granulosum and stratum corneum or horny layer (Figure 5). The stratum corneum i s composed of an i n t e r d i g i t a t e d network of b i o l o g i c a l l y i n a c t i v e epidermal c e l l s . Within the c e l l s , k eratin filaments 60-80 X i n diameter are d i s t r i b u t e d i n an amorphous matrix of l i p i d s and non-fibrous protein. At the macromolecular l e v e l , hydration of the i n t e r c e l l u l a r keratin i n the stratum corneum produces a stable two phase system — a network of non-polar l i p i d dispersed i n a continuous aqueous polar medium (Scheuplein and Blank, 19 71). The lower epidermal layers contain c e l l s i n varying stages of d i f f e r e n t i a t i o n and k e r a t i n i z a t i o n . Epidermal c e l l s produced by mitotic d i v i s i o n i n the stratum germinativum, or basal c e l l layer 23. stratum corneum. stratum granulosum-stratum spinosum-stratum germinativum sebaceous gland-hair follicle--sweat gland -subcutaneous fat ' FIGURE 5: Diagrammatic representation of human skin structure migrate upward, ke r a t i n i z e and are shed as c o r n i f i e d flakes Recent evidence suggests that the time for t o t a l epidermal renewal i s between 5 2 and 7 5 days i n normal skin and between 8 and 10 days i n p s o r i a t i c (Halprin, 1972). The mesodermally-derived dermis consists of a network of collagenous, r e t i c u l a r and e l a s t i c f i b e r s i n a mucopolysaccharide ground substance. The blood vessels of the dermis supply nutrients to and transport substances from the overlying layers of the non-vascularized epidermis. Intact skin, once thought to be impervious to a l l external applications, w i l l , to varying degrees, permit passage of most substances (Feldman and Maibach, 1968). T o p i c a l l y applied substances penetrate skin primarily by two routes -- transepidermal and transappendageal (Malkinson 1964). The predominant bulk d i f f u s i o n pathway i s through the c e l l s of the stratum corneum. The skin appendages --sweat glands, h a i r f o l l i c l e s and sebaceous glands -- act as shunts, d i f f u s i o n pathways bypassing the route through the epidermal c e l l s . Except f o r molecules which penetrate the stratum corneum slowly, the small t o t a l area of the — 3 9 2 skin appendages (10 cm /cm ) minimizes t h e i r e f f e c t on skin permeability (Scheuplein and Blank, 19 71). Before steady state d i f f u s i o n i s established shunt d i f f u s i o n may be more s i g n i f i c a n t (Blank, Scheuplein and MacFarlane, 1967). 2. THE "BARRIER" Relative to the stratum corneum, the viable e p i -dermis and the dermis are permeable to most substances. For example, the d i f f u s i o n a l resistance of the horny layer to water i s approximately 1000 times greater than the resistance of the lower layers of the epidermis or dermis. D i f f u s i o n through the stratum corneum i s the p r i n c i p a l rate l i m i t i n g step i n percutaneous absorption. Although investigators searched for a s p e c i f i c " b a r r i e r " within the epidermis, the best evidence indicates the horny layer i s uniformly impermeable. The p r i n c i p a l " b a r r i e r " function of the epidermis resides i n the entire thickness of the stratum corneum (Scheuplein and Blank, 1971). Modern th e o r i s t s doubt the existence of a c r i t i c a l membrane or " b a r r i e r layer" at the base of the stratum corneum (Tregear, 1966; Idson, 1971b). The r e l a t i v e l y a c e l l u l a r dermis presents l i t t l e b a r r i e r to penetration. However, the dermis may bind some substances i n a manner analgous to plasma protein binding (Feldman and Maibach, 1968). Griesemer (1960) suggested that the mucopolysaccharides of the ground substance may absorb polar compounds. 3. MATHEMATICAL MODELS FOR PERCUTANEOUS ABSORPTION The passage of drugs across skin i s a passive d i f f u s i o n process (Ostrenga ejt a_l. , 1971a; Idson, 1971a) governed by Fick's f i r s t law of d i f f u s i o n : = " D A (Equation 1) dt dx The amount of substance, dq_ d i f f u s i n g i n time i n t e r v a l , dt_ across a plane of area, A i s d i r e c t l y pro-portional to the concentration gradient across the plane, dc/dx and the d i f f u s i o n c o e f f i c i e n t , D. With t h i s basis, Higuchi (19 60) expressed the simplest model for the percutaneous absorption process: dq = Kjn C QA D ^ g (Equation 2) The amount of substance d i f f u s i n g through skin area, A and thickness, 6_ i s d i r e c t l y proportional to the v e h i c l e - s k i n p a r t i t i o n c o e f f i c i e n t , K m, the d i f f u s i v i t y of the drug i n the skin, D and the concentration applied to the surface •m of the skin, C Q. A more correct expression presents the d i f f u s i o n rate i n terms of thermodynamic a c t i v i t y of the penetrant i n the vehicle rather than concentration: g£ = * D A (Equation 3) dt y o where a i s the thermodynamic a c t i v i t y of the drug i n the vehicle and y_, the a c t i v i t y c o e f f i c i e n t i n the b a r r i e r . The d r i v i n g force for drug d i f f u s i o n i s the thermodynamic po t e n t i a l gradient across the skin b a r r i e r between the applied vehicle and the deeper tissues (Higuchi, 1960). Because skin i s i n essence, a "thick membrane", the d i f f u s i o n process cannot be characterized by a single parameter. A s i g n i f i c a n t amount of d i f f u s i n g substance accumulates within the membrane before penetration reaches a steady state (Ainsworth, 1960). Treherne (1956) noted there was always a delay period during which penetration rate increased from zero toward a steady state value. The steady state penetration rate i s the basic parameter required for measurement and comparison of skin permea-b i l i t y (Tregear, 1966). Crank (19 56) presents an equation for the passage of substances through a composite membrane of n number of component sheets of thickness, . The equation: Q _ Dt 2 9 ( - l ) n - D n 2 T T 2 t / 1 2 „ . . f h . Tr ~ —T - ^p> o ~ (1-e ) Equation (4) 1 C o i 2 1 7 2 1 n 2 encompasses the non-linear lag period as well as steady state d i f f u s i o n . Assuming a p a r t i t i o n c o e f f i c i e n t of unity, the quantity, Q passing through a unit area of membrane af t e r time, t i s related to the d i f f u s i o n constant, D and the concentration at the surface of the membrane, C Q. As t approaches i n f i n i t y and d i f f u s i o n rate approaches steady state, the exponential term becomes n e g l i g i b l e . ? oo n 1 TT Since £ (-1) — = j ^ " , as t -»• 0 0 a graph of Q versus t n approaches the l i n e : DC p 2 Q = - ~ (t - ~ ) (Equation 5) 2 with an intercept on the t-axis of I /6D; i . e . , the lag period. 28. To describe the d i f f u s i o n of drugs through skin, the equation i s usually s i m p l i f i e d to: Q = k A C ( t - T + t " t / x ) (Equation 6) P 6 r e l a t i n g the quantity passing through the skin b a r r i e r at time, t to a permeability constant, k^, the concentration gradient between the vehicle and the lower layers of the skin, AC and the lag time, x (Ainsworth, 196 0; Treherne, 1956). Therefore, at steady state: Q = k AC (t - T ) (Equation 7) P K D and k = m m p — ^ (Equation 8) with. K^, the vehicle-membrane p a r t i t i o n c o e f f i c i e n t , D m, the d i f f u s i v i t y of the drug i n the membrane and cS_, the thickness of the membrane (Scheuplein ejt a l . , 1969). The lag time i s a function of membrane thickness, 6_ and d i f f u s i v i t y of the penetrant, (Ostrenga ejt a l . , 1971a; Flynn and Roseman, 1971): 6 2 T = gjj— (Equation 9) Using dimethylpolysiloxane membranes, Flynn ~ et a l . , (1972) derived a mathematical expression f o r d i f f u s i o n layer control of b a r r i e r f l u x . Penetrant f l u x through a membrane sandwiched between two l i q u i d phases i s co n t r o l l e d by the chemical p o t e n t i a l gradient i n the d i f f u s i o n layers. Thus, the permeability constant i s re l a t e d to the d i f f u s i v i t y 29. of the penetrant i n the d i f f u s i o n layer, D a q and the t h i c k -ness of the d i f f u s i o n l a y e r , h rather than the d i f f u s i v i t y i n and thickness of the membrane, D m and &_ as i n equation 8 (Figure 6). The t o t a l amount d i f f u s i n g into compartment V, Mv i s calculated from: M = Daq A Co Ct - Km 6 h a q (1 - e - 2 Daq t/K 6 h )] v 2 D m a q aq aq (Equation 10) D i f f u s i o n layer control occurs when K m D m £h aq >> "SD^. 4. FACTORS AFFECTING PERCUTANEOUS ABSORPTION a) Skin v a r i a t i o n The permeability of mammalian skin varies with species, age and body region. Human skin i s generally less permeable than r a b b i t , rodent or pig skin (Tregear, 1966; Bartek et a l . , 1972). However, the r e l a t i o n s h i p between species i s not consistent for d i f f e r e n t substances (Idson, 1971a). Foetal and infant skin i s more permeable than adult skin. In the aged, dermal atrophy and epidermal changes may a f f e c t absorption (Idson, 1971a). Within any given i n d i v i d u a l , percutaneous absorp-t i o n varies from one s i t e to another. The v a r i a t i o n i n permeability r e f l e c t s the regional differences i n structure and chemistry of human stratum corneum (Scheuplein and Blank, 1971). For example, the order of permeability of "^C-hydrocortisone through skin from various body regions 30. c o g "c OJ u c o u vehicl :nembrane "K \ \ \ receptor -> FIGURE 6a: Concentration p r o f i l e through a membrane b a r r i e r under membrane flux control (Higuchi, 1960). 1 1 1 1 1 1 membrane — & IV (*C^---> sink '°q. FIGURE 6b: Concentration p r o f i l e through a membrane-diffusion layer b a r r i e r under d i f f u s i o n layer flux control (Flynn et a l . , 1972 ). i s scrotum > forehead > scalp and a x i l l a > back > forearm > palm (Feldman and Maibach, 1968). Despite the greater thickness of palmar and plantar c a l l u s (400-60uu) compared to the back, arms and abdomen (10-20u), t h i s tissue i s generally an i n f e r i o r b a r r i e r . For the passage of simple, small molecules through skin, Scheuplein and Blank (19 71) have suggested the following order of decreasing permea-b i l i t y : plantar > palmar > dorsum of hand > s c r o t a l and postauricular > a x i l l a and scalp > arms, legs, trunk. Although blood flow through the dermal vessels may a f f e c t the rate of clearance of drugs from the sub-epidermal regions and, t h e o r e t i c a l l y , the rate of absorption s f o r most substances the l i m i t i n g factor i s d i f f u s i o n across the stratum corneum (Tregear, 1968); Scheuplein and Blank, 1971). The degree of hydration of the stratum corneum also a f f e c t s the permeability of skin. The presence of moisture increases the d i f f u s i o n rate of a l l substances penetrating skin (Idson, 1971a). The greater the aqueous s o l u b i l i t y of a compound; the greater the enhancement of permeability by hydration of the horny layer (Wurster, 1965). The enhancement of absorption by occlusive dressings has been linked to t h e i r hydrating e f f e c t on the stratum corneum. b) C h a r a c t e r i s t i c s of the penetrant The a c t i v a t i o n energy for d i f f u s i o n ; i . e . , the energy l e v e l to which a molecule must be raised before i t can break r e s t r a i n i n g bonds and d i f f u s e , i s a function of the c h a r a c t e r i s t i c s of the penetrant as well as the d i f f u s i o n pathway (Blank et a l . , 19 67). Over a small range of molecular 'size, permeability c o e f f i c i e n t s cannot be correlated with the size of the penetrating molecule (Tregear, 1966). Over a wider range, small molecules generally penetrate more ra p i d l y than large molecules (Barrett, 1969; Idson, 1971a). Polar and non-polar compounds di f f u s e through the stratum corneum by d i f f e r e n t mechanisms. Polar substances appear to penetrate through the immobilized water of the stratum corneum; non-polar through the non-aqueous matrix (Blank e t a^ L. , 1967). The a c t i v a t i o n energy for d i f f u s i o n increases as the molecules become more polar (Blank e_t a l . , 1967; Scheuplein and Blank, 1971). The s o l u b i l i t y c h a r a c t e r i s t i c s prerequisite for percutaneous penetration are uncertain (Tregear, 1966). The penetration flux increases as the s o l u b i l i t y of the penetrant i n the stratum corneum increases (Blank and Scheuplein, 1969). Because of the biphasic character of b i o l o g i c a l membranes, oil/water p a r t i t i o n c o e f f i c i e n t s strongly influence passive d i f f u s i o n . Investigators have correlated permeability constants of numerous compounds with t h e i r p a r t i t i o n between water and non-polar solvents (Idson, 1971a). It has been suggested that an oil/water p a r t i t i o n c o e f f i c i e n t of unity i s the most favourable f o r skin penetration (Treherne, 1956). On the other hand, some ions only barely soluble i n organic solvents and some organic compounds with n e g l i g i b l e aqueous s o l u b i l i t y pene-trat e skin more ra p i d l y than predicted by t h i s theory (Tregear, 1966). Lien et aJL. (1971) have reported quan-t i t a t i v e c orrelations of percutaneous absorption with o i l / water p a r t i t i o n c o e f f i c i e n t s . The log of the permeability constant of alcohols and steroids i s l i n e a r l y dependent on the log of the p a r t i t i o n c o e f f i c i e n t i n three solvent systems -- octanol/water, o l i v e oil/water and amyl caproate/ water. For the percutaneous absorption of alcohols, the log of octanol/water and o l i v e oil/water p a r t i t i o n c o e f f i c i e n t s correlate with permeability constants as well as stratum corneum/water p a r t i t i o n , with c o r r e l a t i o n c o e f f i c i e n t s (r) of 0.979, 0.977 and 0.986 respe c t i v e l y . For the per-cutaneous absorption of s t e r o i d s , both amyl caproate and stratum corneum/water p a r t i t i o n y i e l d high correlations (r = 0.93). However, hexadecane/water p a r t i t i o n gives a r e l a t i v e l y poor c o r r e l a t i o n (r = 0.865), (Lien ejt a l . , 1971). c) Vehicle e f f e c t s B i o a v a i l a b i l i t y of a t o p i c a l l y applied drug depends both on release from the vehicle and permeability through the skin (Idson, 1971a; Ostrenga et aJL. , 1971a). Although composition of the vehicle can a f f e c t both processes, no vehicle w i l l carry through the epidermis a compound otherwise incapable of penetration'(Malkinson, 1964). The r e l a t i v e magnitudes of rate constants for d i f f u s i o n of a drug within the vehicle, p a r t i t i o n between vehicle and skin, and d i f f u s i o n through the skin b a r r i e r determine the rate con-t r o l l i n g step i n percutaneous absorption. For the penetra-t i o n of most substances through i n t a c t skin, the rate c o n t r o l l i n g step i s i n the skin b a r r i e r (Higuchi, 1960; Ostrenga et a l . , 1971a). However, vehicle composition can f a c i l i t a t e skin penetration by modifying a drug's d i f f u s i o n c o e f f i c i e n t i n the b a r r i e r , the vehicle skin p a r t i t i o n c o e f f i c i e n t and s o l u b i l i t y i n the vehicle (Ostrenga ejt al_. , 1971b). The s o l u b i l i t y of a compound i n the chosen vehicle determines the concentration of d i f f u s i b l e drug. Ostrenga et a l . (1971c) were able to e s t a b l i s h a rank order c o r r e l a t i o n between release, % drug s o l u b i l i z e d and vaso-c o n s t r i c t o r response of a stero i d i n a series of creams. The amount of drug absorbed from an ointment suspension i s related to the s o l u b i l i t y i n the external phase of the ointment, thus: Q = (2 A D„ C c t ) 1 / 2 (Equation 11) where, Q i s the amount absorbed at time, t , A the concen-t r a t i o n of drug, C the s o l u b i l i t y of drug i n the external phase of the ointment and D v the d i f f u s i o n constant of the drug i n the external phase (Higuchi, 1960). The d i f f u s i o n constant of a medicament i n the vehicle i s inversely proportional to the v i s c o s i t y of the base (Higuchi, 1960). Although the simple Stokes-Einstein equation, j) - kt , V STWT) (Equation 12) where k i s the Boltsman constant, r the molecular radius and n_ the v i s c o s i t y , may not hold r i g i d l y f o r complex ointment bases, a decrease i n v i s c o s i t y should increase the d i f f u s i o n constant for the suspended drug (Wurster, 1965). To provide adequate release, a vehicle must p a r t i a l l y dissolve the drug, but not to the extent that the drug remains p r e f e r e n t i a l l y i n the base (Dempski et a l . 1969); i . e . , vehicle/stratum corneum p a r t i t i o n must be adequate. High a f f i n i t y of a vehicle for the drug decreases i t s thermodynamic a c t i v i t y and thus the dr i v i n g force f o r penetration (Higuchi, 1960). The product of p a r t i t i o n c o e f f i c i e n t and concentration of d i f f u s i b l e drug i n the vehicle may be a useful parameter i n predicting the optimal vehicle composition for c l i n i c a l e f f i c a c y (Ostrenga et al^. , 1971b). Vehicle components can modify the d i f f u s i v i t y of a drug i n the skin b a r r i e r by a l t e r i n g the b a r r i e r i t s e l f . The so-called penetration "accelerants" such as urea, DMSO, DMA, DMF and surfactants, combine with or dissolve i n the substance of the skin b a r r i e r (Idson, 1971a). DMSO, DMF and DMA, strongly hygroscopic substances may increase permeability by increasing the hydration of the stratum corneum. Reversible configurational changes i n skin protein structure by sub s t i t u t i o n of DMSO fo r bound water, with resultant swelling, may form channels through the stratum corneum matrix (Idson, 1971a). Propylene g l y c o l , on the other hand, by dehydration, apparently renders the b a r r i e r less p l i a b l e and less permeable to steroids (Ostrenga et a l . , 1971b). Surfactants damage skin and as a r e s u l t produce profound changes i n skin permeability. Non-ionic surfactants have less e f f e c t on the stratum corneum than e i t h e r c a t i o n i c s or anionics (Scheuplein and Blank; 1971). 1% sodium l a u r y l sulphate, an anionic surfactant, produces large increases <^ -— i n water permeability. Damage induced increases continuously with, exposure to the surfactant. Extended treatment may produce i r r e v e r s i b l e changes by denaturation of skin protein. Disruption of the h e l i c a l structure of ker a t i n may extend the protein filaments and expand the structure of the membrane (Scheuplein and Ross, 1970). Organic solvents such as chloroform and ether extract s i g n i f i c a n t amounts of l i p i d s from the stratum corneum. Removal of portions of t h i s l i p i d matrix produces a porous, non-selective membrane (Scheuplein and Blank, 1971). The higher molecular weight, less v o l a t i l e organic solvents such as alcohols, o l i v e o i l and isopropyl palmitate do not appear to damage the skin b a r r i e r (Scheuplein and Blank, 1971). STATEMENT OF PROBLEM Despite the c l i n i c a l effectiveness of systemic MTX i n p s o r i a s i s , r e s u l t s with t o p i c a l MTX have been d i s -appointing. If t o p i c a l MTX were e f f e c t i v e , t h i s mode of administration should d e l i v e r the required concentration of drug s p e c i f i c a l l y to the diseased skin and circumvent the t o x i c systemic side e f f e c t s of o r a l and parenteral dosage. The few papers which have dealt with the c l i n i c a l e f f i c a c y of t o p i c a l MTX i n ps o r i a s i s are confusing. Some investigators maintain that i t i s e f f e c t i v e , others that i t i s e f f e c t i v e when combined with systemic MTX, and s t i l l others that i t i s t o t a l l y i n e f f e c t i v e . Unsuccessful therapy with t o p i c a l MTX has prompted several theories to explain the lack of effectiveness. A recurring theme i s the unfavourable penetration characteris-t i c s of MTX. Suggestions range from "MTX does not penetrate skin" to "MTX penetrates skin too r a p i d l y " . However, l i t t l e d e f i n i t i v e work has been attempted i n t h i s area. To c l a r i f y the controversy over the c l i n i c a l e f f i c a c y and percutaneous absorption of t o p i c a l MTX i n p s o r i a s i s , i t was decided to determine: 1) the c l i n i c a l e f f i c a c y of t o p i c a l MTX -- 0.2% cream, a concentration reported to be e f f e c t i v e by Fry and McMinn (19 67); 2) the c l i n i c a l e f f i c a c y of l o c a l l y administered MTX 38. deposited below the stratum corneum or "percutaneous b a r r i e r " ; i . e . , i n j e c t e d i n t r a l e s i o n a l l y ; 3) i n vivo, i n experimental animals — h a i r l e s s mice — the blood, skin and l i v e r l e v e l s reached by applying MTX t o p i c a l l y ; 4) i n vivo, i n p s o r i a t i c patients, the blood and skin l e v e l s reached by applying MTX t o p i c a l l y ; 5) more completely, i n v i t r o , the percutaneous d i f f u s i o n k i n e t i c s of MTX — a lipophobic weak ac i d ; i . e . , a. how much MTX can penetrate skin i n a reasonable period, f o r example 24 hours? b. does i o n i z a t i o n of the weak acid a f f e c t the penetration rate? i . e . , what i s the e f f e c t of changing the pH of the vehicle? c. i s the penetration rate r e l a t e d to the p a r t i -t i o n c o e f f i c i e n t of MTX between o i l (octanol) and water? d. what concentrations are reached within the skin a f t e r t o p i c a l application? i . e . , i s there any p o s s i b i l i t y that concentrations can be reached i n the skin by t o p i c a l a p p l i c a t i o n that approach those achieved by the thera-p e u t i c a l l y e f f e c t i v e systemic route? EXPERIMENTAL I. METHODOLOGY A. ANALYTICAL METHODS FOR MTX 1. Fluorometry MTX was analyzed i n plasma, skin and l i v e r using Freeman's (1957) fluorometric method for the measurement of amethopterin i n plasma. Wavelength scans revealed e x c i t a t i o n and emission peaks at 350 and 462 nm respectively (Figure 7). Standard curves were l i n e a r i n the concentra-t i o n range of 0.01 to 8.0 yg/ml (Figure 8). Plasma (0.2 - 0.5 ml), whole mouse l i v e r s and skin (0.2 mm deep dermatome s l i c e s ) were l y o p h i l i z e d , weighed and homogenized or d i l u t e d with 4.0 ml of d i s t i l l e d water. Protein was removed from tissu e homogenates by centrifugation a f t e r addition of t r i c h l o r o a c e t i c acid (1 ml, 15%). A 4.0 ml aliquot of the cl e a r supernatant was withdrawn and neutra-l i z e d by the addition of sodium hydroxide (0.65 ml, 3.24%). A s l i g h t l y a c i d i c pH (5.4) was maintained by an acetate buffer (0.25 ml, 5 M). MTX within the samples was oxidized by potassium permanganate (0.05 ml, 4%) for 5 minutes. Hydrogen peroxide (0.10 ml, 3%) reduced the excess permanganate and rendered the solution colourless. Fluores-cence was read before oxidation and 3 minutes a f t e r addition of hydrogen peroxide. Concentration of MTX was proportional to the change i n r e l a t i v e fluorescence i n t e n s i t y (AR.F.I.) I I \ ' / \ ' / V I t N / X 300 340 380 420 460 500 WAVELENGTH ( n m ) Excitation spectra Emission spectra at an emission X 462 nm at an excitation A 350 nm for OXIDIZED MTX and UNOXID1ZED MTX FIGURE 7: Fluorescence spectra of MTX (0.4 yg/ml) 4 1 . co a -Z UJ z C O N C E N T R A T I O N O F M T X ( y g / m l ) FIGURE 8: Fluorometric standard curve for MTX (Figure 8). Separate standard curves were prepared for each tissue assayed; i . e . , plasma, skin and l i v e r , using a series of standard solutions and a weighed amount of tissue containing no MTX. Standard curves f o r each tissue were l i n e a r but slopes and intercepts were not equivalent. There-fore, c a l c u l a t i o n of concentrations i n plasma, skin or l i v e r samples was always made using the regression l i n e slope and intercept of the appropriate standard curve. Recovery of MTX from skin was estimated by analy-zing concentrations a f t e r intradermal i n j e c t i o n of known amounts of MTX into pieces of excised circumcision skin. The average recovery was 107%. From standard curves, minimal l e v e l s of MTX detectable f l u o r o m e t r i c a l l y were estimated as 0.0 5 ug MTX per skin sample or m i l l i l i t r e of plasma. Minimal l e v e l s were estimated as those giving a fluorescence i n t e n s i t y three times higher than a blank solu t i o n . 2. Spectrophotometry To analyze the higher concentrations of MTX for p a r t i t i o n c o e f f i c i e n t and s o l u b i l i t y studies, a simpler, more d i r e c t spectrophotometric method was adequate. Spectral scans (Figure 9) revealed several absorbance peaks i n the u l t r a v i o l e t region. However, only the absorbance peak at 305 nm (FigurelO)was unaffected both i n p o s i t i o n and i n t e n s i t y by pH changes from 2.3 to 8.9. The changing amplitude of other peaks presumably r e f l e c t s the d i f f e r e n t i a l 43. 200 250 300 350 W A V E L E N G T H ( nm ) at pH 4.35 and pH 8.99 FIGURE 9: UV absorption spectra of MTX at pH 4.3 and 8.9. FIGURE 10: E f f e c t of pH on abs o r p t i v i t y of MTX at three d i f f e r e n t wavelengths. (210 nm • — • , 260 nm A — A , and 305 nm • — O ) . a b s o r p t i v i t y of the various species of MTX formed as the pH i s altered; i . e . , molecular, mono-anionic and di-anionic. At 30 5 nm, standard curves were l i n e a r at concentrations of 0.004 to 0.002 gm/1 and yielded an absorptivity value of 50.67 8 f o r duplicate samples at 6 d i f f e r e n t concentrations at 8 d i f f e r e n t pH values (Figure 11). Linear regression analysis of pooled samples at the 8 d i f f e r e n t pH values o yielded a c o e f f i c i e n t of determination (r ) of 0.991 for the standard curve. 3. Liquid S c i n t i l l a t i o n Counting To analyze r e l i a b l y the small amounts of MTX i n skin or penetrating through skin from t o p i c a l a p p l i c a t i o n , radiotracer techniques were necessary. 0.5 ml d i f f u s i o n f l u i d samples or NCS-digested skin samples were prepared with a dioxane-based s c i n t i l l a t i o n f l u i d (Comaish and J u h l i n , 1969) to give a f i n a l counting volume of 15 ml. Samples were counted for a minimum of three cycles at 10 minutes per sample. In the Picker Nuclear Liquimat, most e f f i c i e n t counting for t r i t i u m i n the dioxane-based s c i n t i l l a t i o n f l u i d was determined at channel settings of 25 for the lower discriminator energy l e v e l and 350 for the upper. Sample counts were averaged and corrected to disintegrations per minute (dpm) by comparison to a standard quench correction curve (Figure 12) for an external Cs source. Counting e f f i c i e n c y f o r 0.5 ml d i f f u s i o n f l u i d samples averaged approximately 38% and for 50 mg NCS-digested skin, approximately 27%. aoi ojoa C O N C E N T R A T I O N O F MTX ( gm/ I itre ) FIGURE 11: Spectrophotometric standard curve for MTX from UV absorbance at three d i f f e r e n t pH values. (pH 7.9 • , pH 5.3 o and pK 2.3 A ) . O iff 1 1 1 1 1 2 0 I O 13/1 EXTERNAL STANDARD Cs C P M x 10~3 FIGURE Quench correction curve external!- 3? Cs standard Liquimat. for t r i t i u m with an in the Picker Nuclear 48. In the Nuclear Chicago Isocap 300, samples were counted using an external standard r a t i o program fo r t r i t i u m as supplied by the manufacturer of the instrument. Using t h i s program, the quench correction curve for the 1 o q external Ba source of the Isocap 300 was lxnear (Figure 13). E f f i c i e n c y of counted samples could be calculated d i r e c t l y from the l i n e a r regression l i n e . Counting e f f i c i e n c y for 0.5 ml d i f f u s i o n f l u i d samples averaged 45%. B. ANALYSIS OF DHFR ACTIVITY 1. Spectrophotometry The spectrophotometric method used for analysis of DHFR a c t i v i t y i s a well established a n a l y t i c a l procedure (Bertino and Fischer, 1964; Baker et a l . , 1964). The decrease i n u l t r a v i o l e t absorbance of incubation mixtures containing DHF substrate, NADPH co-factor and appropriate buffer ingredients was monitored at 340nm using the Beckman DB-GT spectrophotometer, with a temperature controlled c e l l chamber and recorder. The molar abs o r p t i v i t y change for the enzyme catalyzed reaction represents a t o t a l absorptivity change for conversion of substrate to product; i . e . , DHF to THF, and oxidation of the co-factor NADPH to NADP (Figure 14). Standard curves for each species — DHF, THF, NADPH and NADP -- determined i n duplicate at 6 to 7 concentrations obeyed Beer's law at 340 nm; i . e . , curves were l i n e a r and passed through the o r i g i n (Figure 15). 49. O 1 2 3 4 E X T E R N A L S T A N D A R D RATIO FIGURE 13: Quench correction curve for t r i t i u m using an 13 3 external standard ( Ba) r a t i o method in the Nuclear Chicago Isocap 300. 50. DIHYDROFOLATE TETRAHYDROFOLATE NADPH NADP I ABSORBANCE ( 340nm) due to DHF • THF & NADPH • NADP FIGURE 14: Reactions involved i n the spectrophotometric assay for dihydrofolate reductase a c t i v i t y . C O N C E N T R A T I O N ( m moles/litre) FIGURE 15a: Spectrophotometric standard curves for DHF (o o)*and THF (• •) at 340 nm To convert enzyme a c t i v i t y recorded as decrease i n absorbance per time to micromoles of substrate reduced per time, the t o t a l molar absorptivity change for the reaction was calcu-lated from i n d i v i d u a l standard curves. The molar absorp-t i v i t i e s determined for each species from the slope of standard curves were: mM e NADPH = 5. 220 mM NADP = 0. 0694 £mM DHF = 7. 828 emM THF = 1. 392 Thus, the t o t a l molar abso r p t i v i t y change f o r the enzyme reaction due to conversion of substrate to product (Ae 1 1^ = -6.436) and f o r oxidation of co-factor (Ae™1'1 = -5.151) was Ae™*1 = -11.59. In p a r t i a l l y p u r i f i e d enzyme extracts, NADPH diaphorase a c t i v i t y was frequently present. As a r e s u l t , incubation without substrate produced decreasing UV absor-bance at 34 0 nm due merely to oxidation of NADPH, not coupled to reduction of DHF or DHFR a c t i v i t y . Corrections for a substrate blank were therefore necessary for a l l enzyme reactions monitored spectrophotometrically. Conversion of substrate did not occur without the presence of NADPH. 2. Fluorometry Since diaphorase a c t i v i t y of p a r t i a l l y p u r i f i e d enzyme extracts frequently masked low a c t i v i t y of DHFR i n spectrophotometric a n a l y s i s , a more sensi t i v e and s p e c i f i c method was devised based on the reported (Uyeda and Rabinowitz, 1963) fluorescence of THF. Fluorescence spectra revealed d i s t i n c t peaks for each species involved i n the enzyme reaction. Fluorescence of DHF was maximal at exc i t a t i o n and emission wavelengths of 342 and 425 nm respectively; THF at 302 nm and 364 nm; NADPH at 375 and 458 nm. Linear standard curves were obtained for THF at 6 concentrations ranging from 1 to 10 mM. However, a pH value of 3 for optimum fluorescence of THF (Uyeda and Rabinowitz, 196 3) was unfavourable for the enzyme reaction and a pH of 7.4 f o r optimum enzyme a c t i v i t y was unfavourable for fluorescence of THF. The enzyme reaction could be monitored by follow-ing the increase i n fluorescence at 302 nm ex c i t a t i o n wavelength and 364 nm emission wavelength only a f t e r stopping the reaction and a c i d i f y i n g the mixture with t r i c h l o r o a c e t i c acid. Time curves for the enzyme reaction prepared by stopping the reaction a f t e r minute i n t e r v a l s were not l i n e a r for the i n i t i a l incubation period . Although enzyme a c t i v i t y could be estimated by stopping the reaction at a fixed' time (3 minutes) i n i t i a l v e l o c i t i e s of the DHFR reaction were not obtained. EXPERIMENTAL II . EXPERIMENTS AND RESULTS A. ACTIVITY OF DHFR AND ITS INHIBITION BY MTX The s i t e of action of MTX i n p s o r i a s i s i s not known with c e r t a i n t y . The two most probable s i t e s are the epidermis and the l i v e r . The l o c a t i o n of the s i t e of action w i l l depend on whether the p s o r i a t i c epidermis synthesizes i t s own supplies of reduced folat e s i n s i t u or whether i t receives reduced folat e s from the l i v e r v i a the c i r c u l a t i o n . I f MTX i s to act l o c a l l y i n the pro-l i f e r a t i n g c e l l s of the p s o r i a t i c epidermis, a functional enzyme apparatus, i . e . , DHFR must be present. Thus, attempts were made to i s o l a t e and p u r i f y DHFR from human s u r g i c a l skin according to the technique of Bertino and Fischer (1964). Epidermis (0.2 mm deep dermatome s l i c e s ) was homogenized with 10 volumes of d i s t i l l e d water, centrifuged (10,000 g for 30 minutes), a c i d i f i e d and dialyzed against d i s t i l l e d water. The dialysate was centrifuged (10,000 g for 10 minutes) and the supernatant used f o r enzyme analysis. Using a f l u o r -ometric method, DHFR a c t i v i t y and MTX i n h i b i t i o n of such a c t i v i t y could be detected i n homogenates of human su r g i c a l skin. The v e l o c i t i e s of the enzyme reaction measured a f t e r an incubation period of 3 minutes were not i n i t i a l v e l o c i t i e s and therefore did not permit c a l c u l a t i o n of a K value. A pH value of 3 f o r optimum fluorescence m of THF (Uyeda and Rabinowitz, 1963) was unfavourable for the enzyme reaction and consequently enzyme progress curves could not be obtained using a fluorometric assay. Levels of enzyme a c t i v i t y were too low to obtain r e l i a b l e estimates of a c t i v i t y spectrophotometrically. An a l t e r n a t i v e p u r i f i c a t i o n procedure for DHFR was tested using commercially prepared chicken l i v e r acetone powder. P a r t i a l l y p u r i f i e d DHFR was extracted from chicken l i v e r acetone powder by C e l i t e adsorption and ammonium sulphate f r a c t i o n a t i o n according to the method of Baker et a l . (1964). A l l enzyme reactions were performed at 37°C and pH 7.4 (0.05 M t r i s buffer with 10 mM mercaptoethanol and 1 mM versene). The DHF substrate was prepared as a 1.86 mM suspension i n 0.005 N HCI con-t a i n i n g 0.1 M mercaptoethanol, stored at 3-4°C and d i l u t e d d a i l y f o r use at varying concentrations. NADPH, stored as a 3.72 mM solution i n 0.01 M sodium hydroxide, was dil u t e d to 0.744 mM with buffer f o r d a i l y use. The course of the enzyme reaction was followed spectrophotometrically by the decrease i n absorbance at 34 0 nm. Buffer (2.7 ml), DHF (0.1 ml) and enzyme (0.1ml) were eq u i l i b r a t e d i n the chamber of the spectrophotometer. The enzyme reaction was started with addition of NADPH (0.1 ml). Because of the a c t i v i t y of the enzyme prepara-t i o n , scale expansion, both on the recorder and spectro-photometer, was used to obtain a r e l i a b l e estimate of the slope of the enzyme progress curve. The change i n absorbance was calculated d i r e c t l y from the recording, corrected for a blank (containing only NADPH and enzyme) and converted to i n i t i a l v e l o c i t y i n terms of micromoles of substrate converted per minute. The Michaelis constant (K ) for the DHFR reaction m was determined for a substrate concentration range of Q 0.93 to 15.5 x 10~ M DHF. The K value determined from m each of the three standard enzyme a c t i v i t y plots — Lineweaver-Burk (1/V vs. 1/S), Hofstee (S/V vs. S) and Eadie (V vs. V/S) plots — varied s l i g h t l y (Table 1). The average obtained from the three methods of p l o t t i n g was 6.60 x 10~ 7 M ( + 0.27 x IO" 7 S.E.). To determine the ef f e c t s of MTX on the enzyme preparation, MTX (0.917 x 10~ 8 M) was incubated with the enzyme for 5 minutes p r i o r to the addition of substrate. The i n h i b i t i o n constant (K^) was calculated from the appropriate change i n slope or intercept of the various enzyme pl o t s . I n h i b i t i o n appeared to be competitive at pH 7.4 since addition of MTX to the incubation mixture altered the Michaelis constant while the maximal rate remained e s s e n t i a l l y unchanged (Figures 16, 17, 18). To determine the k i n e t i c s of i n h i b i t i o n more accurately, i n h i b i t i o n of the enzyme preparation by varying concentra-tions of MTX was measured. A Dixon plot (Bertino et a l . , 1964) of r e c i p r o c a l v e l o c i t y versus i n h i b i t o r concentration for MTX i n h i b i t i o n at two substrate concentrations (3.1 and TABLE I Kinetic Constants for Chicken Liver Dihydrofolate Reductase at pH 7.4 Determined With and Without the Presence of MTX Control DHFR Ac t i v i t y MTX In h i b i t i o n of DHFR A c t i v i t y K m 7 (moles/litre x 10 ) V max (u moles/min) h g (moles/litre x 10 ) (u moles/min) Lineweaver-Burk 6.70 + 0.384 2.80 + 0.446 0.915 + 0.015 3.41 + 0.040 Eadie 6.26 + 0.411 2.75 + 0.471 0.895 + 0.015 3.47 + 0.075 Hofstee 4.00 + 0.769 2.83 + 0.612 0.945 + 0.025 3.39+ 0.010 Dixon 1.11 FIGURE 16: Lineweaver-Burk plot of chicken l i v e r dihydro-folate reductase a c t i v i t y with (o o) and without (• •) 9.17 x 10" 9 M MTX. 60. I i i i i i i i i O 0.6 LO 1.4 VELOCITY j [ DHF] ( m in 1 ) FIGURE 17: Eadie plot of chicken l i v e r dihydrofolate reductase a c t i v i t y with (o o) and without (• •) 9.17 x 10" 9 M MTX. 61. 1 I I I I L j L O 4 8 1 2 1 6 [DHF] j jmoles/ litre FIGURE 18: Hofstee plot of chicken l i v e r dihydrofolate reductase a c t i v i t y with (o o) and without (• •) 9.17 x 10" 9 H MTX. 9.3 x 10 M DHF produced two l i n e s which intersected at a K i value of 1.1 x I O - 9 M (Figure 19). To determine the tightness of binding of MTX to DHFR the Ackermann-Potter technique was used (Ackermann and Potter, 1949). Reaction v e l o c i t i e s were measured for varying concentrations of enzyme (0.05 to 0.30 ml) i n the presence of 0.917 x 10~ 8 M MTX and/or 9.3 x 10~ 6 M DHF. At pH 7.4, MTX i n h i b i t i o n of DHFR was r e v e r s i b l e . In the -9 presence of 0.917 x 10 M MTX, changes i n enzyme concentration produced a l i n e a r increase i n v e l o c i t y . A plot of v e l o c i t y of reaction versus enzyme concentration ( i . e . , the Ackermann-Potter plot) did not intercept the enzyme axis (Figure 20). The " i n h i b i t e d curve" had a less e r slope than the control curve i n t e r s e c t i n g the l a t t e r at the o r i g i n . B. IN VIVO DISTRIBUTION OF MTX 1. Intraperitoneal absorption i n h a i r l e s s mice To provide a point of comparison f o r l a t e r experiments with t o p i c a l l y applied MTX, the drug was administered systemically to h a i r l e s s mice by i n t r a p e r i -toneal i n j e c t i o n . 22 male and female h a i r l e s s mice (HRS/J strain) weighing approximately 20 gm were injected i n t r a -p e r i t o n e a l l y with MTX (1.25 mg/kg). Plasma l e v e l s were determined fl u o r o m e t r i c a l l y at 20, 40, 60 and 180 minutes a f t e r administration. Liver and skin samples were obtained from three mice two hours a f t e r i n j e c t i o n . 63. FIGURE 19: Dixon plot of MTX i n h i b i t i o n of chicken l i v e r dihydrofolate reductase with 3.1 x 10~ M (« •) and 9.3 x 10~ 6 M (o o) DHF. 64. ENZYME EXTRACT ( ml ) FIGURE 20: Ackermann-Potter plot of chicken l i v e r dihydro-_ g folate reductase a c t i v i t y at 9.3 x 10 M DHF (• •) and i n h i b i t e d by 9.17 x 10~ 9 M MTX ( o — o ) . Peak plasma leve l s (0.752 yg/ml ±0.114 S.E.) were reached 40 minutes a f t e r i n j e c t i o n of a 1.25 mg/kg dose and f e l l to 0.201 ug/ml by 180 minutes (Figure 21). Mouse l i v e r accumulated s i g n i f i c a n t amounts of MTX (0.160 ug/10 mg l y o p h i l i z e d l i v e r ) . Skin lev e l s could not be detected 2 hours a f t e r MTX administration. 2. Oral absorption i n p s o r i a t i c patients 2 5 mg of MTX -- a frequently used therapeutic dose i n the therapy of p s o r i a s i s -- was administered o r a l l y as ten 2.5 mg t a b l e t s to each of three p s o r i a t i c patients. Plasma l e v e l s were determined i n duplicate 0.5 ml samples at 0, 1/2, 1, 2, 3, 4, 6, 8 and 24 hours. 0.2 mm deep dermatome s l i c e s of p s o r i a t i c epidermis were obtained 24 hours a f t e r MTX administration. The patients studied were not receiving regular medication of drugs such as s a l i c y -lates , a n t i - d i a b e t i c s , sulphonamides and d i u r e t i c s which might i n t e r a c t with MTX. Peak plasma l e v e l s at three to eight hours ranged from 0.630 ug/ml to 0.920 yg/ml (Figure 22). A f t e r a single 25 mg o r a l dose, MTX remained i n the plasma i n appreciable amounts during a 24 hour period with l e v e l s ranging from 0.128 to 0.382 yg/ml. Although the 25 mg weekly dose of MTX. was c l i n i c a l l y e f f e c t i v e i n these three patients, skin l e v e l s of MTX could not be detected f l u o r o m e t r i c a l l y . FIGURE 21: Plasma levels of MTX in h a i r l e s s mice followin i n t r a p e r i t o n e a l i n j e c t i o n of a 1.25 mg/kg dose 67 . T I M E ( h r . ) FIGURE Plasma leve l s of MTX i n three following a 25 mg o r a l dose. p s o r i a t i c patients 3. Percutaneous absorption i n h a i r l e s s mice and p s o r i a t i c patients To determine the extent of absorption and d i s -t r i b u t i o n of t o p i c a l l y applied MTX, 5% MTX i n vanishing cream (20 mg of cream per mouse) was applied f o r three hours under occlusive dressing (Saranwrap ) to f i v e mice anaesthetized with pentobarbital (60 mg/kg body weight). Since chronic t o x i c i t y was not a consideration i n t h i s short term study with experimental animals, a r e l a t i v e l y high concentration of MTX (5%) could be applied without adverse e f f e c t s and thus provide MTX concentrations which were r e a d i l y assayed using the standard fluorometric procedure. To prevent mice from l i c k i n g the s i t e s of ointment ap p l i c a t i o n — and, i n e f f e c t , absorbing the drug o r a l l y -- the animals were kept under anaesthesia through-out the tes t period. MTX le v e l s were determined i n the plasma and skin of f i v e mice and i n the l i v e r s of three mice. Although MTX could not be detected i n the plasma a f t e r a 30 minute app l i c a t i o n of 5% MTX cream, samples obtained a f t e r a three hour ap p l i c a t i o n showed detectable l e v e l s of MTX. The plasma l e v e l s reached were comparable to, and l i v e r l e v e l s (0.0454 ug MTX/mg l i v e r ± 0.0064 S.E.) three times higher than, those obtained a f t e r i n t r a p e r i t o n e a l i n j e c t i o n (Figure 23). Skin l e v e l s a f t e r a 3 hour app l i c a t i o n averaged 0.0628 ug/mg (± 0.0094 S.E.) l y o p h i l i z e d skin. 69 . PLASMA LIVER SKIN jjg/ml. >jg/10mg ;jg/10 mg FIGURE 23: Comparison o f plasma, l i v e r and s k i n l e v e l s o f MTX i n h a i r l e s s mice f o l l o w i n g i n t r a -p e r i t o n e a l i n j e c t i o n (1.25 mg/kg dose) and t o p i c a l a p p l i c a t i o n (20 mg o f a 5% cream). To determine i f MTX was bound by any s p e c i f i c 3 region of the skin, penetration of H-MTX through h a i r l e s s mouse skin was also followed autoradiographically i n seven animals. A 1.5 cm area of skin was enc i r c l e d by a foam pad cemented to the dorsal surface of the anaesthetized animal. 0.06 ml of a 3H-MTX solution of 0.05% sodium bicarbonate (50 uCi/2.5 ug/ml) was pipetted into the c i r c u l a r well formed by the pad and the area occluded with Blenderm tape. F u l l thickness skin samples were removed . from mice every h a l f hour for up to three hours, washed and fixed i n cold acetone. To monitor background r a d i a t i o n , control samples of skin from mice not exposed to MTX were subjected to the same procedure. 4 u thick sections were cut from Paraplast embedded sections using a Spencer 820 American Optical Microtome. Sections coated with Kodak NTB emulsion were exposed for 2 weeks at r e f r i g e r a t o r temperatures, developed, fi x e d and stained with t o l u i d i n e blue ( C u l l i n g , 1963).. From one-half to two hours a f t e r application of t r i t i a t e d MTX soluti o n to h a i r l e s s mice, radioactive l a b e l l i n g of skin sections was no greater than that of control sections (Figure 24 a,b). By 2 1/2 hours, uniform l a b e l l i n g of autoradiographs of h a i r l e s s mouse skin 3 (Figure 24 c) with H-MTX could be r e a d i l y detected throughout the section. Since MTX could be analyzed f l u o r o m e t r i c a l l y i n 71. FIGURE 24a: C o n t r o l a u t o r a d i o g r a p h o f h a i r l e s s mouse s k i n ( T o l u i d i n e b l u e ; x 254). 72. -FIGURE 24b: A u t o r a d i o g r a p h o f h a i r l e s s mouse s k i n one h o u r a f t e r a p p l i c a t i o n o f H-MTX ( T o l u i d i n e b l u e ; x 2 5 4 ) . 73. 4 • •» "is1* ^ FIGURE 24c A u t o r a d i o g r a p h o f h a i r l e s s mouse s k i n two and a h a l f hours a f t e r a p p l i c a t i o n o f FJ-MTX ( T o l u i d i n e b l u e ; x 254). mouse skin and human plasma, attempts were made to analyze MTX i n skin and plasma of p s o r i a t i c patients a f t e r t o p i c a l a p p l i c a t i o n of the drug. Approximately 330 mg of 0.2% MTX cream -- the dose reported by Fry and McMinn (1967) to be c l i n i c a l l y e f f e c t i v e i n psoratic patients -- was applied under occlusion f o r f i v e days to three p s o r i a t i c patients. After f i v e days of t o p i c a l cream ap p l i c a t i o n neither plasma nor skin l e v e l s of MTX could be detected. C. CLINICAL EFFICACY OF LOCAL MTX IN PSORIASIS To determine the c l i n i c a l e f f i c a c y of l o c a l l y administered MTX i n p s o r i a s i s two therapeutic t r i a l s were conducted: the f i r s t , a short-term t r i a l with t o p i c a l a p p l i c a t i o n of MTX cream, a therapy regimen reported as c l i n i c a l l y e f f e c t i v e by Fry and McMinn (1967), and the second, a more prolonged t r i a l with both t o p i c a l applica t i o n and i n t r a l e s i o n a l i n j e c t i o n of MTX. STUDY 1: MTX cream (0.2%) was applied under occlusion f o r f i v e to seven days to nine p s o r i a t i c patients 2 Approximately 330 mg of cream was applied to 16 cm lesions i n each patient. Vanishing cream base alone was applied to alternate lesions as a c o n t r o l . Patients were observed d a i l y f o r c l i n i c a l improvement. Two patients concurrently receiving o r a l MTX (25 mg weekly), but with r e c a l c i t r a n t l e s i o n s , were also included i n the study. STUDY I I : Since a frequent c r i t i c i s m of thera-peutic t r i a l s with t o p i c a l MTX i n p s o r i a s i s i s the short duration of therapy, nine other patients were h o s p i t a l i z e d and each treated with MTX f o r three weeks i n the following manner. Five separate p s o r i a t i c plaques on each patient were treated with one of the following therapy regimens: a) 0.1 ml of a 0.5% MTX sodium solution injected i n t r a -l e s i o n a l l y every 12 hours for one 36 hour period weekly (each dose containing 0.5 mg MTX). b) 0.1 ml of a 0.5% MTX Na-0.3% calcium leucovorin solution injected i n t r a l e s i o n a l l y every 12 hours for one 36 hour period weekly (each dose containing 0.5 mg MTX and 0.3 mg calcium leucovorin). c) 0.1 ml of s t e r i l e saline as a control i n t r a l e s i o n a l i n j e c t i o n . d) Daily a p p l i c a t i o n of 5 mg MTX t o p i c a l l y under occlusion (a 20 cm area was outlined with white soft p a r a f f i n and 0.5 ml of a 1.0% MTX suspension i n 9 5% ethanol was applied. Afte r evaporation of the solvent, the area was occluded with Saranwrap and Dermical tape). e) Continuous occlusion of the skin, without t o p i c a l a p p l i c a t i o n f o r contr o l . Laboratory t e s t s — white blood c e l l count, blood urea nitrogen, u r i n a l y s i s , BSP retention, SGOT, al k a l i n e phosphatase and serum f o l a t e l e v e l s -- were performed before MTX treatment to assess l i v e r and kidney function. SGOT and alk a l i n e phosphatase determinations were repeated twice a week, one and four days a f t e r i n t r a l e s i o n a l therapy to monitor possible side e f f e c t s . Observations were made twice a week and photographs taken before and a f t e r therapy Biweekly observations made by dermatologists recorded f l a t t e n i n g of le s i o n s , decreasing erythema and s c a l i n g , changes i n plaque size and colour, and appearance of any ulcerations. On termination of therapy, an o v e r a l l evaluation of patient progress was made by compiling i n d i v i d u a l observations and comparing before and a f t e r treatment photographs. Because of the unpredictable nature of p s o r i a s i s , i t s frequent remissions and exacerbations, therapeutic e f f e c t of treated areas was always evaluated on the basis of comparison to c o n t r o l , untreated areas. Since patients i n the study were h o s p i t a l i z e d f o r treatment of severe generalized p s o r i a s i s , regions not involved i n l o c a l MTX therapy were treated d a i l y with sub-erythema doses of u l t r a v i o l e t l i g h t and 0.1% dithranol paste. Areas treated with MTX were shielded during u l t r a -v i o l e t l i g h t exposure by black p l a s t i c . The therapeutic e f f e c t of l o c a l MTX i n p s o r i a t i c patients was n e g l i g i b l e . Of the nine patients treated with 0.2% MTX cream under occlusion (Study I ) , eight showed no apparent c l i n i c a l change. In one patient, erythema, s u p e r f i c i a l erosions and burning sensations occurred i n the MTX-occluded lesions a f t e r s i x days of therapy. Neither of the two patients receiving o r a l MTX concurrently showed any improvement i n the t o p i c a l l y treated lesions already refra c t o r y to systemic MTX therapy. Neither more prolonged t o p i c a l treatment nor i n t r a l e s i o n a l i n j e c t i o n of MTX (Study II) produced any con-si s t e n t improvement of p s o r i a s i s . Eight of the nine patients completed the t r i a l with no s i g n i f i c a n t improvement in the areas treated t o p i c a l l y or i n t r a l e s i o n a l l y . In only one patient did i n t r a l e s i o n a l MTX unquestionably c l e a r the p s o r i a t i c patch so treated. In other instances, no greater degree of f l a t t e n i n g of lesions or decrease i n erythema and scaling occurred i n MTX-treated than i n control areas. P s o r i a t i c patches distant from those involved i n MTX t r e a t -ment also improved. In addition, both occluded and non-occluded control areas, which were shielded from u l t r a v i o l e t l i g h t and dithranol treatment showed improvement. With the exception of one patient, no gross abnormalities i n SGOT or al k a l i n e phosphatase l e v e l s were noted throughout the study. In one patient a d e f i n i t e pattern was apparent. SGOT le v e l s showed a s l i g h t , but consistent, increase (from 20 to 60 units) a f t e r completing each series of three i n t r a l e s i o n a l i n j e c t i o n s . This e f f e c t i s presumably due to the small amounts of MTX absorbed from the i n t r a l e s i o n a l i n j e c t i o n s i t e . Serum fo l a t e l e v e l s were above 3 ng/ml i n seven out of nine patients, but less than 1 ng/ml i n two patients. Such serum f o l a t e l e v e l s 78. indicate f o l a t e deficiency. The single instance of improve-ment i n the p s o r i a t i c lesions treated occurred i n one of two fol a t e d e f i c i e n t patients. Since no improvement was noted eit h e r a f t e r i n t r a -l e s i o n a l i n j e c t i o n of MTX alone or a f t e r a combined i n j e c t i o n of MTX and calcium leucovorin, no conclusions could be drawn concerning the i n t e r a c t i o n of l o c a l l y administered MTX and i t s "antidote", calcium leucovorin. D. DETERMINATION OF SOLUBILITY AND PARTITION COEFFICIENT OF MTX. <-Physical chemical properties of drugs play an important part i n determining t h e i r a b i l i t y to penetrate b i o l o g i c a l membrances such as the skin. Since MTX i s a weak aci d , and thus physical chemical properties such as solu-b i l i t y and p a r t i t i o n c o e f f i c i e n t would be affected by pH and degrees of i o n i z a t i o n , determination of these properties at only one pH value would therefore be inadequate. Both s o l u b i l i t y and p a r t i t i o n c o e f f i c i e n t were therefore determined i n duplicate at 8 d i f f e r e n t pH values ranging from 2.47 to 8.99. Both parameters were also determined i n 0.5% bicarbonate s o l u t i o n . 1. S o l u b i l i t y An excess of MTX was shaken with buffers of varying pH and water-saturated octanol i n a 25°C (+ 0.1°) water bath. Samples were withdrawn at 3 and 6 days, f i l t e r e d through a 0.45 u M i l l i p o r e f i l t e r and d i l u t e d appropriately f o r UV analysis at 305 nm. No s i g n i f i c a n t difference was noted i n the concen t r a t i o n of MTX s o l u b i l i z e d a f t e r three and six days, there-fore the system was assumed to be at equilibrium. The reported s o l u b i l i t y values are therefore the pooled r e s u l t s fo r both three and six day analyses. MTX was only very s l i g h t l y soluble except i n a l k a l i n e solutions. As pH increased and therefore i o n i z a t i o n of the weakly a c i d i c MTX, s o l u b i l i t y generally increased (Table I I , Figure 25). The large amount of MTX required to saturate the pH 8.99 system made i t impractical to determine s o l u b i l i t y at t h i s pH. 2. P a r t i t i o n C o e f f i c i e n t Preliminary experiments had shown that MTX reached equilibrium d i s t r i b u t i o n i n the two phase octanol/ water system by four days. Therefore, 10 ml of MTX solutio n (10 and 20 mg/100 ml) i n buffers of varying pH were tumbled for four days with 10 ml of water-saturated octanol i n amber b o t t l e s . Since MTX has been reported (Freeman, 19 57) as stable f o r 55 days at room temperature when prepared as a 5 mg% s o l u t i o n , possible decomposition . of the drug during t h i s time period was not a s i g n i f i c a n t consideration. A f t e r four days, octanol and aqueous phases were allowed to separate and samples withdrawn from both layers. UV absorbance of octanol samples was read d i r e c t l y Aqueous samples were d i l u t e d (1:25) fo r UV analysis at 305 nm. P a r t i t i o n c o e f f i c i e n t s were calculated from the TABLE II Effect of pH on S o l u b i l i t y of Methotrexate pH of S o l u b i l i t y of Methotrexate Buffered Vehicle (gm/litre)(+ S.E. for N = 4) 2 .47 0.7079 + 0.0053 3. 49 0.3023 + 0.0111 4 . 35 0.3475 + 0.0085 5. 31 0.8142 + .0. 0138 6.45 0.7537 + 0 . 0145 7.12 1.2135 + 0.0070 7.90 1.3541 + 0.0226 * 8.15 11.769 + 0.306 •: 0.5% bicarbonate solution FIGURE 25: E f f e c t of pK on s o l u b i l i t y of MTX r a t i o of octanol to aqueous concentration of MTX. The t o t a l amount of MTX analyzed i n the two phase system a f t e r four days was not s i g n i f i c a n t l y d i f f e r e n t from that added to the system i n i t i a l l y . As a n t i c i p a t e d , no s i g n i f i c a n t decomposition had taken place at room tempera-ture during t h i s time period. As the pH of the aqueous phase was increased, the octanol/water p a r t i t i o n c o e f f i c i e n t of MTX decreased (Table I I I , Figure 26). Even at optimum values (pH 3.49), the p a r t i t i o n c o e f f i c i e n t was s t i l l low — 0.034. Doubling the concentration of MTX i n the aqueous phase from 10 to 20 mg/100 ml did not change the p a r t i t i o n c o e f f i c i e n t . Data from both aqueous phase concentrations were therefore pooled to obtain the reported means. P a r t i t i o n c o e f f i c i e n t at pH 2.47 was lower than and s o l u b i l i t y higher than at pH 3.49. Nash of Lederle Laboratories (personal communication) reported that minimum s o l u b i l i t y of MTX occurred at i t s i s o e l e c t r i c point between pH 2 and 3. At low pH, MTX i s apparently capable of i o n i z i n g further as a weak base; i . e . , the amino groups, probably of the pteridine nucleus, assume a p o s i t i v e charge. Therefore, i n considering the c o r r e l a t i o n of physical chemical parameters with the degree of i o n i z a t i o n of MTX, data at pH 2.47 was not included. Calculation of the degree of i o n i z a t i o n (see Appendix III) at t h i s pH, based on the known pK values (4.3 and 5.5, Nash, personal communication) without consideration of a t h i r d i o n i z a t i o n constant would introduce s i g n i f i c a n t e r r o r s . TABLE III Ef f e c t of pH on Octanol/Water P a r t i t i o n C o e f f i c i e n t of Methotrexate pH of Octanol/Water P a r t i t i o n Aqueous Phase C o e f f i c i e n t of Methotrexate (+ S.E. for N=4) 2.47 0.02192 + 0.00055 3.49 0.03381 + 0.00035 4.35 0.02970 + 0.00031 5. 31 0.02052 + 0.00074 6.45 0.01259 + 0.00106 7.12 0.00637 + 0.00015 7 . 90 , 0.00538 + 0.00007 * 8.15 0.00492 + 0.00001 8.99 0.00367 + 0.00066 * 5% bicarbonate solution FIGURE E f f e c t of pH on octanol/water p a r t i t i o n c o e f f i c i e n t of MTX. Octanol/water p a r t i t i o n c o e f f i c i e n t (K°) was r w related to the f r a c t i o n of unionized drug (a^) by a power b law function of the form: y = a x Thus: K° = 0.0311 a ° ' 1 2 9 w 2 or a l t e r n a t i v e l y : log K° = 0.129 log a - 1.506 6 w & 2 with a c o r r e l a t i o n c o e f f i c i e n t (r) of 0.9 80 and a o c o e f f i c i e n t of determination (r ) of 0.961 as determined using a non-linear regression analysis program on the Wang 600 programmable c a l c u l a t o r (Figure 27). E. IN VITRO PERCUTANEOUS PENETRATION From i h vivo r e s u l t s and preliminary tests i n V i t r o , i t was apparent that fluorometric methods were not s u f f i c i e n t l y s e n s i t i v e to determine the k i n e t i c s of MTX penetration through the skin. Because of c e r t a i n por-t i o n of MTX administered to patients i s retained i n the l i v e r f o r as long as 116 days (Charache et aJL. , 1960), i t was considered inadvisable to study percutaneous penetra-t i o n of r a d i o a c t i v e l y l a b e l l e d MTX i n vivo i n p s o r i a t i c patients. Using radiotracer techniques i n v i t r o , however penetration could be accurately followed at hourly i n t e r v a l s f o r any period of time. 1. Of t r i t i a t e d water Since buffer pH affected s o l u b i l i t y and p a r t i t i o n c o e f f i c i e n t of MTX, i t was of in t e r e s t to determine the ef f e c t of vehicle pH on percutaneous penetration of the 86. - L O G FIGURE 27: Log-log plat of octanol/water p a r t i t i o n c o e f f i c i e n t (K°) as a function of f r a c t i o n w of MTX unionized (a_) at varying pH. drug. However, before any changes i n permeability constant could be d e f i n i t e l y linked to pH-dependent MTX parameters, i t had to be determined i f the pH range and buffers used affected skin permeability i n general. To monitor the e f f e c t of buffer pH on skin permeability, d i f f u s i o n of t r i t i a t e d water through samples of f u l l thickness h a i r l e s s mouse skin was determined for a series of f i v e buffers ranging i n pH from 2.47 to 7.90. Samples of f u l l thickness h a i r l e s s mouse skin, removed from the dorsal surface of the animal and devoid of any subcutaneous t i s s u e , were stored at 0°C p r i o r to use. Skin samples were sandwiched between the Teflon discs of the d i f f u s i o n c e l l and clamped t i g h t l y i n place atop the ground glass surface of the receptor chamber. 8 ml of 0.9% sodium chloride solution was pipetted into the lower chamber. The c e l l s were c a r e f u l l y inverted to d i s p e l any a i r bubbles below the skin surface and to check for any overt leaks around the edge of the skin membrane. o.4 ml of t r i t i a t e d water d i l u t e d with buffer solutions to give a s p e c i f i c a c t i v i t y of 1 uCi/0.1 ml ( i . e . , 2.22 x IO 6 dpm/0.1 ml) was pipetted into the upper r e s e r v o i r . Samples were occluded with Blenderm tape and the side arm stoppered to prevent evaporation. 0.5 ml samples of receptor chamber f l u i d were withdrawn every h a l f hour for a f i v e hour period and added to 14.5 ml of dioxane-based s c i n t i l l a t i o n f l u i d . Receptor volume was renewed by addition of 0.5 ml of s a l i n e . 88. D i f f u s i o n curves, plotted as dpm/crn^ versus time, were l i n e a r within one hour i n a l l cases (Figure 28) in d i c a t i n g no progressive damage to the skin. Permeability constants (k ) were calculated by di v i d i n g the slope of the P l i n e a r portion of the d i f f u s i o n curve, as calculated from l i n e a r regression a nalysis, by the a c t i v i t y of the t r i t i a t e d 3 water i n dpm/cm . The units of the permeability constant are thus cm/time. The permeability constants determined at f i v e d i f f e r e n t pH values ranging from 2.4-7 to 7.9 0 did not d i f f e r s i g n i f i c a n t l y (Table IV) when subjected to a two-t a i l e d t - t e s t for unpaired samples at a l e v e l of s i g n i f i -cance p = 0.05. The permeability constant for d i f f u s i o n of t r i t i a t e d water through h a i r l e s s mouse skin i n v i t r o averaged 2.83 + 0.369 x 10 cm/sec. f o r the pooled r e s u l t s of 21 d i f f u s i o n runs. 2. Of MTX MTX solutions i n buffer (0.4 ml) or creams were applied to the exposed epidermal surfaces of h a i r l e s s mouse skin and human skin(removed from the abdomen at autopsy), assembled i n the d i f f u s i o n c e l l as described for t r i t i a t e d water d i f f u s i o n experiments. 0.5 ml samples of receptor chamber f l u i d were withdrawn at hourly i n t e r v a l s f o r a 30 hour period. A f t e r 30 hours, the d i f f u s i o n c e l l was dismantled and the exposed area of skin dissected out. The c i r c u l a r fragment of tissue was thoroughly rinsed and 89. TABLE IV Comparison of Permeability Constants for Diffus i o n of T r i t i a t e d Water through Hairless Mouse Skin i n  v i t r o from Buffers of Varying pH pH of Buffer p N . -1 i n 3 cm hr x 10 2.47 1.274 + 0.413 4 4.35 0.891 + 0.128 5 5.31 0.913 + 0.165 5 6.45 1.127 + 0.576 4 7.90 0.918 + 0.009 3 Mean 1.018 + 0.133 21 90. 0 1 2 3 4 5 T IME ( hr) FIGURE 28: Cumulative d i f f u s i o n of t r i t i a t e d water through h a i r l e s s mouse skin in v i t r o at pH 7.90 (o o) and 2.47 (• • ) . stripped with Schotch tape to remove any s u p e r f i c i a l MTX. A 50-60 mg sample of skin was digested with 1.0 ml NCS s o l u b i l i z e r f o r approximately 2-3 hours at 55°C. When digestion was complete, 0.0 3 ml g l a c i a l a c e tic acid was added, as recommended by the manufacturer, to decrease the chemiluminescence of ti s s u e samples. For l i q u i d s c i n t i l l a -t i o n counting, 14 ml of dioxane-based s c i n t i l l a t i o n f l u i d was added. Typical penetration curves for MTX through excised skin samples showed an i n i t i a l lag period (x) during which the penetration rate increased gradually to the steady state rate (J ) (Figure 29). Substituting the s concentration of MTX int o equation 7, the permeability constant could be thus calculated from the l i n e a r portion of the curve. To check the i d e n t i t y of the r a d i o a c t i v i t y l a b e l l e d substance d i f f u s i n g through the ski n , samples of receptor f l u i d were subjected to t h i n layer chromatography i n two solvent systems — pH 7.4 phosphate buffer (0.1M) and isopropanol/water (2:1). 82-90% of the r a d i o a c t i v i t y was recovered from an area corresponding to the R^ . value f o r standard preparations of non-radioactive MTX — 0.65-0.72 f o r isopropanol/water and 0.56-0.60 f o r phosphate buffer. Although some t r i t i u m exchange may occur during the time of the d i f f u s i o n process, the radioactive compound pene t r a t i n g through the skin i s pri m a r i l y MTX. 9 2 . 3 -d"!"'* 5 I O 1 5 ZO 2 5 3 0 TIME ( hr ) FIGURE 29: Representative percutaneous penetration curve of MTX -- cumulative d i f f u s i o n of MTX through h a i r -less mouse skin in v i t r o from a 0.2 5% suspension i n pH 3.49 buffer. a) Comparison of human autopsy and h a i r l e s s mouse skin In v i t r o , i n both f u l l thickness h a i r l e s s mouse and human autopsy skin, only a small percentage of the t o t a l applied dose of MTX (1 mg/0.4 ml) penetrated within a 30 hour period. From a 0.25% solution of MTX i n sodium bicarbonate (pH 8.15), 0.05% of the t o p i c a l l y applied 3 H-MTX had penetrated a f t e r t h i s time period. Although flux through human skin varied more than through mouse skin, the average penetration rates were not s i g n i f i c a n t l y d i f f e r e n t , 0.0338 (+ 0.0076 S.E.) and 0.0276 (± 0.0008) ug/hr respectively (Table V, Figures 30 and 31). The greater v a r i a t i o n i n human skin permeability i s probably due to the greater b i o l o g i c a l v a r i a t i o n i n an unselected patient population than i n an inbred s t r a i n of laboratory animals. b) Penetration through h a i r l e s s mouse skin from suspensions  of varying pH and vanishing cream Since penetration of MTX through h a i r l e s s mouse skin was comparable both i n rate of penetration and lag time to human abdominal skin and provided a more reproducible model system, the e f f e c t of pH on penetration rate was studied with mouse skin only. As the pH of the buffered vehicle was increased from 3.49 to 8.15, flux of MTX through 2 ? the skin decreased from 0.226 ug/cm /hr to 0.0267 ug/cm /hr (Table VI). Penetration rates and permeability constants at the f i v e d i f f e r e n t pH values were s i g n i f i c a n t l y d i f f e r e n t using a two-tailed t - t e s t for unpaired samples at a sign i f i c a n c e l e v e l p = 0.05. However lag times averaging TABLE V Comparison of Diffusi o n of MTX through Hairless Mouse and Human Autopsy Skin i n v i t r o from a 0.25% solution (pH 8.15) N -2 -1 (yg cm hr ) (hr) Human Autopsy Skin 0.0339 11.9 4 + 0.0076 + 1.4 Hairless Mouse Skin 0.0276 11.7 4 + 0.0008 + 0.3 T IME ( hr ) FIGURE 30: Cumulative d i f f u s i o n of MTX through h a i r l e s s mouse skin i n v i t r o from a 0.25% solution i n 0.5% bicarbonate CpH 8.15) (composite of 4 d i f f u s i o n runs + S.E.). FIGURE 31: C u m u l a t i v e d i f f u s i o n o f MTX t h r o u g h f u l l t h i c k n e s s h u m a n a u t o p s y s k i n i n v i t r o f r o m a 0.25% s o l u t i o n i n 0.5% b i c a r b o n a t e T p H 8.15) ( c o m p o s i t e o f 4 d i f f u s i o n r u n s + S.E.) 97. TABLE VI Ef f e c t of Vehicle pH on In Vit r o Percutaneous Penetration of MTX through Hairless Mouse Skin. oH of J k T Skin Concentra-Buffered g 2 p ^ ti o n of MTX at Vehicle ug/cm /hr (cm/hr x 10 ) (hr) 30 hrs (ug/mg) 2.47 0.238 + 0.058 0.95 + 0.23 1.75 + 0.22 0.123 + 0.048 3.49 0.226 + 0.020 0.90 + 0.20 1.27 + 0.19 0.139 + 0.035 4.35 0.187 + 0.010 0.75 + 0.04 1.20 + 0.31 0.171 + 0.033 5.31 0.134 + 0.003 0.54 + 0.01 1.51 + 0.28 0.155 + 0.036 6.45 0.0829+ 0.009 0.33 + 0.04 1.58 + 0.23 0.106 + 0.025 8. 15 0. 0276+ 0. 0008 0.11 + 0.003 U..7 + 0.33 98. 1.20 to 1.75 hours were not s i g n i f i c a n t l y d i f f e r e n t except at pH 8.15 where the lag time increased to 11.7 hours. Steady state flux from a 0.25% cream averaged 0.129 yg/cm2/hr (+ 0.057 S.E.) for f i v e d i f f u s i o n runs. Lag times averaging 4.4 hours were greater than those obtained f o r penetration from the series of 0.25% suspen-sions (Table VII). This may r e f l e c t the increased thickness of the e f f e c t i v e ' d i f f u s i o n layer' by a p p l i c a t i o n of a cream. Although the pH of the cream was estimated as approximately neutral using Universal Indicator, penetration rates related more c l o s e l y to those of a pH 5.3 suspension. However, i t i s d i f f i c u l t to accurately determine the pH of the external aqueous phase of such an emulsion cream and actual pH may be more a c i d i c than predicted. d) Retention of MTX by skin following t o p i c a l a p p l i c a t i o n  m vitro. Skin samples removed from d i f f u s i o n c e l l s a f t e r 30 hours contained MTX i n concentrations ranging from 0.049 to 0.278 yg/mg fresh t i s s u e . No s i g n i f i c a n t difference could be detected between concentrations of MTX i n skin samples with vehicles of varying pH (Table V) using a two-t a i l e d t - t e s t . Neither was any c o r r e l a t i o n apparent between the steady state flux through the skin and the amount of MTX ,retained within the"membrane. Concentrations of drug achieved In v i t r o by application of a 0.25% cream were i n the same range as those 99. TABLE VII Eff e c t of Vehicle Composition and Concentration on In Vitro Percutaneous Penetration of MTX through Hairless Mouse Skin J k x Cone. MTX i n s p 2 4 N ug/cm /hr cm/hr x 10 hr Skin ug/mg 0.25% MTX Cream 0.129 + 0.057 0.52 + 0.23 4.36 + 0.75 0.0369 + 0.0141 5 1% MTX Cream 0.512 + 0.226 0.51 + 0.23 3.60 + 0.54 0.0695 + 0.0133 5 0.2 5% MTX Suspension (pH 5.3) 0.134 + 0.003 0.54 + 0.01 1.51 + 0.28 0.155 + 0.036 7 1% MTX Suspension (pH*5.3) 0.578 + 0.065 0.58 + 0.07 1.53 + 0.20 0.257 + -.054 4 100. obtained i n vivo i n h a i r l e s s mice by application of a 5% cream (Figure 18). Since 1 gm of l y o p h i l i z e d skin was determined to be equivalent to 2.67 gm of fresh t i s s u e , correcting i n v i t r o data (Table VI) f o r t h i s weight change produced an average concentration of 0.0985 + 0.0376 ug/mg l y o p h i l i z e d weight as compared to 0.0628 + 0.0210 Ug/mg in vivo. Less MTX was retained by the skin a f t e r a p p l i c a -t i o n of creams than a f t e r a p p l i c a t i o n of aqueous suspensions, e) E f f e c t of concentration on i n v i t r o penetration of MTX Since no s i g n i f i c a n t difference was noted between concentrations of MTX retained by mouse skin a f t e r applica-t i o n of 0.2 5% MTX cream i n v i t r o and 5.0% cream i n vivo, i n v i t r o experiments were conducted to determine the e f f e c t of concentration on penetration rates of MTX and the amount of drug retained by the skin. A 1.0% pH 5.3 suspension and cream, representing a f o u r - f o l d increase i n the concentration of MTX over the 0.25% v e h i c l e s , were also applied to h a i r l e s s mouse skin in_ v i t r o . The four-f o l d increase i n MTX concentrations i n both cream base and pH 5.3 aqueous suspension produced corresponding increases i n steady state penetration rates. Permeability constants, calculated by d i v i d i n g the steady rate flux by concentration of the penetrant i n the v e h i c l e , were equivalent (Table VII). Although the concentration of MTX dissolved i n the vehicle was not changed by increasing the t o t a l concentrations from 0.2 5 to 1.0%, penetration rates were enhanced. However, despite the increase i n penetration 101. rates, the concentrations of MTX retained by the skin were not s i g n i f i c a n t l y d i f f e r e n t (using a two-tailed t - t e s t at p = 0.05) when 0.25 and 1.0% MTX preparations were applied to h a i r l e s s mouse skin i n v i t r o . f) Penetration of MTX from intracutaneous i n j e c t i o n  i n v i t r o . Since rapid t r a n s i t time of i n t r a l e s i o n a l l y i n j e c -ted MTX has been implicated (Comaish and'Juhlin, 1969) i n the i n a c t i v i t y of t h i s mode of MTX administration, penetration k i n e t i c s of intracutaneously injected MTX were also determined i n v i t r o . Because h a i r l e s s mouse skin i s too t h i n to perform intracutaneous i n j e c t i o n s , human autopsy skin was used. 0.5 mg of MTX i n 0.1 ml of 0.5% sodium bicarbonate -- the same dose administered to p s o r i a t i c patients i n Study II — was injected intracutan-eously i n v i t r o into samples of f u l l thickness human autopsy skin assembled i n d i f f u s i o n c e l l s . Within eight hours, 2 8% of the dose had penetrated through the skin into the d i f f u s i o n f l u i d . Cumulative d i f f u s i o n curves (Figure 32) were hyperbolic. I n i t i a l rapid d i f f u s i o n rates declined to approach a steady state rate. The decline of d i f f u s i o n rate i s probably due to the decreasing concen-t r a t i o n gradient as a r e s u l t of penetration of a s i g n i f i c a n t percentage of the injected dose and/or non-sink conditions;;, thus produced; i.e.,the concentration of the lower chamber can no longer be considered as i n s i g n i f i c a n t when compared to the concentration on top of the membrane. 102 . FIGURE 32: Cumulative d i f f u s i o n of MTX through human autopsy skin in v i t r o from an intracutaneous i n j e c t i o n of 0.5 mg MTX i n a 0.5% bicarbonate solution (composite of three samples + S.E.). 103. DISCUSSION A. ACTIVITY OF DHFR AND ITS INHIBITION BY MTX Despite several attempts to i s o l a t e and obtain k i n e t i c data from human skin DHFR, re s u l t s were unsuccess-f u l due probably to the low le v e l s of DHFR i n normal skin (Grignani et_ a l . , 1967) and the extreme l a b i l i t y of the enzyme (Newbold, personal communication). More successful experiments using DHFR extracted from chicken l i v e r acetone powder produced a k i n e t i c constant, K m of 6.60 x 10~ 7 M which compared favourably _ 7 with l i t e r a t u r e reports of 4 x 10 M (Perkins and Bertino, 1966) and 5 x 10~ 7 M (Osborn et a l . , 1958). At p h y s i o l o g i c a l pH, MTX produced competitive r e v e r s i b l e i n h i b i t i o n of p a r t i a l l y p u r i f i e d DHFR extracted from chicken l i v e r acetone powder. Although the tightness of binding of MTX to enzyme i s increased at pH 5.9, rendering the i n h i b i t i o n of DHFR v i r t u a l l y i r r e v e r s i b l e (Bertino et a l . , 1964), the si g n i f i c a n c e of t h i s phenomenon i n vivo i s unknown. The i n h i b i t i o n constant, calculated from the — 9 three standard enzyme plots averaged 0.918 x 10 M (+ 0.013 x 10~ 9 S.E.). The Dixon plot yielded a comparable value — 1.1 x IO" 9 M. The K. value for MTX i n h i b i t i o n l of DHFR has been reported as approximately 1 0 - 9 M for most dihydrofolate reductases (Huennekens et a l . , 1971). MTX i n h i b i t i o n of DHFR at pH 7.4 has been reported as both competitive (Bertino e_t a l . , 1964) and non-competitive (Osborn et a l . , 1958). However, l a t e r investigators have suggested that f o l a t e antagonists such as MTX and the sub-strate DHF bind to the same s i t e on the enzyme. Also, fluorometric experiments have shown that MTX displaces DHF from i t s complex with the enzyme (Huennekens ejt a l . , 1971). From these experiments and i n keeping with the findings of t h i s t h e s i s , a competitive rather than a non-competitive i n h i b i t i o n mechanism would seem more probable. B. CORRELATION OF pH EFFECTS ON IN VITRO PERCUTANEOUS PENETRATION OF MTX WITH PARTITION COEFFICIENT AND IONIZATION. As demonstrated by Lien et aJL. , (19 71) f o r percutaneous absorption of alcohols and steroids through human epidermis, the log of the permeability constant correlates well with the log of lipid/water p a r t i t i o n c o e f f i c i e n t s . Lien et a_l. , (1971) also calculated equations c o r r e l a t i n g absorption of weak acids and bases through the buccal mucosa with both log p a r t i t i o n c o e f f i c i e n t and log a c i d / s a l t r a t i o . No data i s available to correlate the penetration of s i m i l a r i o n i z i n g substances through the skin to p a r t i t i o n c o e f f i c i e n t and degree of i o n i z a t i o n . General statements merely quote the greater permeability of s a l i c y l i c acid over sodium s a l i c y l a t e (Scheuplein and Blank, 1971). Preliminary experiments with d i f f u s i o n of t r i t i a t e d 105. water through h a i r l e s s mouse skin using buffers of varying pH revealed that for the pH range used there was no e f f e c t of pH on skin permeability. The permeability constant f o r t r i t i a t e d water d i f f u s i o n (2.83 x 10 cm/sec) was si m i l a r to that reported by Tregear (1966) (3.3 x 10" 7 cm/sec) f o r d i f f u s i o n through the skin of the in t a c t animal. Moreover, the k was si m i l a r to that reported for human stratum P corneum — 2.8 x 10" 7 cm/sec (Scheuplein, 1965), again demonstrating that h a i r l e s s mouse skin i s a s a t i s f a c t o r y model for studying penetration of many substances through human skin. Comparing the penetration of MTX through h a i r l e s s mouse and human autopsy skin i n v i t r o at pH 8.15 revealed no s i g n i f i c a n t difference i n either penetration rate or lag time f o r the two skin types. Since the lag time for d i f f u s i o n of substances through membranes i s related both to the thickness of the b a r r i e r and to the d i f f u s i v i t y of the penetrant i n the b a r r i e r , t h i s would imply -- at least for the present system -- that f o r MTX penetration, human autopsy skin and h a i r l e s s mouse skin behave s i m i l a r l y . Since pH of buffer vehicles did not a f f e c t skin i n t e g r i t y , changes i n penetration rates of MTX from pH 3.49 to 8.15 can be related to changes i n physical chemical parameters of the drug i t s e l f . For the percutaneous penetration of MTX from vehicles ranging i n pH from 3.49 to 8.15, log-log plots of steady state flux as a function of f r a c t i o n of unionized drug and concentration of dissolved, 106. unionized drug were l i n e a r (Table VIII; Figure 33). The relationships can be expressed as power law functions f i t t e d by non-linear regression analysis. Thus: 0.131 . 0 J = 0.212 a0 with vl - 0.995, S 0.166 9 and J =0.259 [MTX°] with r = 0.985. s L i t t l e change was noted i n lag time f o r penetration at pH values ranging from 3.3 to 6.4. However, increasing the pH of the vehicle to 8.15 resulted i n a s i g n i f i c a n t increase i n t h i s parameter. This may indicate that from a pH 8.15 v e h i c l e , MTX penetrated through the skin by an alternate, more devious route than from an a c i d i c vehicle. Middleton (1969) has suggested that ions penetrate skin by passage between rather than through the c e l l s of the epidermis. This may be the case f o r MTX at pH 8.15 where l i t t l e drug i s unionized. A more c i r c u i t o u s i n t e r c e l l u l a r route may explain the prolonged lag time at an a l k a l i n e pH. In addition, steady state flux i s apparently a l i n e a r function of octanol/water p a r t i t i o n c o e f f i c i e n t over the pH range tested, such that: J = 6.657 K° - 0.0037 s w with r 2 equal to 0.997 (Table VIII; Figure 34). Permeability constants were c h a r a c t e r i s t i c of a -4 slowly penetrating molecule (0.11 - 0.95 x 10 cm/hr). At optimum penetration (pH 3.49), only 0.5% of the t o t a l dose penetrates within 30 hours. S u p e r f i c i a l l y t h i s would seem an apparent contradiction of Newbold and Stoughton's (1972) recently reported r e s u l t s . According to t h e i r study 107. TABLE VIII Steady State Diffusion Rate, S o l u b i l i t y , P a r t i t i o n C o e f f i c i e n t and Fraction of Unionized MTX at vary-ing pH P H a2 • C s K J s 2 N (gm/litre) (ug/cm /hr) 3.49 0.8643 0.3021 0.03381 0.226 6 4.35 0.3434 0.3475 0.02970 0.187 5 5.31 0.0561 0.8142 0.02052 0.134 7 6.45 0.71xl0 - 3 0.7537 0.01259 0.0829 7 8.15 1.52xl0~ 7 11.769 0.00497 0.0267 4 108. FIGURE 33a: Log-log plot of steady state flux ( J g ) as a function of f r a c t i o n of MTX unionized ( a 9 ) . 109. / FIGURE 33b: Log-log plot of steady state flux (J ) as a function of concentration of dissolved unionized drug ([MTX 0]). 110. FIGURE 34: Steady state f l u x of MTX through h a i r l e s s skin in v i t r o at varying pH as a function octanol/water p a r t i t i o n c o e f f i c i e n t . mouse of 111. MTX i s absorbed through both mouse and human skin i n v i t r o i n r e l a t i v e l y large amounts -- up to 16% of the applied dose a f t e r 20 hours. However, ce r t a i n discrepancies are apparent i n the reported data. Although the authors state that 0.01 ml of 0.5% and 2.5% MTX solutions were applied, t h e i r tabulated figures are of applications of 0.1 and 0.5 mg, thus implying concentrations of 1.0 and 5.0%. Surface area of the skin exposed i s variously reported as 1.5 cm and 1.2H craS However, i f a p l a s t i c r i n g 1.5 cm i n diameter i s used to enclose the skin 2 samples, the surface area exposed should be 1.76 cm . An important point to consider when comparing the present r e s u l t s to those of Newbold and Stoughton (1972) i s the method of determining and reporting penetration of the drug. This study did not use a k i n e t i c approach; i . e . penetration rates were not determined. Penetration c h a r a c t e r i s t i c s are based on single point measurements af t e r only one time i n t e r v a l -- 20 hours. Since, i n t h e i r case, the t o t a l amount of MTX applied to the surface of the skin i s extremely small -- 0.5 to 32 ug, reporting r e s u l t s as percentage of dose penetrated (2.5 to 16.4%) exaggerates the permeability of skin to MTX. By computing a penetration rate from the reported data at a comparable concentration (0.32% i n t h e i r case) and correcting for 2 the larger surface area exposed (1.76 cm ), the flux --2 0.087 ug/cm /hr -- i s comparable to the re s u l t s presented i n t h i s thesis (Table VIII). The authors also suggest that a greater f r a c t i o n of the dose of MTX applied was absorbed with the lower amounts (0.5 ug) than with the higher amounts (32 ug) applied. On closer examination, t h i s phenomenon i s most l i k e l y an ar t e f a c t of the method of data presentation. I f the f l u x of MTX i s estimated from t h e i r reported 20 hour d i f f u s i o n data by c a l c u l a t i n g the amount penetrated from the reported percentage and the amount applied and di v i d i n g by the time allowed f o r d i f f u s i o n , a plot of fl u x versus concentration of penetrant i s l i n e a r (Figure 35); i . e . , penetration of MTX obeys Fides' law. Although the data as reported by Newbold and Stoughton (1972) suggests that the e f f i c i e n c y of MTX penetration i s impaired when large amounts of the drug are applied, the more important parameter — penetration rate a c t u a l l y increases as greater amounts of MTX are applied to the skin. Such i s the case f o r data reported i n t h i s thesis (Table VII). Penetration rates f o r MTX from vehicles containing 1% of the drug were greater than from those containing 0.25%. C. IN VIVO DISTRIBUTION OF MTX T o p i c a l l y applied MTX penetrates h a i r l e s s mouse skin and accumulates i n l i v e r and plasma i n concentrations comparable to those obtained a f t e r systemic administration. From autoradiographic experiments, MTX retained by the skin following t o p i c a l a p p l i c a t i o n i s apparently not concentrated i n any s p e c i f i c area, but rather i s randomly d i s t r i b u t e d throughout both epidermal and dermal layers. 113. FIGURE 35: Flux of MTX through h a i r l e s s mouse (• •) , and human leg skin (o o) from dimethylacetamide solutions as a function of concentration of penetrant (Data from Nev/bold and Stoughton, 1972). The high l i v e r l e v e l s following t o p i c a l a p p l i c a t i o n of MTX cream may r e f l e c t the cumulative absorption of the larger t o t a l amount of MTX applied t o p i c a l l y (1 mg) than in j e c t e d i n t r a p e r i t o n e a l l y (0.025 mg). From t h i s i n v e s t i g a t i o n i n mice, one could conclude that t o p i c a l application of high concentrations of MTX might y i e l d the side e f f e c t s of systemic dosage. In the p s o r i a t i c patients tested, neither skin nor plasma l e v e l s could be detected a f t e r a 5 day t o p i c a l a p p l i c a t i o n of 0.2% MTX cream. I t i s conceivable that plasma concentrations are too low to be detected since the cream i s applied to a r e l a t i v e l y small percentage of the t o t a l body surface. It i s s u r p r i s i n g , however, that no skin l e v e l s could be detected a f t e r 5 days of continuous ointment ap p l i c a t i o n . Several explanations are possible: a) e i t h e r penetration c h a r a c t e r i s t i c s of human skin are s u f f i c i e n t d i f f e r e n t from h a i r l e s s mouse skin that MTX i s simply not absorbed, or b) MTX i s absorbed so r a p i d l y through the epidermis that a detectable concentration never accumulates i n the skin, or c) such small amounts of MTX penetrate the epidermis that the fluorometric method i s not s u f f i c i e n t l y s e n s i t i v e to detect l e v e l s that are present. These p o s s i b i l i t i e s w i l l be considered further i n the l i g h t of i n v i t r o penetration data. 115 . Peak plasma l e v e l s (0.630-0.920 ug/ml) reached i n the three p s o r i a t i c patients following a 25 mg o r a l dose of MTX are i n keeping with previous reports of 0.740-1.55 ug/ml (Freeman, 1958; Halprin et_ a l . , 1971). Patients i n t h i s study with peak le v e l s at six and eight hours are consistent with Kettel's et a l . (1968) "slow absorbers". Detectable plasma concentrations (0 .128-0 . 382 yg/ml) persisted f or 24 hours a f t e r a single 25 mg dose of MTX. D. CLINICAL EFFICACY OF LOCAL MTX IN PSORIASIS The therapeutic e f f e c t of l o c a l MTX i n the present studies with p s o r i a t i c patients was n e g l i g i b l e . More prolonged MTX treatment does not r e s u l t i n any c l i n i c a l improvement (Study I I ) . In 17 out of 18 p s o r i a t i c patients treated, neither t o p i c a l nor i n t r a l e s i o n a l MTX produced any c l i n i c a l improvement which could be distinguished from the control treatments. The t r i a l of i n t r a l e s i o n a l MTX was undertaken not as a p r a c t i c a l approach to the therapy of a generalized skin disease, but to bypass the "cutaneous penetration b a r r i e r " . D i s t r i b u t i o n studies of systemically administered MTX have shown peak concentrations of approximately 60 ng/gm of skin a f t e r 5 mg of MTX intravenously ( S u l l i v a n et a l . , 1966; Anderson et a l . , 1970) and 450-850 ng/gm of skin a f t e r a 50 mg dose (Liguori et a l . , 1962). 0.25 mg of MTX 3 injected intradermally i n h i b i t s incorporation of H-UdR into epidermal DNA of p s o r i a t i c skin f or 12 hours (Weinstein et a l . , 1971). Yet d i r e c t i n t r a l e s i o n a l i n j e c t i o n of a 116. greater amount — 0.5 mg at 12 hour i n t e r v a l s for 36 hours, the length of the p s o r i a t i c c e l l cycle — produced no c l i n i c a l improvement i n our patients a f t e r three weeks of therapy. Both t o p i c a l and i n t r a l e s i o n a l MTX, even f o r protracted periods, was i n e f f e c t i v e i n 17 out of 18 p s o r i a t i c patients treated. Despite hypotheses that a penetration b a r r i e r to MTX might explain the ineffectiveness of t o p i c a l therapy, the i n a c t i v i t y of even i n t r a l e s i o n a l MTX would suggest a more complex theory i s necessary. Percutaneous penetration c h a r a c t e r i s t i c s were further delineated by i n v i t r o studies. E. PERCUTANEOUS PENETRATION AS RELATED TO THE INEFFECTIVENESS OF TOPICAL MTX Although the physical chemical properties of MTX are not i d e a l for passive d i f f u s i o n through b i o l o g i c a l mem-branes, the ineffectiveness of t o p i c a l MTX i n p s o r i a s i s i s probably due to more than a penetration b a r r i e r . Assuming i n v i t r o penetration data may be applied to the i n vivo s i t u a t i o n , evidence suggested that the a v a i l a b i l i t y of MTX to the skin from t o p i c a l a p p l i c a t i o n should at le a s t be equiva-l e n t to that from systemic administration. A f t e r systemic administration of a 50 mg dose, Li g u o r i et al^. (1962) report skin l e v e l s of only 0.45-0.85 ug/gm. Whereas, a f t e r a 30 hour t o p i c a l a p p l i c a t i o n of 0.25% MTX cream i n v i t r o , l e v e l s of 36.9 ug/gm were reached. In v i t r o comparison of MTX penetration through h a i r l e s s mouse and human skin suggests that permeability c h a r a c t e r i s t i c s of the two membranes are not 117. s u f f i c i e n t l y d i f f e r e n t to account for the d i s p a r i t y i n i n vivo analyses. Failure to detect MTX f l u o r o m e t r i c a l l y a f t e r t o p i c a l application i n p s o r i a t i c patients was probably due to i n s u f f i c i e n t amounts of MTX i n the small a n a l y t i c a l samples obtained. With a more se n s i t i v e method, i t might be possible to detect MTX i n the skin of p s o r i a t i c patients a f t e r t o p i c a l a p p l i c a t i o n . Since i n v i t r o experiments suggested that the capacity of the skin membrane to r e t a i n MTX i s l i m i t e d , i t i s doubtful that increasing the concentration of MTX for t o p i c a l a p p l i c a -t i o n would provide any greater l o c a l concentration of the drug i n the p s o r i a t i c epidermis. Comaish and J u h l i n (1969) have treated p s o r i a t i c patients t o p i c a l l y with MTX concentrations of up to 10% without any consistent therapeutic improvement. Although higher concentrations of MTX than the 0.2% employed here might ultimately have some therapeutic e f f e c t i n p s o r i a s i s , i t i s questionable whether the e f f e c t would be due to the l o c a l i z e d action of the drug on the skin c e l l s or to a more generalized action. I f such i s the case, t o p i c a l therapy of p s o r i a s i s with MTX would provide no advantage over systemic administration of the drug. C l i n i c a l l y neither t o p i c a l nor i n t r a l e s i o n a l MTX was e f f e c t i v e i n 17 out of 18 p s o r i a t i c patients treated. Thus, depositing MTX below the stratum corneum by i n t r a l e s i o n a l i n j e c t i o n did not increase therapeutic e f f i c a c y . However, t h i s may be due to rapid d i s p e r s a l of MTX from the i n j e c t i o n s i t e . 118. In v i t r o t ests of penetration from intracutaneous i n j e c t i o n are hampered by the accumulation of drug i n the lower chamber as time progresses. The slowing of penetration rates with time would probably not occur i n vivo because of the constant removal of diffused drug by the c i r c u l a t i o n . F. SITE OF ACTION OF MTX IN PSORIASIS The lack of effectiveness of l o c a l MTX, despite i n t r a l e s i o n a l i n j e c t i o n and i n the presence of low serum fo l a t e ( i n one p a t i e n t ) , suggests that MTX might act elsewhere than d i r e c t l y i n the skin. Although d i r e c t biochemical e f f e c t s of MTX have been demonstrated i n v i t r o (Marks et a l . , 1971) and i n vivo (Weinstein el: a l . , 1971) , neither i n h i b i t i o n of THF production nor UdR incorporation correlate with c l i n i c a l e f f e c t s . The ultimate step governing the e f f e c t of MTX i n p s o r i a s i s i s probably not i n h i b i t i o n of THF production, but i n h i b i t i o n of thymidylate incorporation i n t o DNA. Investiga-tions into the mechanism of action of MTX i n p s o r i a s i s have considered only one source of thymidylate, de novo synthesis. However, two sources e x i s t — de novo synthesis and a salvage pathway. Cleaver (1967) suggested that the body may salvage TMP by incorporating DNA from dead c e l l s within the same tis s u e or throughout the whole organism and by absorbing DNA of sloughed g a s t r o i n t e s t i n a l c e l l s f o r l o c a l synthesis or d i s t r i b u t i o n throughout the body. In v i t r o studies on L - c e l l cultures by Borsa and Whitmore (19 69a) demonstrated that TMP i n s u f f i c i e n t concentrations can overcome the MTX induced block of c e l l p r o l i f e r a t i o n . These experiments suggest that 119. pools of metabolites may be available f o r synthesis of DNA by tissues despite the presence of MTX. Only a f t e r these supplies are depleted would c e l l u l a r p r o l i f e r a t i o n be com-p l e t e l y i n h i b i t e d . The unusual amount of nuclear debris present i n the p s o r i a t i c epidermis could provide a large pool of per-3 formed thymidylate. Despite MTX i n h i b i t i o n of H-UdR inco r -poration into DNA of rat skin, White (1971a,b) found l i t t l e change i n the amount of DNA per gram of skin. The salvage pathway may provide adequate thymidylate f o r continuation of' DNA synthesis and epidermal c e l l r e p l i c a t i o n even i n the face of complete i n h i b i t i o n of skin dihydrofolate reductase by MTX. With continuing systemic administration of MTX, there may be decreasing supplies of salvaged thymidylate because of the universal "slow down" of epidermopoiesis, or i t may become indispensable to other tissues and be shunted away from the epidermis. The r a p i d l y d i v i d i n g p s o r i a t i c epidermis would be very susceptible to even a minor shortage of metabolites. A more d i r e c t look must be taken at the epidermal synthesis of DNA i n p s o r i a t i c s treated systemically with MTX. I f the mechanism of action of MTX i n p s o r i a s i s i s r e l a t e d to mitot i c suppression, i t should be possible to demonstrate a closer c o r r e l a t i o n between i n h i b i t i o n of DNA synthesis and improve-ment of p s o r i a s i s i n patients treated with MTX. I f such a c o r r e l a t i o n i s l a c k i n g , the answer must be sought among other p s o r i a t i c c h a r a c t e r i s t i c s ; e.g., the absent granular l a y e r , the postulated (Stankler, 1970) c i r c u l a t i n g f a c t o r and dermal inflammation. 120. SUMMARY A. INHIBITION OF DIHYDROFOLATE REDUCTASE Low le v e l s of DHFR a c t i v i t y could be detected f l u o r o m e t r i c a l l y i n homogenates of human s u r g i c a l skin. Enzyme a c t i v i t y was too low to be followed spectrophoto-m e t r i c a l l y and thus, k i n e t i c constants could not be estimated for human skin enzyme. However, i n v i t r o , at p h y s i o l o g i c a l pH 7.4, MTX was a competitive i n h i b i t o r of chicken l i v e r _q dihydrofolate reductase (IO = 0.97 x 10 M). B. IN VIVO DISTRIBUTION In vivo i n h a i r l e s s mice, MTX was r a p i d l y absorbed both from i n t r a p e r i t o n e a l i n j e c t i o n (a 1.25 mg/kg dose) and t o p i c a l a p p l i c a t i o n (a 5% cream). Mouse l i v e r accumulated s i g n i f i c a n t amounts of MTX from both routes of administration. Although skin l e v e l s of the drug were undetectable a f t e r i n t r a p e r i t o n e a l i n j e c t i o n , t o p i c a l a p p l i c a t i o n of MTX ointment yielded 0.0628 ug MTX/mg l y o p h i l i z e d skin. Peak plasma l e v e l s a f t e r o r a l administration of 25 mg of MTX to p s o r i a t i c patients ranged from 0.630 to 0.920 yg MTX/ml plasma, at 3 to 8 hours. S i g n i f i c a n t l e v e l s persisted f o r 24 hours. After 5 days of ointment a p p l i c a t i o n (0.2% MTX cream) to p s o r i a t i c patients, neither skin nor plasma l e v e l s could be detected. 121. C. CLINICAL EFFICACY MTX, injected intradermally (o.5 mg for 3 consecu-t i v e 12 hour i n t e r v a l s each week) and applied t o p i c a l l y (o.2% cream or a 5 mg deposit from alcohol suspension) was c l i n i c a l l y i n e f f e c t i v e i n 17 out of 18 p s o r i a t i c patients treated. D. PHYSICAL CHEMICAL PROPERTIES MTX was only poorly soluble i n a l l but a l k a l i n e solutions. S o l u b i l i t y generally increased as the pH of buffers was increased. On the other hand, octanol-water p a r t i t i o n c o e f f i c i e n t decreased as the pH of the aqueous phase increased. The p a r t i t i o n c o e f f i c i e n t was apparently r e l a t e d to the f r a c t i o n of unionized drug by a power law function. P a r t i t i o n between o i l and water was i n e f f i c i e n t , with a maximum c o e f f i c i e n t of only 0.034. E. IN VITRO PERCUTANEOUS PENETRATION In v i t r o , at pH 8.15, MTX penetrated through both h a i r l e s s mouse and human skin at comparable rates. Percu-taneous penetration of MTX through h a i r l e s s mouse skin i n v i t r o was affected by vehicle pH. The e f f e c t of pH on penetration was correlated with changes i n octanol-water p a r t i t i o n c o e f f i c i e n t , f r a c t i o n of drug unionized and con-centration of dissolved unionized drug. The permeability constant for d i f f u s i o n of MTX through h a i r l e s s mouse skin i n v i t r o from a cream base was 0.52 x 10 cm/hr. Increasing the concentration of MTX from 0.2 5 to 1.0% increased the penetration rate of MTX but not the concentration of MTX retained by the skin. Concentrations of MTX retained by h a i r l e s s mouse skin i n v i t r o — 0.09 85 ug/mg l y o p h i l i z e d weight (corrected) — were i n the same range as those obtained i n vivo — (0.0628 yg/mg). In v i t r o , 28% of MTX injected intracutaneously penetrated through samples of f u l l thickness human autopsy skin within 8 hours. 123. CONCLUSIONS Local treatment of p s o r i a s i s f o r up to three weeks with MTX, e i t h e r t o p i c a l l y (0.2% cream) or i n t r a -l e s i o n a l l y (0.5 mg) i s c l i n i c a l l y i n e f f e c t i v e . Since i n v i t r o studies suggest that penetration from t o p i c a l a p p l i c a t i o n i s adequate to provide concentrations of MTX equivalent to those reaching the skin by the c l i n i c a l l y e f f e c t i v e systemic route and since i n t r a l e s i o n a l l y injected MTX i s i n e f f e c t i v e despite evidence of dihydro-f o l a t e reductase i n h i b i t i o n demonstrated by other workers, the s i t e of action of MTX i n p s o r i a s i s may involve tissues other than the affected skin. 124. APPENDIX I MATERIALS 1. Methotrexate, 4-amino-N -methylpteroylglutamic acid (Lederle Laboratories D i v i s i o n , American Cyanamid Company, Pearl River, N.Y.) Methotrexate powder Methotrexate t a b l e t s , 2.5 mg Methotrexate sodium, parenteral, 5 mg v i a l s 2. Methotrexate (3',5'-3H) sodium, 30 mCi/mg, 99% + pure radiochemically, l y o p h i l i z e d under nitrogen (Dhom Products Limited, 11120 Cumpston Street, North Hollywood, C a l i f o r n i a ) . 3. Calcium leucovorin i n j e c t a b l e , 3 mg calcium leucovorin per cc. (Lederle Laboratories D i v i s i o n ) . 4. Vanishing cream base (Remington's Practice of Pharmacy, eds. Martin, E.W., Cook, E.F., Leuallen, E.E., et a l ; 12th e d i t i o n , p. 1803, Mack Publishing Company, Easton, Pennsylvania, 1961) Stearic acid 18 gm Potassium carbonate 1 gm Glycerin 5 gm D i s t i l l e d water 7 6 gm This simple emulsion base cream i s made by melting the s t e a r i c acid and adding a heated solution of potassium carbonate, g l y c e r i n and vrater. The mixture i s maintained at 85°C and s t i r r e d for 10 minutes to ensure complete saponi f i c a t i o n and absence of free a l k a l i . The ointment i s then made up to 100 gm with d i s t i l l e d water. 5. T r i t i a t e d MTX ointments The l y o p h i l i z e d powder of 3H-MTX (250 yCi) was reconstituted with 0.4 ml of d i s t i l l e d water. An 0.1 ml aliquot of the radioactive solution was pipetted onto a glass slab containing the required amount of "cold" MTX and a c i d i f i e d with 0.0 0 5M HCI. For example, to prepare 1.0 gm of a 1.0% MTX ointment with a s p e c i f i c a c t i v i t y of 10 yCi/mg MTX ( i . e . 22.2 x 10 6 dpm/mg) requires 10 mg "cold" MTX, 0.1 ml 3H-MTX solution and 0.9 9 gm vanishing cream. The acid solution i s evaporated by a current of cold a i r and the remaining powder incorporated into the vanishing cream base by l e v i g a t i o n . The s p e c i f i c a c t i v i t y of each batch of cream was tested by dispersing an accurately weighted amount of the prepared cream i n 100 ml of water and dioxane and counting a 0.5 ml aliquot of the solution i n 14.5 ml of s c i n t i l l a t o r . As for radioactive solutions, ointments were stored at r e f r i g e r a t o r tempera-tures i n t i g h t l y closed containers protected from l i g h t and a i r . 126 . 6. T r i t i a t e d water, t r i t i u m (hydrogen 3) i n j e c t i o n , 5 uCi/ ml (Amersham Searle, Arlington Heights, I l l i n o i s ) . 7. P0P0P,l,4-bis [2-(5-phenyloxazolyl)]-benzene. (Sigma Chemical Company, 3500 de Kalb Street, St. Louis, Missouri). 8. PPO,2,5-diphenyloxazole (Sigma Chemical Company). 9. Napthalene, r e c r y s t a l l i z e d (Eastman Kodak Company, Rochester, New York). 10. NADP, nicotinamide adenine dinucleotide phosphate (Sigma Chemical Company). 11. NADPH, nicotinamide adenine dinucleotide phosphate Type I I , enzymatically reduced form (Sigma Chemical Company). 12. Dihydrofolic acid (Sigma Chemical Company). 13. Tetrahydrofolic acid (Sigma Chemical Company). 14. 2-mercaptoethanol (Sigma Chemical Company). 15. Chicken l i v e r acetone powder (Sigma Chemical Company). 127. 16. C e l i t e , a n a l y t i c a l f i l t e r aid (Johns-Manville). 17. Medical Anti-foam A, Simethicone (Dow-Corning Corpora-t i o n , Midland, Michigan). 18. Kodak NTB emulsion, D-19 developer powder and T r i -Chem Pak (Eastman Kodak Company, Rochester, New York). 19. Paraplast embedding medium (Sherwood Medical Industries Inc., St. Louis, Missouri). 128. SOLVENTS AND REAGENTS 1. Octanol-1 (Fisher S c i e n t i f i c Company, F a i r Lawn, New Jersey). 2. p-Dioxane, s c i n t i l l a t i o n q u a l i t y (Matheson, Coleman and B e l l , Los Angeles, C a l i f o r n i a ) . 3. Dioxane s c i n t i l l a t i o n solvent (Comaish and J u h l i n , 1969): PPO 7 gm POPOP 0.3 gm Naphthalene 10 0 gm dioxane, q.s 1000 ml was prepared f r e s h l y each week. 4. NCS , tissue s o l u b i l i z e r , 0.6 N solution i n toluene (Amersham Searle, Arlington Heights, I l l i n o i s ) . 5. Low i o n i c strength buffers (Perrin, 1963), i o n i c strength 0.01: a) pH 2.3 .... 47.4 ml 0.1 M c i t r i c acid plus approximately 6.7 ml 0.1 sodium hydro-xide , q.s. to 100 ml with d i s t i l l e d water. b) pH 3.3 .... 10.0 ml 0. IM c i t r i c acid plus approxi-mately 6.5 ml 0.1 M sodium hydroxide,q.s. to 100 ml with d i s t i l l e d water. 129. c) pH 4.3 .... 36.2 ml 0.1 M a c e t i c acid plus approxi-mately 9.9 ml 0.1 M sodium hydroxide, q.s. to 100 ml with d i s t i l l e d water. d) pH 5.3 .... 12.8 ml 0.1 M acetic acid plus approxi-mately 9.9 ml 0.1 sodium hydroxide, q.s. to 100 ml with d i s t i l l e d water. e) pH 6.3 .... 7.8 ml 0. IM sodium dihydrogen phosphate plus approximately 0.8 ml 0.1 M sodium hydroxide, q.s. to 100 ml with d i s t i l l e d water. f) pH 7.3 4.2 ml 0.1 M sodium dihydrogen phosphate plus approximately 2.3 ml 0.1 M sodium hydroxide, q.s. to 100 ml with d i s t i l l e d water. g) pH 7.8 .... 13.8 ml 0.1 M T r i s plus approximately 10.0 ml 0.1 M hydrochloric a c i d , q.s. to 10 0 ml with d i s t i l l e d water. h) pH 8.8 .... 46.4 ml 0.1 M T r i s plus approximately 10.0 ml 0.1 M hydrochloric a c i d , q.s. to 100 ml with d i s t i l l e d water. The f i n a l pH of buffer solutions v.-as measured to three decimal places using an expanded scale Radiometer pH meter. 130 . APPENDIX II APPARATUS AMINCO-BOWMAN SPECTROPHOTOFLUOROMETER, Model 4-8202, equipped with a xenon lamp source, 1P21 phototube, s o l i d state xenon lamp D.C. power supply unit and photomultiplier (American Instrument Company, 8030 Georgia Avenue, S i l v e r Spring, Maryland). BECKMAN DB-GT SPECTROPHOTOMETER and 10" l i n e a r recorder equipped with 1P2 8A photomultiploer, deuterium and tungsten sources (Beckman Instruments, Inc., F u l l e r t o n , C a l i f o r n i a ) . CONSTANT TEMPERATURE BATH, Model Ml, (Cannon Instrument Company, Boalsburg, Pennsylvania) c i r c u l a t i n g water bath for constant temperature control of spectro-photometer chamber. R PICKER NUCLEAR LIQUIMAT , Model 650-503, 8-counting assembly with automatic external standardization using 13 7 a shielded Cs source (Picker Nuclear, 127 5 Mamaro-neck Avenue, White P l a i n s , New York). NUCLEAR CHICAGO ISOCAP/300, ambient temperature, auto-matic programming l i q u i d s c i n t i l l a t i o n counter with 13 3 Ba external standard (Nuclear Chicago, Des Plames, I l l i n o i s ) . 131. 6. DERMATOME, a modified Castroviejo Electro-Keratotome (Storz Instrument Company, St. Louis, Missouri) equipped with a 10 mm wide blade and 0.2 mm depth spacer (Stewart and Runikis, 1967). 7. SKIN DIFFUSION CELL designed by Coldman et a l . (1969) consisting of a lower glass receptor chamber (capacity, 8.0 ml) with a side arm and magnetic s t i r r i n g disc attached to a polyethylene s a i l , t e f l o n discs and clamps to hold the skin i n place and expose 2 a central c i r c u l a r area of 1 cm with the upper Teflon disc forming a r e s e r v o i r (0.4 ml capacity) for the applied vehicle, (Figure 36). 8. MAGNI-WHIRLR constant temperature bath (Blue M E l e c t r i c Company, Blue Island, I l l i n o i s ) . 9. RADIOMETER pH METER, Type PHM 26 equipped with standard glass and calomel electrodes and b u i l t - i n p r e c i s i o n scale expander (Radiometer A/S, Copenhagen, Denmark). 10. SPENCER 820 AMERICAN OPTICAL MICROTOME (Instrument D i v i s i o n of American O p t i c a l , B e l l e v i l l e , Ontario). 132. FIGURE 36: Diagrammatic representation of the d i f f u s i o n c e l l . APPENDIX III CALCULATION OF THE FRACTION OF UNIONIZED MTX To calculate the f r a c t i o n of MTX present as each species -- d i p r o t i c , unionized (o^) , monoprotic (a^) and aprotic (ct^) the e q u i l i b r i a are (Butler, 1 9 6 4 ) : [H +] [MTXl] = K a [MTX] ( 1 ) a i [H +] [MTX ] = K [MTX -] ( 2 ) a 2 The mass balance on MTX i s : C = [MTX] + [MTX~] + [MTX'] (3) where C i s the a n a l y t i c a l concentration of methotrexate. Since [H +] and C are known, there are a t o t a l of three equations i n three unknowns. The f r a c t i o n of unionized MTX present i s the r a t i o of the concentration of the unionized species to the a n a l y t i c a l concentration, c 2 . [ > ™ oo The r e c i p r o c a l of the f r a c t i o n of unionized drug can be calculated by d i v i d i n g each term of equation (3) by [MTX], thus : :L_ _C_ 1 +[MTX~] [MTX~] . . a 2 " [MTX] = [MTX ] [ MTX] K b ) Substituting from equilibrium equations ( 1 ) and ( 2 ) into equation ( 5 ) y i e l d s : 134. K K K 1 1 + 1 a x a 2 = + — L (6) 2 + +2 CH ] [H +] Thus a 2 , the f r a c t i o n of unionized drug can be calculated from i t s r e c i p r o c a l using equation (6), the reported pK a values -- 4.3 and 5.5 (Nash, personal communication) and the measured pH of the buffer solutions. REFERENCES Ackermann, W.W. and Potter, V.R. (1949). Enzyme i n h i b i t i o n in r e l a t i o n to chemotheraDV, Proc. Soc. Exp. B i o l . Med. T2_, 1-9. Ainsworth, M. (19 60). Methods for measuring percutaneous absorption, J. Soc. Cosmet. Chem. 11, 69-85. Allenby, C F . (1966). Relapsing pustular eruption of hands and feet treated with l o c a l methotrexate, B r i t . J. 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