Percutaneous Penetration and Anti-Inflammatory Activity Of
PERCUTANEOUS PENETRATION AND ANTI-INFLAMMATORY ACTIVITY OF
DESFLUOROTRIAMCINOLONE ACETONIDE
by
SUBHASH CHANDER VERMA
B. Pharm., M. Pharm. (Pharm. Tech.) Gujarat University, India
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE IN PHARMACY
in the Division
of
Pharmaceutics
of the
Faculty of Pharmaceutical Sciences
We accept this thesis as conforming to the
required standard
THE UNIVERSITY OF BRITISH COLUMBIA
March 1972 In presenting this thesis In partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make It freely available for reference and study, I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, 8, Canada. i
ABSTRACT
Desonide, a new topical anti-inflammatory and anti• pruritic steroid, has been investigated for its clinical, vasoconstrictor and in vitro percutaneous penetration prop• erties, and compared to betamethasone 17-valerate, triam• cinolone acetonide and hydrocortisone. The clinical and vasoconstrictor bioassay tests place desonide quantitatively among the most effective topical anti-inflammatory agents, possibly because of its relatively rapid skin penetration rate,
The significance of the study is i (a) it provides definitive data on topical anti-inflammatory effectiveness of desonide and (b) it reveals that, contrary to current opinion, fluorination of the steroid molecule may be unnecessary for topical anti-inflammatory activity, and that
9 oC-fluorination in prednisolone acetonides impedes rather than favours their skin penetration rates.
New data on octanol/water partition coefficients and an unsuccessful effort of adopting the Martin (1968) oxime derivative spectrophotofluorometric technique for desonide assays are also included.
Supervisor. 11
TABLE OF CONTENTS
Page
I. INTRODUCTION 1
II. LITERATURE SURVEY 5
A. Inflammation and Anti-inflammatory Steroids 5
1. Inflammation 7
2. Anti-inflammatory mechanism of corticosteroids 10
3. Structure-activity relationships 14
4. Topical anti-inflammatory cortico• steroids 19
B. Skin Penetration 24
1. Biophysical factors in skin
penetration 24
a. Avenues of penetration 24
b. Barrier characteristics 28
c. Factors influencing penetration 30
d. Physical chemistry of diffusion 33
2; Methods for measuring percutaneous absorption 41 3. Isolation of epidermal sheets from
human autopsy skin 43
C. • Analytical Methods 47
1. Spectrophotometric 47
2. Fluorometric 47
3. Other methods (GLC and radioisotopic) 48
4. Vasoconstriction assay 49 iii
Page III. STATEMENT OF PROBLEM 51 IV. EXPERIMENTAL METHODS 52 A. Apparatus 52 1. Poulsen skin diffusion cell 52
2. Spectrophotofluorometer (SPF) 52 3. Picker nuclear liquimat 55 4. Spectrophotometer (Hitachi & Beckman) 55 5. Dermatome 55 B. Procedures 55 1. Analysis of steroids 55 a) Spectrophotofluorometric analysis of desonide 55 b) Spectrophotometric 58 2. Solubility and partition coefficient determination 58
3. In^vitro penetration studies 60 a) Preparation of membranes for diffusion cell 60 i) Full thickness of mouse skin 60
ii) Epidermal sheets from human autopsy 60 b) To check intactness of epidermal sheets by tritiated water 62 c) In^vitro steroid penetration 63
4. Vasoconstriction test 64 iv
Page
V. RESULTS AND DISCUSSION 66
A. Spectrophotofluorometric Method of the Estimation of Desonide 66
B. Spectrophotometry 67
C. Solubility and Partition Coefficient 68
D. Reliability of Heat Separation Method for the Removal of Epidermis from Autopsy Skin 74
E. Test for Intactness of Skin with Tritiated Water 74-
P. Percutaneous Penetration of Steroids 79
G. Vasoconstriction Bioassay 83
H. Clinical Studies of Effectiveness of
Desonide 91
VI. SUMMARY AND CONCLUSION 99
VII. BIBLIOGRAPHY .102
APPENDIX 111 V
LIST OF TABLES
Page
1. Summary of the inflammatory response 9 2. Enhancement factors for various functional groups
of corticosteroids 20 3. Anti-inflammatory congeners of hydrocortisone 22 4. Solubility of steroids in two different solvents 71 5. Octanol/water and ether/water partition coefficient of several steroids 72 6. Rate of diffusion of tritiated water through human abdominal skin 77 7. Rate of diffusion of tritiated water through human epidermis and hairless mice skin 78 8. Cumulative penetration of desonide and triam• cinolone acetonide through human abdominal skin at room temperature. The steroids were applied in drug deposit form 84 9.. Cumulative penetration of betamethasone 17- valerate, desonide, triamcinolone acetonide and hydrocortisone, through human abdominal skin. The steroids were in 40# ethanol 87
10. Cumulative penetration of betamethasone 17- valerate, desonide, triamcinolone acetonide and hydrocortisone, through human skin. The steroids were in hydrophllic ointment base, 89 11. Vasoconstriction produced by triamcinolone acetonide and desonide 92 12. Effectiveness of desonide vs betamethasone 17- valerate, in contact dermatitis 95 13. Effectiveness of desonide vs betamethasone valer• ate in atopic dermatitis 96 14. Effectiveness of desonide vs betamethasone valer• ate in psoriasis 97 vi
LIST OF FIGURES
Page
1. Structure of an anti-inflammatory corticosteroid 11
2. Possible mode of interaction of serotonin and histamine with Cortisol receptor(s) 13
3. Numbering and sites modified in hydrocortisone molecule 17
4. Structure of four anti-inflammatory steroids studied 17(a)
5. Anatomical zones encountered in percutaneous absorption 25
6. Possible avenues of penetration through the unbroken skin 26
7. Diagrammatic representation of epidermis ultra structure 29
8. Representative penetration profile for drug diffusing through human skin 40
9. Assembled side view of Poulsen's skin diffusion
cell 53
10. Different parts of the diffusion cell 54
11. Top view of the diffusion cell, showing the
exposed epidermal area 54
12. Removal of epidermis from autopsy skin 6l
13. Standard curve for corticosteroids in normal saline 69 14. Relationship of partition coefficient to cumulative penetration 73 15. Dermatome and heat separated; sections of human epidermis 75
16. Steroid penetration through human abdominal skin. The steroids were in drug deposit form 85 17. Amount of desonide and triamcinolone penetrated through human skin
18. Steroid penetration through human epidermis at room temperature. The steroids were in 40$ ethanol
19. Steroid penetration through human epidermis at room temperature. The steroids were in hydro- philic ointment base
20. Vasoconstriction bioassay, log concentration vs % dose response ACKNOWLEDGEMENTS
To Dr. J.O. Runikis, thesis supervisor, for his encourage• ment throughout the course of this study.
To Dr. William D. Stewart, for his advice in the clinical aspect of this thesis.
To Kiss Sylvia Wallace, for her very valuable comments and constructive criticism.
To Dr. T.H. Brown, Dr. A.M. Goodeve and Mrs. Pat Haugen for their valuable discussions.
To my wife, Asha, for her unceasing encouragement, which helped me in completing this project.
To Dean 3.E. Riedel especially, who extended his advice and support to me, and provided new ideas in the organization of this thesis. To my beloved parents whose inspiration has made this work
possible. I INTRODUCTION
The Introduction of topical hydrocortisone in derma• tology by Sulzburger and Witten (1952), shortly after• wards followed by more effective hydrocortisone derivatives, particularly fluorinated derivatives, were major therapeutic events in the treatment of non-infective inflammatory and pruritic skin conditions. Their effectiveness surpassed by far other treatments. Since inflammation is the commonest pathological process encountered in dermatology (Calnan,
1970) and since the more potent topical corticosteroids are effective against other diseases like psoriasis, their eco• nomic importance is as great as their therapeutic importance.
Today, as a consequence, there is a drive on the part of manufacturers of topical medications, and also derma- tological investigators to develop more potent topical anti• inflammatory corticosteroids in more diverse dosage forms.
This drive for 'more* coupled to a lack of clear understand• ing of the etiology of the inflammatory process or the phys• ical-chemical and bio-chemical factors determining the topical effectiveness of the corticosteroids, has produced a situation where the newer, more potent, and more toxic fluorinated corticosteroids, are used to the exclusion of the less potent corticosteroids, and pharmacies have to stock more than 30 corticosteroids in several hundred dose- vehicle combinations.
Generalizations ranging from probably erroneous to demonstrably erroneous have become widespread among the med• ical and pharmaceutical practitioners. A literature survey was undertaken to identify what need there is for use of more potent and more toxic fluorinated corticosteroids, what
Information there exists on the biological equivalence of the multitude of topical corticosteroid preparations and to inquire into experimental possibilities to provide objective information on the relative effectiveness of these prepara• tions. The survey revealed: long-term use of highly potent topical compounds can cause significant systemic absorption and adverse effects like increased epidermal fragility, telangiectasis, striae, ecchymoses and fibrablastic repair.
Waddington et al (I968) reported that the use of fluorinated steroids should be avoided in the treatment of infantile eczema. Epstein ^t_al (1963), Grlce (I966), Ive and Marks
(1968) reported that long-term use of potent flourinated corticosteroids cause atrophy of the skin. Sneddon (1969) has reported adverse effects after prolonged topical applica• tion of betamethasone in patients suffering from rosacea.
New non-fluorlnated steroid 'Desonide' (1'6'oC- hydroxy prednisolone - 16,17 aee&onide or desfluorotriamcinolone acetonide) has been developed by Miles Laboratories, Inc. and promoted as a potent anti-inflammatory steroid. It dif• fers from its fluorinated analog, triamcinolone acetonide, only 3
by the absence of 9 Mantica (1970) has studied the chemical and physical char• acteristics of desonide. Mascitelli-Coriandoli (1970) have studied the biochemical and pharmacological activity of desonide. Phillips et al (1971) from Miles Laboratories have reported on the physical, pharmacological and toxlco- logical properties. Their studies showed desonide to be as effective as fluorinated compounds and having lower toxicity systemically, but they had few data on topical activity. Polano (1970) has shown that another non-fluorinated steroid hydrocortisone butyrate, is as effective topically as triam• cinolone acetonide. A double blind comparison carried out by Reid and Brooks (1968) of 3 corticosteroids, revealed that hydrocortisone a non-fluorinated steroid in a very large number of eczema patients, was as effective as triamcinolone acetonide or fluocinolone acetonide, both fluorinated ster• oids. These results conflict with the widely accepted general• ization that fluorlnation increases antIT inflammatory activity to a degree where fluorinated corticosteroids, as a group, are more effective topical anti-inflammatory agents than non- fluorinated steroids. The generalization may be incorrect and deserves investigation. No study has so far been reported involving the penetra- 4 tion of desonide through human skin. The rate of penetra• tion of desonide through human skin shall be compared to triamcinolone acetonide, betamethasone «r> 1? - valerate and hydrocortisone. Vasoconstriction test shall be carried out to compare desonide and triamcinolone acetonide in their abilities to blanch the skin. The combination of in-vltro studies and human biological testing provides a valuable correlation between theory and therapy. This work should be regarded as a preparatory study for a comprehensive investigation into the biological and thera• peutic equivalence of topical anti-inflammatory corticoster• oids. 2 II LITERATURE SURVEY A. Inflammation and Ant1-Inflammatory Steroids Corticosteroids are used primarily for the non-specific, paliative treatment of a large array of dermatoses with an inflammatory component in which the primary etlologic factor is unknown or unassailable. Corticosteroids are indicated for use in diseases as varied as hand eczemas, contact der• matitis, pemphigus, psoriasis, exfoliative dermatitis (Brown, 1970). The mechanism and causes of inflammation in these diseases are complex and vary with the disease. It is ex• pected that the effectiveness of different, drugs, including different derivatives of corticosteroids, varies accordingly. The observation, that the topical administration of potent fluorinated corticosteroid is effective in psoriasis, whereas topical hydrocortisone is not, is an example. To evaluate the relative effectiveness of topical cortico• steroids and the role of fluorination in enhancing that effec• tiveness, it is necessary to find model inflammation systems which imitate topical corticosteroid action in the clinical situation. In absence of a known etiologic factor, the search must be for models which include some steps in the inflamma• tory process of the disease which is blocked by the drugs. As pointed out by Weiner and Pillero (1970), there is too much of a rush to evaluate drugs in favourite anti-inflammatory 6 models. This understandable pattern tends to direct the de• velopment of anti-inflammatory drug design along a line which will select agents working by the same mechanism as previously discovered drugs which perhaps correlate too well with each other and not well enough with clinical applications. That such has been the case in the development of corticosteroids is hardly in doubt. Since the discovery by Pried and Sabo (1953) of greater systemic anti-inflammatory effectiveness of 9 The following section emphasizes the events in inflamma• tion which have been thought to be blocked by topical cortico• steroids and are of value in the interpretation of their effectiveness. Less attention has been devoted to events which may be of importance in the development of the theory of inflammation but whose relationships to the activity of corticosteroids have not been clearly demonstrated. The ex• cellent reviews of^ffiibMson^' and Angell (1971) and Weiner and Pillero (1970) should be here consulted. These reviews and that of Wlnkelman (197Dt together with the older publica• tions of Dougherty and Frying (1955)t form the basis of the following survey of the nature of inflammation. 1. Inflammation Inflammation is a dynamic process which follows a vari• ety of pathways. There is agreement only on a descriptive definition of inflammation. Attempts to define inflammation in mechanistic physical-chemical or biological terms have failed (Florey, 195*+). The current definition of inflamma• tion is as follows: "Inflammation is a reactive train of morphologic and biochemical events affecting both blood ves• sels and cells which occurs in the vital tissues surrounding a site of injury" ( BObinson, 1971). This definition includes the classical clinical description of the successive steps In inflammation as heat, redness, pain and swelling, due to the progressive development of changes in vascular perme- ability, cellular migration, edema formation and ultimately leading to either the removal of the inflammatory stimuli or cellular death. The timing of these events and the bio• chemical mediators thought to be responsible are summarized in Table 1. The timing of the changes described in Table 1 varies with the severity and nature of the injury. The in• jurious stimuli may be physical e.g. a foreign body such as a cotton pellet or croton oil, chemical, e.g. such as an allergic response to a sensitizing drug, or microbiological. The following detailed events have at one time or another acquired prominence in attempts to explain the mode of action of corticosteroids. Histamine appears to be active principally during the early phase of inflammatory response, when it induces arterio• lar dilation and the first wave of increased permeability following injury, affecting chiefly the venules. Serotonin (5 - hydroxy - tryptamine) distributed widely in most cells and platelets can produce vasodilation. Proteoses and poly• peptides (the kinins) are important mediators. They are among the most potent vasodilators known. They are capable of causing striking increase in venular and capillary perme• ability. Moreover kinins have shown to attract leukocytes and alone can evoke many of the cardinal manifestations of inflammation (Menkln, 1956). Table i. Summary of the Inflammatory Response Temporal Hemodynamic Permeability White Cell Visible Sequence Mediator Site Changes Changes Changes Change Immediate (transient): Va s oc ons t r i c t i ve 0 to 5 min. Neurogenic Arterioles ischemia None None Blanching Early Phase: 5 to 30 min. Histamine Serotonin Arterioles Vasodilation Increased None Kinins Proteases Rubor Miles factor Capillaries New channels Increased None (redness) opened Calor Globulin per• Venules Engorgement- Increases- Pavementing (heat) meability overall increase endothelial Adhesion Dolor factor in blood flow joints opened Beginning (pain) Complement emigration Tumor esterases (swelling) Other mediators Delayed Phase: I to 2 hr. ?Lysolecithin ?Protein prod• Emigration ucts Venules Engorgement- As above Leukocyte em- Capillaries overall increase in Increased Perivascular formation igrating blood flow leukocyte of fluid factors aggregation and cellu• lar exudate \o ( Prom : Robinson, and Angell, 1971 ) 10 Among hemodynamic changes there is an increase in the permeability of vessels and leukocytic change. Another point in inflammation is the destruction of connective tis• sue. Frying and Dougherty (1955) proposed that the inten• sity of inflammation is enhanced by the destruction of fibroblasts. In this respect the destruction of one cell adds to the amount of the phlogogenic substance produced which leads to the destruction of another cell. The stages of inflammation (Winkelmann, 1971) have been studied by combined structural and pharmacologic-physiologic methods. Many investigators prefer to classify inflammation in two stages, an early stage related to venous permeability and a later stage to cellular damage. 2. Anti-inflammatory mechanism of corticosteroids Anti-inflammatory mechanisms can be considered from the viewpoints of biological and structure-activity mechanisms. The proposed structure-activity relationship and the chem• istry of corticosteroids are discussed in the next chapter. Corticosteroids exert their anti-inflammatory effect^by in• terrupting the chain of reactions of cellular destruction, that is triggered by the initiating stimulus. It has been suggested that the anti-inflammatory effect of the cortico- steroids is related to their effect on the metabolism of selected cells participating in the process of inflammation. Dougherty and Schneebeli (1955) propose that an anti-in• flammatory hormone probably acts by stabilizing the imperme• able state of the cell membrane, thus preventing cellular destruction. The concept that the fibroblast is the primary cell involved in inflammation and that it is the primary target of hydrocortisone activity is further substantiated by the observed structure-activity relationship of anti-In• flammatory steroids and their fibroblast inhibiting ability. Steroids that have shown to have an anti-inflammatory activ• ity in human skin can be represented by the following struc• ture: -c- 0 Fig; 1. /± ~ Pregnene - ll|^17cC -diol - 3,20 dione Berliner and Ruhmann (I967) indicated a direct relationship between the structure of steroids, their fibroblast inhibit• ing potency, and their ability to suppress Inflammation. Schayer (1964) indicated that the principal physiolog• ical function of corticosteroids in Inflammation would In• volve a vasoconstrictive effect as well as a moderation of the histamine action. Corticosteroids stabilize endothelial cells against changes induced by histamine. Cline and Melman (1966) explained that anti-inflammatory steroids control in• flammation by preventing the release of active kinin by pre• venting the interaction between kalllkrein and its substrate. Greaves and Shuster (1970) explained that the anti-inflamma• tory activity of corticosteroids may be due to reduced forma• tion of kinins. Corticosteroids inhibit the delayed inflam• matory response to streptokinase, a powerful activator of kinin forming enzymes. The mechanism of anti-Inflammatory activity of these compounds, lie, not in the action of kinin on blood vessels, but on enzymic formation of kinin. Keir (I967) proposed a model Fig. 2 explaining how the binding of Cortisol with hypothetical receptors may inter• fere with serotonin and histamine, functioning as an antago-^ nist to these amine inflammation mediators. The manner in which steroids interact with their recep• tors was suggested by Bush (I962). He proposed that intrinsic action results not from a chemical reaction but from rela• tively firm "physical" association with their receptors that involve little or no movement of the parts of the molecules -13 Pig. 2 . Illustration of possible mode of interaction of serotonin and histamine with Cortisol re• ceptors). From Kier, L.B.: J. Med. Chem., 11, 915 (1968). — 14 that are in close apposition. Wolf and collaborators ( 1964 ) suggested that in the steroid-receptor complex the steroid is in contact with the receptor surface in two discrete areas: the0 -face of ring A, B and C and °C -face of ring D. They claimed that the effect of the steroid is to induce a conformational change in the receptor, since no chemical reaction as such takes place. The -face" theory has been widely accepted as the basis for the design of new molecular modifications and to explain differences in pharmacological activities of hydrocortisone congeners (Goldstein, 1968; Wagner, 1971). 3. Structure activity relationships The search for mediators of inflammation, specific tar• get cells and receptors for corticosteroids continues. Little is known on the nature and location of receptors. Recently Lev- I'KSonrr (1972) announced a receptor for glucocortlcocoid ac• tivity of hydrocortisone in the acid protein of cells. It has been proposed that anti-inflammatory effects of cortico• steroids depend on local action of steroids. The understand• ing of molecular mechanisms by which corticosteroids exert their characteristic effects on the inhibition of inflammatory processes in either the early inflammatory stage (edema, fibrin 15 deposition, capillary dilation, migration of phagocytes into the inflamed area) and the later manifestations (capillary proliferation, fibroblast proliferation, deposition of col• lagen and cicatrization) is incomplete. In view of lack of specific knowledge of receptor site, structure activity re• lationship (abbreviated SAR) studies have been generally directed towards the evaluation of biological effects e.g. thymus involution and liver glycogen neogenesis assays in animals. Structure-activity relationship studies however cannot deepen the understanding of molecular mechanisms by which corticosteroids act in man. For that the isolation and identification of receptors are needed., Therefore con• clusion from SAR studies must be regarded as speculative. The SAR studies however have provided useful information in research for new and more effective steroids. Basically, SAR studies are done as follows: A suitable biological effect of a drug is chosen. A proto• type drug, which elicits the characteristic effect, is then modified systematically in its molecular structure. Sub• stituents are added or subtracted at various positions and in different steric configuration. A series of such chemi• cally related drugs is known as a congeneric series. By testing the congeners and observing how the substituent groups of each member affect the biological potency, it is possible 16 to postulate an imaginary receptor surface and draw con• clusions about the precise mode of combination of a drug with its receptor surface (GoldsteinJlS?). The main limita• tions of SAR in the evaluation of corticosteroids are in the arbitrary choice of the biological effect employed to evaluate substituent group-effect and the speculative nature of receptors. The SAR studies cannot distinguish readily be• tween substituent group effects which are due to altered affinity of a drug for the receptor or altered transport properties. In no case SAR data preclude that there could not be an alternative group which would increase the effectiveness still more, and there is always the uncertainty that conclu• sions drawn for one biological effect may mot hold for an• other. The structure of the prototype, or parent, molecule of anti-inflammatory steroid is hydrocortisone. The numbering and the sites most frequently modified for increased anti• inflammatory activity are given in Fig. 3 The role of various substituent groups influencing the anti-inflammatory is thought to be as follows: They all contain the 4-ere3-one system in ring A, an oxygen function (either keto or hydroxyl) at G'^lll in ring C, angular methyl groups at C-10 and C-13, and a dihydroxyacetone func- 17 Fig. 3 . Substituent groups increasing the anti-inflammatory- activity of hydrocortisone. CH OH I 2 Desonide Betamethasone 17-valerate Pig. 4 '. The structure of four steroids investigated in this study. 18 tion at C-17 in ring D. The ring structure and the func• tional groups mentioned must remain essentially intact if the compound is to have anti-inflammatory activity. Molec• ular modifications to enhance the anti-inflammatory effect have taken place in the shadowed area, shown on the hydro• cortisone structure. The following changes, either singly or in combination, appear to be the most efficacious in In• creasing the anti-inflammatory activity (Brown, 1970; Berliner, 1968): 1. Introduction of a double bond between C-l and C-2. 2. Introduction of fluoro groups at C-9 and/or C-6 in the alpha configuration. 3. Introduction of an alpha hydroxyl group at C-16. 4. Introduction of methyl group either alpha or beta at C-16. 5. Introduction of the acetonide group on the hydroxyls at C-16 and C-17. 6. Esterification of either or both hydroxyls, in the C-17 side chainr e.g. Structure of side chain: H H — CI — C — CH? -C— C( - CHo -C I— CI — CHw I II I I I I II 3 OH 0 OH OH OH OH OH OH ; ' : — / 17 Ketogenic Steroids 19 The enhancement factors measure the potency increment that a particular function contributes to the compounds activity. The vast majority of structural modifications that result in increased biological activity of steroids probably produce their effect by reducing the rate of elim• ination of steroids from the body, by altering their distri• bution, effective solubility and partition coefficient. Table (2 shows an example of the relative influence of sub- stituent groups in enhancing the anti-inflammatory activity relative to hydrocortisone. Note that the tests are for systemic responses in rats and may lead to wrong conclusions that the enhancement increments are applicable to topical anti-inflammatory activity as well. 4. Topical anti-inflammatory corticosteroids Manufacturers of topical steroid medications and derma- tological investigators are constantly attempting to develop more potent topical anti-inflammatory drugs. This has been achieved by fluorlnation at the 9 °z site on the steroid mole• cule, and more recently by further fluorlnation at the 6 c<- site as well. Added effectiveness is produced by altering side chains at 6,16,17, and 21 positions. Topical cortico• steroids available for dermatologic therapy are considered Table 2 Enhancement Factors for Various Functional Groups of Corticosteroids Functional Glycogen Ant1-inflammatory Effects on group deposition activity urinary sodiuma 9 - Fluoro 10 7-10 +++ 9 OL - Chi or o 3-5 ^ 3 ++ 9 OL - Bromo 0.4b + 12 oc - Fluoro 6-8° 12 oC - Chloro 4C 1 - Dehydro 3-4 3-4 6 - Dehydro 0.5-0.7 + 2 oL - Methyl 3-6 1-4 ++ 6 «c - Methyl 2-3 1-2 l6o6 - Hydroxy 0.4-0.5 0.1-0.2 l?oc - Hydroxy 1-2 4 - 21 - Hydroxy 4-7 25 ++ 21 - Fluoro 2 2 a+ = retention; - = excretion. bIn 1-dehydrosteroids this value is 4. cIn the presence of a 17 oC - hydroxyl group this value is <£ 0.01. . ( From; Burger,A., 1970 ) by medical investigators as belonging to one of the four classes according to halogenation (Bluefarb, 1970). 1. Nonhalogenated corticosteroids: - hydrocortisone, pred• nisolone, desonide. 2. Monofluorinated corticosteroids: - fluorohydrocortisone, fluoromethalone, fluorandrenolone, triamcinolone, dexa- methasone and betamethasone. 3. Difluorinated corticosteroids:- fluocinolone, flumethasone. 4. Chlorinated corticosteroids: - beclomethasone. Other substituent groups than halogens however are also present. Table 3 gives the therapeutically most frequently used congeners of hydrocortisone. Most of them have topical as well as systemic activity. Pew have equal topical and systemic activity and some have equal activity. It is seen that most steroids contain fluorine group in addition to other modifications. This has made it easy to ascribe the main potency enhancing properties to fluorlna• tion since, whether needed or not,fluorine had been added to all potent steroids. In fact many fluorinated steroids have little topical activity as in the cases for Instance, SfV; triamclnolone and fluocinolone. Desonide, containing no fluorine, if proven to be as potent as the most potent topical fluorinated steroids would demonstrate the lack of need for fluorlnation and this would aid Table 3 Anti-Inflammatory Congeners of Hydrocortisone No. Corticosteroid Substituent of Hydrocortisone In Position and abbreviations used —^r- 9oC- 16oC- 160 - 16*-, 17*6- 21 or 17c^- 4,5-double bond In A-Rlng 1. Hydrocortisone (HC) 2. Hydrocortisone acetate (HCOAc) acetate 3. Hydrocortisone butyrate (HCOBu) butyrate 4. Fludrocortisone (9FHC) fluoro 5. Fludrocortisone acetate (9FHC0Ac) fluoro acetate 6. Flurandrenolone fluoro fluoro hydroxy 7. Flurandrenolone acetonide fluoro acetonide 1,2-double bond & 4,5-double bond in A-Rlng 8. Prednisolone 9. Prednisolone acetate acetate 10. Methyl prednisolone methyl 11. Methyl prednisolone acetate methyl acetate 12. Fluorometholone methyl fluoro deoxy 13. Paramethasone acetate fluoro methyl acetate 14. Paramethasone fluoro methyl 15. Dexamethasone fluoro methyl 16. Betamethasone fluoro methyl 17. Betamethasone 17-valerate (B-17-V) fluoro methyl valerate 18. Beclomethasone diproplonate chloro methyl propionate propionate 19* Flumethasone fluoro fluoro methyl 20. Flumethasone plvalate fluoro fluoro methyl trimethyl acetate 21. Desonide (DA) acetonide 22. Triamcinolone acetonide (TA) fluoro acetonide 23. Triamcinolone fluoro hydroxy 24. Fluocinolone fluoro fluoro hydroxy 25. Fluocinolone acetonide (FA) fluoro fluoro acetonide 26. Fluocinolone acetonide acetate (FAOAc) fluoro fluoro acetonide acetate 27. Deprodone propionate deoxy *Fluorometholone has a 17 fl -hydroxy instead of a 17«?C-hydroxy group as the other steroids have. 23 in establishing the truly relevant properties of steroids which determine the topical anti-Inflammatory activity. B. Skin Penetration 1. Biophysical factors in skin penetration The skin is under constant assault by a huge variety of noxious chemicals as well as from substances applied to the skin as cosmetics or medlcinals. The degree of penetra• tion is dependent primarily on physiologic factors of the skin and physical chemical factors due to the penetrant and somewhat secondarily on the vehicle and formulation. (a) Avenues of penetration: Molecules moving from the environment on to the intact skin of living man have three potential portals of entry; via the follicular region, through the sweat ducts, or through the unbroken stratum corneum in between these appendages (Idson, 1968). Grasso (1971) has explained the role of skin appendages in per• cutaneous absorption. According to him, skin appendages form the major pathway for absorption^ for a brief but vari• able period, Immediately after percutaneous application of the test substance. After this period percutaneous absorp• tion occurs mainly through the stratum corneum, but a sub• stantial proportion of the test material passes through the appendages. Skin appendages influence percutaneous absorption via the stratum corneum through their secretions, in particular 2-5 Pig. 5» Anatomical zones encountered by a penetrating substance in transit from the skin surface to the blood vessels (from Griesemer, R.D., J. Soc. Cosmetic Chemists, 11, 80 (i960)). CO Betweon the Cells • Through the Celli ot the of the Stratum Corneum. Sirolum Corneum, Through the Wall* Through the ol the • Sebaceoum Hair Follicle Gland Through the B Sweat Gland FN--'-' Fig. 6. Possible avenues of penetration into and through the unbroken skin (from Griesemer, R.D., J. Soc. Cosmetic Chemists, 11, 81 (I960)). 27 by altering the state of hydration of stratum corneum and by altering skin pH. The epidermis presents a surface area 100 or 1000 times greater than other routes of absorption. The appendages, sweat glands and hair follicles probably contribute only 0.1 to 1.0 per cent of the area of the skin. The two principal secretions from the skin appendages (sebum and sweat) affect the lipid and water content of the stratum corneum, indirectly affecting percutaneous absorption. With the exception of sodium ions and water which may be actively diffused into the skin,most substances are thought to penetrate the skin by passive diffusion, and the degree of absorption Is primarily a function of the combined properties of the drug preparation and of the skin. Scheuplein (1965)])maintains that it is wrong to assume that a principal route of penetration exists without a re• quirement to further specify other conditions. He proposes two stages in the diffusion of drugs. An initial stage when diffusion occurs primarily via follicles and ducts and later stage or steady state diffusion primarily through intact stratum corneum. Once a substance passes through stratum corneum, there is apparently no significant hindrance to penetration through the remaining epidermal layers and the dermis. 28 (b) Barrier characteristics: The stratum corneum is the rate limiting barrier which restricts inward and out• ward movement of chemical substances. Structurally, the stratum corneum is a heterogeneous tissue composed of flat• tened keratinised cells, the outer layers of which are less densely packed than those adjacent to the underlying granu• lar layer. This greater impermeability in the lower horny layer has led to the suggestion that a separate barrier ex• ists at this level, the so-called "stratum conjunctivum" (Idson, 1971). No real evidence exists for the localization of the functional barrier. Analysis of penetration data, evidence from controlled stripping experiments and the de• tailed picture of the stratum corneum gained from electron microscopy, all support the idea that the barrier to penetra• tion consists of a keratln-phospholipld complex In the dead and relatively dry cells of the entire stratum corneum. The proposed mechanisms by which the stratum corneum serves as a barrier also vary. Rein (1924) originated the concept of the barrier being electronegative thereby attracting cations and repelling anions. According to Rothman (1956) the bar• rier layer has the characteristics of an electric double layer, the outer horny layer having a strongly acid, and the inner epidermal layer, a slightly alkaline reaction while the proteins of the Interposed membrane layer are at the Fig. 7' Diagrammatic representation of epidermal ultrastructure (from Selby, C.C.J J. Soc. Cosmetic Chemist, 2*594, 1956). SMx Stratum malpighii SL: Stratum lucldum SG: Stratum granulosum SC: Stratum corneum 30 isoelectric point. Tregear (1962) conceived the resistance of the barrier to be a physical property. There is a limited knowledge of the composition of the barrier. The main cellular components are the proteins, lipids and water combined into an ordered structure. Skin with a disrupted epidermal barrier will al• low up to 80 per cent of hydrocortisone to pass into the dermis. With a functionally intact epidermis the absorption of the steroid is about 1 per cent (Keipert, 1971). (c) Factors influencing penetration: Factors influenc• ing penetration have been reviewed by Shelmire (i960), Higuchi (I960), Wagner (1961), Barr (1962), Tregear (1966), Blank and Scheuplein (1969), Sarkany and Hadgraft (I969), Scheuplein (1971). These factors can be divided into different cate• gories: a) nature of the skin, b) nature of the drug, c) ve• hicle, as follows: a) Nature of skin: i Species difference ii Age of the skin iii Skin temperature iv The state of the skin (normal, abraded, diseased) v Pretreatment of skin 31 vi The degree of hydration of skin vii Skin appendages b) Nature of drug: 1 Molecular size and shape ii Dissolution characteristics of drug iii Polymorphic forms iv Particle size, in case of drugs in suspension or emulsion ^ v Solubility and partition coefficient vi Thermodynamic properties of drugs vii Concentration of the medicament c) Nature of vehicle: i Partition coefficient vehicle/stratum corneum ii Moisture in vehicle and skin barrier iii Nature of the base of the vehicle iv Presence of surfactants in vehicle v Viscosity of the vehicle d) Miscellaneous factors of importance are: i Area of application, contact time and frequency of reapplication ii Route of absorption iii Thickness of skin barrier phase iv Manner of application as under occlusion McKenzie and Stoughton have shown that the penetration 32 of corticosteroids may be increased one hundredfold by oc• cluding the site of application. Occluding the skin prevents evaporation of water that passes across the epidermis, and the watery secretions of sweat ducts leading to an increase in the water content of the stratum corneum, thus increasing percutaneous absorption. Wet epidermis may show up to lOOOx greater diffusion constant (Scheuplein, 1965). Vickers (1963) has demonstrated that occlusion not only enhances penetration of corticosteroids, but creates a depot of corticosteroids in the stratum corneum. A large reservoir f»or the gluco- cortlcosteroids oan be established in the stratum corneum very rapidly by using dimethysulfoxide (DMSO) as a vehicle. "State of hydration" of skin is very important. According to Blank (1965)t dry stratum corneum presents a very high resis• tance to penetrations. It has been claimed that skin pH can be made less acidic if the water content is increased (Blank, 1965). The shift in pH can affect the absorption of some chemical agents, but not steroids which are non-electrolytes. Fritsch and Stoughton (i960) found a tenfold Increase in penetration when the environmental temperature was raised from 10° to 37° and about a tenfold Increase in the completely hydrated skin as compared to a relative humidity of 50 per cent. 33 (d) Physical chemistry of diffusion: The transport of drugs across the skin barrier may be considered as a process of passive diffusion. The flux, J, for transport across a membrane is proportional to the product of force and concentration. dx J = gRgT fL =$&~JJ (1) " -DC ~ ™ a ( u = uo + RT In a) (2) d In a (3) (4) -D dC = —5J~ (for a constant y ) (5) In the above equations: (Ostrenga, 1971) D is the diffusion coefficient for the drug in the barrier, R Is the gas constant, T is the absolute temperature, du Is ~ dx the chemical potential gradient across the barrier, a is the thermodynamic activity and J£_ is the activity coefficient. The rate of penetration, dg>, is then given by: dt h ( (6) J1* h Is the effective thickness of the barrier, and is the amount penetrated per unit area. Since the concentration of drug In the membrane surface on the vehicle side, Cg, is vt related to the concentration in the vehicle, Cv, by K = C2/C and if the concentration at the membrane surface on the oppo• site side, C^, is maintained at zero concentration such that the concentration gradient of diffusion, dC/dx, is equal to the ratio of C£» over the thickness of the membrane, Cg/h, then: d£ = D(K)C *t = h" ( 7) dQ can be obtained from the penetration studies by calcu- dt lating the slope in the steady-state region from plots of amount of drug penetrated versus time. Higuchi (i960) in• itiated the basic equations, describing the variables af• fecting the rate of release of solid drugs suspended in vehicles. He pointed out that the driving force behind drug movement through the skin is the difference in the thermodynamic potential between the vehicle and deeper tis• sues. The direction of flow for systems Is always from higher thermodynamic potential to lower thermodynamic po• tential. i)Solubility and partition coefficient characteristics of the penetrant: The aqueous solubility of a drug determines the con• centration presented to the absorption site, and the par• tition coefficient strongly Influences the rate of transport across the absorption site. Katz and Shaikh (1965) indicate that the efficiency of percutaneous absorption may be a function of the product of the partition coefficient and the square root of the aqueous solubility, in agreement with theoretical considerations developed by Higuchl (196,0). The lipid/water partition coefficient per se is not as signifi• cant as the stratum corneum/vehicle partition coefficient. Treherne (1956) related permeability constant to ether/water partition coefficient. A similar relationship between vaso• dilator activity and lipid/water partition coefficient was demonstrated for.a series of esters of nicotinic acid by Stoughton et al (i960). They also found a similar correla• tion between benzene/water partition coefficients and the penetration of epidermis by a series of closely related boronic acid derivatives. The effects of solubility and partition coefficient on skin penetration are perhaps best illustrated with cortico- 36 steroids. Triamcinolone possesses five times the systemic activity of hydrocortisone, but only one-tenth its topical activity. Conversion of triamcinolone to its acetonide yields a more favourable llpid/water partition coefficient and enhances the topical activity one thousandfold (McKenzie, 1962). Similarly, betamethasone has 30 times the systemic activity of hydrocortisone, but only 10 times its topical activity. Conversion to betamethasone-17-valerate increases topical activity over tenfold (Idson, 1971). ii) Molecular characteristics of penetrant: An inverse relationship appears to exist between ab• sorption rate and molecular weight (Tregear, 1964). Small molecules penetrate more rapidly than large molecules, but within a narrow range of molecular size there is little cor• relation between size and penetration rate. The effect on penetration rate of the size and shape of the penetrating molecules can be determined only if the effect of size and shape can be separated from the effect of solubility char• acteristics. According to Blank and Scheuplein (1964) water solubility decreases and lipid solubility Increases with an increase in molecular weight in the series of analogs such as aliphatic alcohols. The rate of penetration increases as the molecular weight increases. But higher molecular weight materials also show variable penetration. Little is known of the effect of the molecular shape, iii) Vehicle effects: The literature on the Influence of vehicles on skin penetration is confusing and often contradictory. A lack of awareness of possible drug-vehicle interaction and the function of the thermodynamics involved in the interpretation of results(, has added to the difficulties. Rothman (1954) reviewed the literature on vehicles up to 195^« and- Barr to 1962. More recent reviews are those of Barret et al. (1969), Munro (1969) and Sarkany and Hadgraft (I969). Phys• iological availability of the topically applied drug depends on both the rate of release from the vehicle and the permea• bility of the skin. Release of a substance will be favoured by the selection of vehicles having a low affinity for the penetrant, or in which the drug is least soluble. This is consistent with the view that the rate of release is governed by the vehicle to receptor phase (stratum corneum) partition coefficient (Schutz, 1951). Another important factor is how firmly or loosely the solute is held by the vehicle. Solutes held firmly by the vehicle, such as when the drug forms a soluble complex with the vehicle, exhibit low activity coefficients; hence the rate of release from such drug-vehicle combinations will be slow. Solutes held "loosely" by the vehicle, exhibit high 38 activity coefficient; therefore the rate of release from such drug-vehicle combination will be faster (Poulsen, 1968). Dempskl (19695) et al„demonstrated that in vitro release of a medicinal agent is a function of the degree of solubil• ity of that agent in both the base and its surrounding media. Penetration of an ointment film such as is applied to skin occurs in two phases. Initially, the penetrating agent par• titions between its carrying medium. This is followed by the second phase of penetration, the diffusion of the penetrat• ing agent through the ointment film. The second phase of the permeation may be further subdivided into two stages. The first stage is the establishment of a uniform concentration gradient of penetrant across the barrier, and second is the constant, uniform diffusion of penetrant through the barrier after the uniform concentration gradient has been established. The second phase is known as steady state diffusion. The steady state diffusion lasts as long as the chemical remains in adequate supply on top surface and Is removed from the lower surface (Lueck, 1957)• Iv) "Accelerant" solvent: An accelerant increases cutaneous permeability by caus• ing the keratin to swell and by leaching out essential struc• tural material from the stratum corneum, thus reducing the diffusional resistance. Varied agents have been reported to 38 activity coefficient; therefore the rate of release from such drug-vehicle combination will be faster (Poulsen, 1968). Dempski (1969.) et al,. demonstrated that in vitro release of a medicinal agent is a function of the degree of solubil• ity of that agent in both the base and its surrounding media. Penetration of an ointment film such as is applied to skin occurs in two phases. Initially, the penetrating agent par• titions between its carrying medium. This is followed by the second phase of penetration, the diffusion of the penetrat• ing agent through the ointment film. The second phase of the permeation may be further subdivided into two stages. The first stage is the establishment of a uniform concentration gradient of penetrant across the barrier, and second is the constant, uniform diffusion of penetrant through the barrier after the uniform concentration gradient has been established. The second phase is known as steady state diffusion. The steady state diffusion lasts as long as the chemical remains in adequate supply on top surface and is removed from the lower surface (Lueck, 1957)• iv) "Accelerant" solvent: An accelerant increases cutaneous permeability by caus• ing the keratin to swell and by leaching out essential struc• tural material from the stratum corneum, thus reducing the diffusional resistance. Varied agents have been reported to 39 have accelerant action, particularly propylene glycol, sur• face active agents, and aprotic materials such as urea, DMSO, DMP, and DMA. Work with these solvents has contributed to a greater understanding of the chemical nature of the skin bar• rier in relation to a specified penetrant and the transport mechanism of various compounds across skin. DMSO, DMP, and DMA are all strongly hygroscopic and hence increase the hydra• tion and therefore its permeability. There are possible re• versible configuration changes in skin protein structure brought about by substitution of integral water molecules by DMSO. DMSO can extract soluble components from the stratum corneum, suggesting ultrastructural modifications consistent with an increase in permeability (Plfbaum, 1968). Surface active agents appear to play a major role in promoting transappendageal absorption. Penetration of cer• tain antimicrobial substances appears to be enhanced by the addition of surface active agents. The penetration of fatty acid soaps varies inversely with pH (Sprott, 1970). v) The pattern of skin penetration: The theoretically expected pattern of skin penetration is as follows (Tregear, 1966): When a substance is applied to skin, alone or in solution, it only reaches the circulation, or in the case of diffusion cell, the washing saline, gradually; the rate of transfer DMSO '(Demethylsulfoxidei)), DMF, (Dimethylformamide), DMA (Dime thy lac etamide) 4U Representative Penetration Profile for Drug Diffusing Through Human Skin Time Pig. : 8 .. ; A generalized penetration curve showing delay time, td and steady state condition. through the skin rises to reach a steady rate. This steady penetration rate is maintained thereafter indefinitely, pro• vided that (a) a constant concentration of the penetrant is maintained on the skin, and (b) the penetrant, or its vehicle, does not attack the skin surface. The time taken to reach this steady state is given by the delay period, td (Pig. 8 )• The steady state penetration, Q_, is obtained from the slope of the straight line portion in a plot of penetration rate vs time. Its units are mass per unit time per area. 2. Methods for measuring percutaneous absorption Blank (I960), Gemmel (1957)t Ainsworth (i960) and Stoughton (1964) have reviewed methods for measuring percu• taneous absorption. Most authors find it convenient to classify these methods as in-vivo, in^vitro and autoradio• graphy methods. Blank (i960) has observed more specifically that most percutaneous absorption investigations have in• volved the measurement of: i) penetration in model systems ii) histological studies iii) the use of tracers 42 The measuring methods using models all are of In vitro type. Membranes composed of cellulose film e.g. dimethyl- polysiloxane (Flynn, 1971) sheep bladder etc., have been used as models, alternatively human and animal excised skin is used. The assumption is made that the process of penetra• tion in the skin is similar to the diffusion through a mem• brane. Various designs of skin diffusion cells have been used for drug penetration studies (Poulsen, 1969). The use of the diffusion cell is very convenient and gives a satisfac• tory representation of the relative ability of drugs to pene• trate the skin. They have a limited value, however, because the models represent either artificial or metabolically in• active systems which lack circulating blood. Histological studies have also been utilized to measure. absorption of medicaments (Duemling, 1941). Goldzieher (1952) used histologic sections in their study of the penetration of estrogens. Kosch (1944) studied the penetration of sali• cylic acid, sulfur, resoroinol and other substances by ex• amining histological sections for evidence of keratolysis. The most common methods of studying percutaneous absorp• tion In vivo have been done by analysis of circulating blood, urine or feces. In certain oases organs or tissues have been excised and analyzed for drug content. Hlynka (I969) studied the intracutaneous absorption of drugs, utilizing the latter 43 approach. Extraneous dyes, fluorescent materials, radioactive tracers, have been utilized for a long time to trace drug penetration inppercutaneous absorption studies. The use of dyes and fluorescent materials in penetration studies is open to criticism. One cannot be certain that the dye or fluorescent material remains with the substance whose pene• tration is being studied. Radioactive tracers have become Increasingly popular for studying percutaneous absorption. The use of radioactive tracers is carried out by one of the following techniques: (1) direct counting at the surface (11) autoradiographic technique (ill) measurement of radioisotope within the body. 3. Isolation of epidermal sheets The closest approximation to In vivo diffusion conditions In model systems is through the use of human autopsy skin for a diffusion membrane either in the form of Isolated epidermal sheets or through the use of whole thickness skin. The iso-1 lated epidermal sheets give more reproducible results. The human epidermal sheets can be removed mechanically, chemically or by heat separation: 44 i) Mechanical method: Wolf (1939) first introduced a cellophane tape strip• ping technique for the removal of epidermal sheets. Van Scott (1952) showed that if a strip of human skin is stretched to approximately double its original length, the epidermis can be stripped away quite easily. Unfortunately, the tension required to achieve this purpose is very little less than the breaking point of the skin. Gilbert et al.? (1963) • proposed an improved technique for the isolation of epidermis from human skin. They found it necessary to stretch the skin to 1.75 times its relaxed length. The epidermis could then be separated by blunt dissection with a suitable instrument. ii) Chemical method: In the leather industry, the epidermis is removed from dermis by efficient chemical methods. Baumberger (1939) used chemical methods for separation of epidermis from der• mis. They used dilute acetic acid, or one normal ammonium hydroxide for 20 minutes at room temperature. They also found sodium carbonate solution to be equally effective al• though slower in action. The effects of the compounds studied was^gthat an acid or a base which can penetrate the tissue with sufficient speed and will not react with the tissue proteins,., can form a compound having an acid dis• sociation constant near the isoelectric point of collagen. *5 This may cause the collagen to swell, permitting the de• tachment of the epidermis. Wood and Bettley (197D removed the stratum corneum by suspending the skin on the glass rods of a chromatogram tank above 0.88 ammonia solution for 60 minutes, after which the stratum corneum was lifted off. According to Medawar (1941), epidermal sheets can be isolated by incubation of the skin with enzymes. He ob• served that epidermal sheets could be prepared from human skin Incubated in Tyrode's solution containing commercial trypsin. Epidermis is thought to be secured to the dermis by elastic fibres and the enzyme elastase breaks down elas• tic fibres, ill) Heat separation: Scheuplein (I965) removed the whole epidermal sheets from autopsy skin by Immersing It in a water bath at 60° for 30 seconds. These membranes carry the intact stratum corneum, and can be obtained in unruptured pieces, Allenby (1969) et al removed epidermal sheets by dipping human autopsy skin (after removing the subcutaneous adipose tissue) in a water bath at 60° for one minute, blotting dry, and peeling the epidermis off with forceps. Marrs (1971) removed the epidermis by immersing the skin in a water bath at 55° for 30 seconds and immediately cooling with ice water. The 46 skin was spread on an Iced petrl-dlsh cover and epidermis was easily peeled away. Stratum corneum Is shown to undergo Irreversible structural changes when heated above 65° (Bernstein, 1970). Depending upon the purpose for which the epidermal sheet Is required, a choice from these various techniques can be made. For the purposes of penetration studies, Scheupleints (I965) method was found to be most useful because It ren• dered large Intact epidermal sheets which could be stored easily. The complex chemical, physical and biological prop• erties of living skin preclude adequate simulation by In vitro models. Therefore, In vivo methods should also be carried out for complete Information. 47 G. Analytical Methods Analysis of corticosteroids in biological samples can be performed by spectrophotometry, fluorometric, gas chroma• tographic and radioisotopic techniques. 1. Spectrophotometric assays 4 Solutions of steroids containing the A - 3 keto group 240-242 nm. and 310 nm. Substituents groups may change the 250 -250 peak intensity wavelengths towards nm. The 240 nmii region can be used for quantitation. The great advantage of this type of assay is its rapidity. The disadvantages are low sensitivity and low specificity. 2. Fluorometric assay Noujaim and Jeffery (1970) have reviewed the spectro- photofluorometric methods for the estimation of corticosteroids. The spectrophotofluorometric (SPF) method identifies and quantitates compounds through either their fluorescent or phosphorescent properties. The SPF method is accurate, 48 precise and specific. Corticosteroids do not possess native fluorescence but can be made to fluoresce by treatment with strong acid. The functional groups responsible for promot• ing the acid-induced fluorescence are the 11 - hydroxyl and the A - 3 - keto groups ( sweat, 1954). Cortisol, cortico- sterone, 20 -|J - hydroxycortisol and their 11 - hydroxy epimers possess these groups. Fluorescence of steroids is further Increased by the presence of a 17 - or 21 - hydroxyl group. The functional groups responsible for inhibiting fluores• cence are the 11: - keto or 16<<- hydroxyl group. Reduction of the double bond at the 4th position ( Shimo, 1967 ) also de• creases- fluorescence. 3. Other methods f (a) Gas liquid chromatography assay: %X£f$ t ) Gas liquid chromatography (GLC) is a useful technique for the analysis of complex mixtures because it affords a means of separation and quantitation in one procedure. The use of GLC with steroids requires columns with low concentrations of stationary phase. Determination of steroids in biological samples necessitates the use of detectors with high sensitiv• ities because of very low concentrations of steroid present. 49 (b) Radioisotopic assay: Noujaim and Jeffery (1971) have recently reviewed the analysis of corticosteroids in biological samples by radioisotopic methods. Radioisotopic methods are reported to be far superior in sensitivity, pre• cision and specificity when compared to nonradioactive tech• niques. Their main disadvantages are expense of the Isotopes, precautionary measures necessary especially in human experi• mentation and their Inapplicability for routine testing of commercially available steroids. 4. Vasoconstriction assay Topical or systemic anti-inflammatory substances, have been evaluated in a variety of procedures, which apparently represent different modes of action and/or different stages of inflammatory process. The most commonly used method is the vasoconstriction test (McKenzle, 1962). The mechanism of steroid-induced dermal vasoconstric• tion remains unexplained. Some authors have proposed that steroids increase the sensitivity of vascular smooth muscles to normal levels of nor-epinephrine (Solomon, 1965). More recent theories include: 1. a direct effect of steroids, similar to sympathomimetics, on sodium transport across smooth muscle, 2. a release of locally bound nor-epinephrine from cutaneous 50 stores, or 3. effects on various enzyme systems involved in the re• lease of a number of substances, including nor-epinephrine, serotonin, bradyklnin, thus interrupting normal vascular tone (Cline, 1966). Still another proposal is that steroids act by decon- gesting the capillaries of the normal papillae. Investigations Into ,the mechanism of ant1-inflammatory action of Cortisol have revealed that steroids may exert their effect by inhibiting the release of plasma kinins and also by preventing interaction between the activated plasma enzymes and their plasma protein substrate (Cline, 1966) which are vasodilating substances. It seems possible that this mechanism explains the excellent agreement between vasoconstriction activity and the anti-inflammatory activity of the steroids. Certain difficulties are encountered with vasoconstric• tion tests,and errors are inherent. Care must be taken in vasoconstriction assays to allow for site to site and indi• vidual to individual variations (McKenzie, I966). Despite the low precision of the assay, most authors agree that it is a very useful and a quite reliable method for screening new compounds before clinical trials. 51 III STATEMENT OF PROBLEM The objectives of this Investigation ares (1) to determine whether the recently introduced non- fluorinated steroid desonide (desfluorotriamcinolone aceto• nide) is clinically as acceptable for the topical treatment of steroid-responsive dermatoses as the fluorinated beta• methasone 17-valerate which is regarded as the therapeutically most potent anti-inflammatory steroid. (2) To explore the possibilities for the development of spec- trophptofluorometrlc technique for the assay of desonide. (3) To develop a convenient and reproducible In vitro method for the measurement of epidermal penetration rates of anti• inflammatory corticosteroids. (4) To interpret the results with the aid of auxiliary parti• tion coefficient and solubility determinations and by compar• ing results with those obtained for two other steroids, hydrocortisone which is known to be less active than triam• cinolone acetonide and betamethasone 17-valerate, which is known to be more active than triamcinolone acetonide. (5) To evaluate the effects of 9-alpha fluorinatlon on the biological activity by determining the ED^Q of the vasocon• strictor test for desonide and its fluorinated analog, triam• cinolone acetonide. IV EXPERIMENTAL METHODS A. Apparatus 1. Poulsen skin diffusion cell The Poulsen skin diffusion cell shown in Fig. 9 , consists of an upper donor chamber in which the penetrating agent is placed and a lower glass chamber with a side arm to allow sampling of the receptor phase. A teflon coated bar attached to a polyethylene sail provides efficient mix.- ing within the lower receptor chamber. Two teflon discs holding the skin are clamped onto the flat ground glass surface at the top of the receptor chamber,/ leaving an ex- posed central circular area of 1.0 cm t. 0.05 cm/through which the penetration is measured. The volume of the re• ceptor chamber is 10 ml, and that of donor chamber 0.4 ml. Fig. 9 .,, shows the assembled diffusion cell. Fig. 10? shows all the different parts of the diffusion cell, and Fig. II shows the top view of the diffusion cell. 2. Spectrophotofluorometer Aminco-Bowman spectrophotofluorometer Model 4-802^ was used. This Instrument consists of a solid state xenon lamp, D.C. power supply unit, I.?> 21 photomultiplier tube of 1 American Instrument Company, Inc., Silver Spring, Maryland. 53 Poulsen Skin Diffusion Cell Fig. 9 A, Skin specimen. B, Teflon pieces holding skin. C, Receptor chamber. D, Side arm. E, Teflon coated Bar. F, Polyethylene sail. G, Clamp. Fig. 11 Showing top view of the diffusion cell, having epidermis exposed, through which penetration occurs. 55 a wave length range of 300 - 700 millimicrons. Slit ar• rangement #3 (33331) was used. The cells were fused quartz of 1.0 cm path length. 3. Picker Nuclear Llqulmat, Model-650-503 was used. Division of Picker X-ray Corporation. Manufactured by: Intertech. Inc., North Haven, Conn., U.S.A. 4. A. Hitachi-Cabman 124 - Spectrophotometer B. Beckman - DGBT Spectrophotometer 5. Dermatome,ccastroviejo electro-keratotome was used in modified form (Stewart and Runlkis, 1967). B. Procedures 1. Analysis of steroids (a) Spectrophotofluormetric analysis of desonide: The fol• lowing procedures were tried: i) Modified Mattingly's method (1962) ii) MacKenzie's method (1967) ill) Modified Martin's method (1968) Mattingly's and MacKenzie's method: The fluorometric method originally established for 56 hydrocortisone was tried for analysis of desonide. The following were the steps involved: (A) 15 ml of methylene chloride was added to 2 ml of deso• nide standard solution (5 ^ug/ml). The extract ion\&was al• lowed to proceed for 20 minutes. (B) The tubes were removed from the shaker and the two layers allowed to separate. The upper layer was discarded and the methylene chloride layer filtered. (C) 10.0 ml of the filtrate was transferred to clean test tubes. 5»° ml of fluorescence reagent was added and mixed thoroughly. The fluorescence of the acid layer was measured at an excitation wavelength of 395 JHftt an emission wavelength of 450 nm with slit arrangement #3 (333D. Step C was also modified slightly by evaporating the methylene chloride in a warm current of nitrogen at 48°C before the addition of fluorescar»^seagent. To the dry tubes 5.0 ml of fluorescence reagent was added, mixed for 30 sec• onds and the fluorescence read immediately. Modified Martin's method: Martin proposed an oxime formation method to differen• tiate Cortisol and corticosterone in human plasma. The oxime formation was very rapid and final fluorescence could be read even after 60 minutes. 57 -C— C + NH9OH NHOH II 0 OH Their method was tried to analyze desonide, as follows: (a) To the standard solution of desonide, 15 ml of methylene chloride was added and steroid extracted by gentle shaking. The tubes were centrifuged and the upper layer discarded. (b) Duplicate 5 ml aliquots of methylene chloride were transferred into clear centrifuge tubes, acidified with a drop of 25$ acetic acid, and evaporated to dryness in a water bath at a temperature not exceeding 40°c. (c) Duplicate tubes were prepared, - the front row serving as a sample, the back row of tubes as "-k.blank. To the front row of tubes, 0.3 ml of blank reagent was added and to the back row of tubes 0.3 ml of hydroxylamine reagent was added. The solutions were mixed and then air- lowed to stand for one hour at room temperature. At the end of one hour 1.7 ml of distilled water was added to all tubes. (d) 10.0 ml of methylene chloride was added to all tubes. The tubes were shaken, centrifuged and the aqueous layer discarded, care being taken to remove all water from the tubes. (e) 1.2 ml of the fluorescence reagent was added. 5« The tubes were shaken vigorously for 30 seconds and the extract allowed to stand for one hour. The fluorescence of the ethanolic sulfuric acid phase was read. (b) Spectrophotometric methods The steroids namely, hydrocortisone, desonide, triam• cinolone acetonide, betamethasone 17-valerate,were dissolved in a minimum quantity of ethanol and diluted with normal saline. The dilutions ranged from 0.5 to 10.0 ug/ml. Spec• tra for all steroids were obtained in the region of 200-340 nm. For analytical purposes the absorbance was measured at the peak for each steroid. 2. Solubility and partition coefficient determinations 1) Solubility determinations The solubilities of hydrocortisone, triamcinolone acetonide, desonide and beta• methasone 17-valerate were determined in distilled water, normal saline and 40$ ethanol at 25°C, according to Dempskl (1970). An excess of steroid was added to approximately 100 ml. of solvent in glass bottles. The bottles were tightly capped and placed on a rotating-bottle apparatus in a 25°C i 0.1 water bath for periods of not less than 24 hours or more than 72 hours. Equilibrium was determined by repetl- 59 tive sampling. Before the bottles were sampled for assay, the rotating apparatus was turned off to allow the excess of steroid to settle in the solvent. The liquid was fil• tered through Millipore filters (0.22 AH pore size) to ensure the absence of any solid particles. The filtrate was di• luted with the appropriate solvent for assay. The UPV, absorbance of each solution was determined and the steroid concentration calculated from previously determined absorp• tivity, ifa 5) values. ii) Partition coefficient: The partition coefficient of hydrocortisone, triamcinolone acetonide, desonide and betamethasone 17-valerate were determined in octanol/water system. 20 mg. of each steroid was weighed and dissolved in 25 ml of octanol. 25 ml water was added to the octanol solutions, the bottles capped tightly and placed on a ro• tating bottle apparatus in a 25 - 0.1°C water bath for 3 days. Equilibrium was determined by repetitive sampling. After the third day, contents of the bottles were poured into separatory funnels and left undisturbed for 2k hours. The aqueous layer was separated and filtered through Whatman #1 filter paper. The steroid concentration in the aqueous layer was calculated from the absorptivity, a, value. The concentration of steroid in octanol could be calculated by 60 subtracting the concentration of steroid in aqueous layer from the original concentration used. .- concentration in organic phase concentration in aqueous phase 3. In vitro penetration studies (a) Preparation of membranes for diffusion cell: i) Pull thickness hairless mouse skin: Hairless mice (HRS/J strain) from Jackson Laboratories, Bar Harbour, U.S.A., were used for tritiated water diffusion studies. The full thickness of the mice skin was used,, age 8 to 32 weeks. ii) Epidermal sheet of human autopsy skin: Human autopsy skin samples were obtained from Vancouver General Hospital. The autopsy skin was from the abdomen of the patients, who were not on steroidal therapy for one week prior to death. The samples were from autopsies done within 24-48 hours after the death of the patients. Epidermal sheets from the autopsy skin were obtained, first by removing the subcutaneous adi• pose tissue. The skin was then immersed in a water bath at 60° for 30 seconds. It was pressed between the two glass sheets. The epidermis was slowly lifted off as shown in Fig. |12 . The epidermal sheets were stored in the refrigerator at Fig. 12 . Showing how the epidermis was removed from the autopsy skin. 62. 4°c, and rehydrated when required for experiment by dipping them in distilled water at 37° for 30 minutes (Comaish,1971 ). (b) To check intactness of epidermal sheets by tritiated water: Hydrated epidermal sheets were sandwiched between the two teflon pieces of the diffusion cell and 8.0 ml of physiological solution was added to the receptor chamber. 0.4 ml (25/# ci) of tritiated water was put on top of the epidermis fixed in the diffusion cell. 0.5 ml samples were withdrawn from the lower chamber at varying time intervals. Each sample was replaced by 0.5 ml of saline in order to keep the volume in the lower chamber constant. The 0.5 ml samples were added to 12.5 ml of the scintillation fluid. The solution in the vials was allowed to stand until the sodium chloride had settled at the bottom of the vials. Appropriate standard and background samples were pre• pared and assayed with each set of unknowns. A known amount of tritiated water was added to a series of previously as• sayed samples, the increase in counts were within experimen• tal error, showing there was no quenching. Rate of diffusion of tritiated water was calculated by using Downes (1967 ) equation. Rate of diffusion = C A x B where is the area of the skin in contact with the solu• tion in the lower chamber, B_ is the specific activity 63 (counts/min/mg.) of water on top of the skin, C_ is the total amount of radioactivity (counts/min.) which has accumulated on the sampled side during one hour. (c) Inevitro steroid penetration: The^penetration of steroids; namely.hydrocortisone, triamcinolone acetonide, desonide.and betamethasone 17-valerate,through human epidermis was studied using the following vehicles: i) Steroid deposit on epidermal sheets: 0.4 ml of the steroid solutions containing 2.0 mg. of hydrocortisone, triam• cinolone acetonide and desonide and betamethasone 17-valerate was slowly deposited on top of the epidermal sheet fixed in the diffusion cell. Ethanol was evaporated by a current of cold air from the blower. ii) Steroids in 40$ ethanol: 0.4 ml of steroid solutions, in 40$ ethanol, representing O.56 mg of each steroid, was pipetted on top of the epidermis. iii) Steroids in hydrophilic ointment base: 0.15 gm/ of the steroid ointments, representing 1.5 mg of each steroid was spread on the epidermal sheet. Penetration studies: The steroids, hydrocortisone, triamcinolone acetonide, desonide, betamethasone 17-valerate in drug deposit, 40$ ethanol and hydrophilic ointment base, were applied on top of the epidermis which was sandwiched between the two teflon 64 pieces, leaving an exposed circular area of 1.0 cm , through which the penetration was measured. 8.0 ml. of the solution of 0.9% NaCl was pipetted into the receptor chamber. The side armswas closed with parafilm, and a glass cover slip was placed on top of the exposed surface to prevent evaporation. 2.0 ml. of sample solution was withdrawn from the lower chamber at regular intervals for analysis. The sample solution was replaced by fresh 2.0 ml. of solution of normal saline in the diffusion cell. 4. Vasoconstriction bioassay The McKenzie-Stoughton vasoconstriction assay as im• proved by Place ( '1970) and his Syntex group of co-workers was used. The test was based on the observation that upon penetration into the skin an anti-inflammatory corticosteroid, above its threshold dose, caused skin blanching due to vaso• constriction. We determined the El|g of desonide and triam• cinolone acetonide as follows; 50 adults with no skin dis• orders were selected, the steroids were applied in ethanol solutions and in creams in tenfold serial dilutions from 0.0001 to 0.1%. The skin was prepared by washing with soap and water. 2 Sixteen squares of one cm area were marked off on both fore- 65 arms by means of a silicone greased stamp. The solutions and the creams were coded by a nonparticipant in the assay. Tables of random numbers were used to assign the® test areas. Solutions and creams were randomized differently. Each arm had a row of four solutions and a row of four creams. Since the steroids at.«higher concentrations show considerable lat• eral spreading, the spacing between test areas had to be about 1.5 cm. Thus, the number of test preparations were equal to the number of test areas so that every test area had an equal chance to receive every test preparation. Solutions were applied in 20 /til. volumes, The solvent was allowed to evaporate and the steroid deposits covered 2 with matching plastic cups. Uniform layers of 20 ng/cm - 20,^/g/cm of solid drugs were thus obtained. The creams were filled flush in plastic cups and pressed firmly on their test areas. Blenderm tape and Saran wrap were used to secure the cups and to occlude the areas for 16 to 24 hours. Two investigators read the responses one hour after the removal of dressings. 66 TfV RESULTS AND DISCUSSION A. Spectrophotofluorometric Methods for the Estimation of Desonide Adaptation of Mattingly's (1962),MacKenzie's (1967), and Martin's methods (1968) for the estimation of hydro• cortisone were tried for desonide. With the first two methods the addition of fluorescence reagent to methylene chloride extract resulted in formation of an emulsion, which was difficult to break. The method had to be modified & slightly in order to avoid the formation of this emulsion. Methylene chloride was first evaporated from the final extract of desonide, and the fluorescence reagent was then added to the dry tubes. The contents were mixed, and the fluorescence was measured. With this modification no emul• sion was formed. However, fluorescence decreased rapidly. In fact, no fluorescence could be recorded after one minute. The modified method was thus found unsatisfactory. Efforts were continued to find an alternative SPP method, and this time, Martin's method was tried. Most of the glucocorticosteroids having a keto group at the 20th position react very rapidly with hydroxylamine hydrochloride to form the respective oxime derivative. Martin has used this technique to analyze Cortisol and corticosterone. 67 Even this method was unsatisfactory for desonide. As Martin observed in his work with Cortisol, immediately after addi• tion of fluorescence reagent, the fluorescence intensity of the hydrocortisone oxime derivative decreased and then leveled off and remained constant for 60 minutes. However, addition of fluorescence reagent to desonide and blank tubes resulted in an increase in fluorescence intensity in both sets. The possibility of using Martin's method for estimation of desonide was : therefore ruled out. Fluorometric methods for estimation of desonide were unsuccessful because sf (a) the presence of -OH group at the C-16 position, does not favour fluorescence ( Shimo, 1967 )? (b) the addition of fluorescence reagent causes rapid hydrolysis of the compound at C-16 and C-17 positions (c) the addition of strong acid causes the formation of different species or decomposition of the drug. Spectrophotofluorometric methods were also unsatisfac• tory for triamcinolone acetonide. B. Spectrophotometric Method Spectrophotometric methods are most frequently used. The two main disadvantages of spectrophotometric assays are 68 low sensitivity and lack of specificity. Fortunately the steroids studied had a high absorption intensity and stable apsorption peaks. There was no interference by other ma• terials in our spectrophotometric assay. The minimum amount of steroid detectable was in the range of 0.5 ug to 2.0 ug, depending upon the steroid and solvent used. This enabled us to measure the steroid diffused through the skin to the lower chamber of the diffusion cell. C. Solubility and Partition Coefficients of Steroids The solubility of a drug in the vehicle determines the concentration presented to the absorption site, and the par• tition coefficient strongly influences the rate of transport across the absorption site. Theoretical considerations de• veloped by Higuchi (I965), Katz and Shaikh (1965) indicated that efficiency of percutaneous absorption may be a function of the product of the partition coefficient and the square root of the aqueous solubility. The water solubility of steroids is given in Table 4. Hydrocortisone has maximum water solubility whereas Beta• methasone 17-valerate has the minimum. It indicates the pro gressive decrease in permeability as the steroid becomes increasingly more polar. o 70 The octanol/water partition coefficients of the steroids were determined. The partition coefficient in this system are reported by us for the first time. The ether/water partition data are taken from Plynn (1971). Ether/water partition coefficient for desonide was calculated from Plynn*s equation. These partition coefficients in both the systems are given in Tabl& 5. Fig. 14 shows the linear relationship of partition coefficient to cumulative penetration. Betamethasone 17-valerate has the highest partition coefficient and hydrocortisone the lowest. It Indicates that the greater the partition coefficient of a drug, the more rapidly it diffuses from aqueous fluids to membranes. Theoretical calculation of ether/water partition co• efficient for desonide according to Plynn (1971). F Partition coefficient of prednisolone (Ky) = 1.13 Value of group constant factor ( £ ) of 16 ©c hydroxy «s 0.45 Partition coefficient of 16 ochydroxy prednisolone 1.13 x 0.45 8 0.$59 Value of group constant factor ( £ ) of 16 and 17 - aceto• nide = 19.3 oc Partition coefficient of 16 - hydroxy, prednisolone, 16-17, acetonide O.559 x 19.3 = 10.708. 71 Table 4 Solubility of steroids In two different solvents Name of the steroid Solubility (mg./ml.) at 25° Distilled water 40% Ethanol Hydrocortisone 0.280 5.48 Desonide 0.075 5.20 Triamcinolone Acetonide 0.012 1.44 Betamethasone 17-valerate 0.005 1.47 0 Solubility of steroids as reported in the literature Hydrocortisone 0.280s Desonide Triamcinalone Acetonide 0.010' Betamethasone 17-valerate aThe Merck Index, eighth ed., 1968, p. 542. ^Malkinson and Klrschenbaum,(1905)• 72 Table 5 Octanol/water and ether/water partition coefficients of several steroids Name of Steroid PC(K|) PC(K^) Hydrocortisone 28.8 1.6 Desonide 61.0 10.8 Trlamcinalone acetonide 88.4 14.6 Betamethasone 17-valerate 149.8 509.0 *Ether/water partition coefficients reported by G.L. Blynn (197D. **Ether/water partition coefficient theoretically calculated from the equation of Elynn (1971). D. Reliability of Heat Separation Method for the Removal of Epidermis from Autopsy Skin The epidermis was removed by a heat separation technique. In order to check if the epidermis separated by heat was dam• aged, the whole skin was exposed to heat for exactly 30 sec• onds at 60°c. Prom one autopsy skin, dermatome sections of about 0.1 mm. thickness were cut and from the rest of the autopsy skin, the epidermis was removed by heat. Sample sections were obtained using a cryostat microtome. Micro• scopic examination of both samples revealed that the epider• mal sheets were not damaged by the heat separation technique (Pig. 15 )• The iheat separation technique is very safe and satisfactory for removing the epidermis from the autopsy skin. Large, unruptured, intact pieces could be obtained. E. Tests for Intactness of Skin with Tritiated Water The intactness of the epidermal sheets was checked with tritiated water. Downes (1967) et al. determined the rate of diffusion of tritiated water through hairless mice skin and autopsy skin. His results showed marked variation, ranging from 0.18 to 1.08 mg/cm /hr. for hairless mice skin Dermatome section of human skin I 1 — " Heat separated epidermal sheet from human autopsy skin Fig. 15. Dermatome and heat separated sections of human epidermis. 76 and 0.01 to O.69 mg/cm /hr. for human autopsy skin. Table 7 shows the rate of diffusion of tritiated water through hair• less mice skin and human epidermis. The results obtained were very close to the reported values. Downes et al com• pared the diffusion rates of tritiated water from the dermal side with those of the epidermal side of the skin. Their results suggested that diffusion in either direction is ap• proximately equal. The variability in the rate of diffusion of water existed not only among specimens of skin from differ• ent body regions of the same animal. Blank (1952), and Mali (1956) have also noted variation in water diffusion rates through different samples of human skin In vitro. Accurate measurements of the rate of diffusion of triti• ated water through human epidermis can be made within a period of two hours. Moreover, the data obtained are highly repro• ducible. Our main object was to check the intactness of the epidermis. If our results of rate of diffusion of tritiated water fell within the reported range, it was assumed that the epidermal sheet under test was intact and could be used for steroid penetration studies. Table 6 Rate of diffusion of tritiated water through human abdominal autopsy skin Specimen number Number of hours Rate of diffusion and age (Years) after death (mg/cm /hour) # age 4 39 8 0.450 t 0.032 12 41 47 0.459 - 0.045 10 56 7 0.387 - 0.051 9 58 24 0.332 - 0.038 6 58 7 0.430 i 0.031 20 70 11 0.451 - 0.047 24 66 44 0.520 - 0.060 29 32 23 0.382 t 0.038 43 68 12 0.463 - 0.037 38 7 24 0.582 - 0.040 Mean (n = 4) - standard deviation. Table 7 Diffusion of tritiated water 2 Rate of diffusion (mg./cm /hr.) Specimen # Hairless mice skin Human epidermis 1 0.38 - 0.032 0.61 - 0.021 2 0.40 i 0.03.8 0.60 t 0.028 3 0.37 - 0.040 0.45 t 0.031 4 0.41 - 0.032 0.43 - 0.037 5 0.50 t 0.028 0.38 ± 0.040 6 O.36 - 0.025 0.44 ± 0.060 Literature values; Rate of diffusion of tritiated water: (a) hairless mice>w, 0.18 - 1.8, (b) autopsy 0.01 - 0.70. mg./cm2/hr. Permeability constant of tritiated water through the -3 human epidermis as reported in the literature: 0.1 - 0.5x10 C/w Permeability constant of tritiated water through human epi- -3 dermis as obtained in our experiments: 0.2 to 0.45 x 10 o~ The rate of diffusion of tritiated water is the mean of four diffusion results from each specimen of skin, hairless mice/human epidermis. 79 Sample calculation for measuring rate of diffusion of tritiated water through human, skin: 1 microcurie (uci) = 2.220 x 10 dpm Efficiency of the machine for tritium, E = 49.17$ Fig. of merit = (E)2 = (49.17)2 = 161.00 B 15 Rate of diffusion of water = C A x B A = 1.0 cm2 B = 69375 counts/min./mg. C = 16085 Rate of diffusion = 16085 = 0.23 mg/cm2/hr. 1.0 x 69375 F. Percutaneous Penetration of Steroids The most important goal in percutaneous research is to express all results in terms of comparable measurements. Typical cumulative penetration curves for betamethasone 17- valerate, desonide and triamcinolone acetonide, hydrocorti• sone are shown in Fig. 16 .17 ,18 . 19 . The^penetra- tion curves show an initial rapid penetration of steroids. This could be due to the fact that shunt diffusion might be dominating in the initial transient stage of diffusion. Penetration slows progressively with time, not quite reach• ing steady state penetration within 25 to 50 hours, the duration of our observations. It has been suggested that for drugs having the same intrinsic pharmacological activity, drug transport rates to active sites determine their rela• tive potency. By this hypothesis, one would.expect beta• methasone 17-valerate to have the fastest penetration rate, hydrocortisone the lowest, with desonide and triamcinolone acetonide giving intermediate penetration. In 25 hours, —1 rather substantial amounts .5 to 3»5%—of the steroid penetrated the skin barrier. Tables 8, 9t 10 and graphs 16, 18, 19 show that desonide penetrated the skin,barrier faster than triamcinolone acetonide. The only structural difference between desonide and triamcinolone acetonide is the absence of a fluorine mole• cule in desonide. The strongly electronegative fluorine atom decreases the redox potential of the ketone-hydroxyl pair stabilizing the 11-betahydroxyl group and making triamcinolone acetonide more polar than desonide. In other words, the presence of fluorine at the 9o^ position in triamcinolone acetonide decreases its lipid solubility. Water solubility of triamcinolone acetonide is less than that of bl desonide (Table 4). This contradicts the theoretical as• sumption that triamcinolone acetonide Is more polar than desonide. One possible explanation of its lower water solu• bility is that an interaction between the C-ll hydroxyl group and an electron on the C-l double bond takes place, forming a reasonably stable six-membered ring structure. Another pos• sibility is that an interaction occurs between the C-ll hydroxyl group and the C-20 carboxyl group, yielding a seven-membered ring. However, this is less likely to occur (Brown, personal communication). The penetration curves (Pig. 16, 18, 19) indicate a pro• gressive decrease in the permeability of steroids as they be• come increasingly polar. More polar and less mobile compounds have lower potencies when applied topically. Our results are in agreement with this. Our penetration curves show that topical activity could be Increased by reducing the polarity of the molecules. This effect has been attained most fre• quently by esterlflcation of hydroxyl groups at C-21 and C-17 or by linking the hydroxyl group at C-16 and C-17 with an acetonide bond (Schlagel, 1965). Desonide shares the ability of triamcinolone to pene• trate epidermis because of the presence of the acetonide moiety. Our results show that of the four ant1-Inflammatory agents studied, betamethasone 17-valerate Is the most effective. followed by desonide, triamcinolone acetonide and hydro• cortisone respectively. The main objective of the work was a comparative eval• uation of steroids with respect to their ability to penetrate $h$ skin barrier. No emphasis was given to the use of different vehicles. Sample calculations: To calculate the cumulative diffusion of drug in time interval, t: Say, in the 1st hour, the sample gave an absorbance of 0.02, C = A/a = 0.02 = .00059 mg/ml (a is calculated from st. curve) 3378" The sample drawn was 2 ml. concentration in 2 ml, .00059 x 2 = 0.00118 The total volume of liquid in the lower chamber of the cell was 8 ml. concentration in 8 ml, 0.00059 x 8 = .00472 mg/8 ml. But 2 ml. of the sample was replaced by fresh 2 ml. of saline solution. Therefore the drug left in the lower chamber of the diffusion cell, was 0.00472 - 0.0018 = 0.0035^ mg. The second sample was drawn at a definite interval and read absorbance, Cone. A/a = 0.02 5 = .00073 mg/ml "33T8 Cone, in 2 ml, 0.0073 x 2 = 0.00146 mg. 83 Cone, in 8 ml, 0.0073 x 8 = 0.00584 mg. Drug left in the lower chamber of the diffusion cell =o 0.00584 -0.00146 .00438 mg. Drug diffused in the 2nd hour would be 0.00584 - 0.00354 mg = 0.0023 mg. Cumulative diffusion in the 2nd hour would be: 0.00472 + 0.0023 = 0.00702 mg. or 7.02 ug. G. Vasoconstriction Bioassay Corticosteroids, above their threshold dose, cause skin blanching due to vasoconstriction. Applied at several con• centrations the test gives the typical sigmoid dose-response curve of a standard bioassay. Using statistical techniques which transform the dose-response curve to a straight line, the ED^Q can be calculated. When two anti-inflammatory steroids give parallel dose-response curves, it is evident that the same mechanism of action is operative (Goldstein, 1968). The ratio of their ED^Q'S expresses their relative potency. Extrapolation to ED<^ gives an estimate of the maximum dose beyond which no greater physiological response — can be achieved. Both desonide ( O P~C7) and triamcinolone acetonide (A—A—A ) g&ve practically coincident dose-response -4 curves and the same ED^ (approximately, 2 x 10 %). The max- Table 8 Cumulative penetration of desonide and triamcinolone acetonide through human abdominal skin (epidermis) at room temperature.. The steroids were applied in drug deposit form. Time interval Amount of drug penetrated (mg./cm x 1(H) Hours Desonide Triamcinolone acetonide 1 10.0 ± 0.50 9.0 + 0.63 + 2 15.5 ± 0.50 13.3 0.50 3 21.7 - 0.63 16.0 + 0.50 5 25.0 t 0.60 1928 + 0.50 + 10 32.2-± 0.60 26.5 0.60 15 40.0 t 0.50 38.5 0.60 20 47.2 t 0.48 42.5 + 0.48 25 52.7 - 0.50 47.2 ± 0.48 + 30 63.5 - 0.37 58.0 0.60 Mean (n = 10) - standard deviation Pig. 17. Penetration of desonide and triamcinolone acetonide through human epidermis (age 58 years, 2k hours after death). v v Desonide • Triamcinolone acetonide Table 9 Cumulative penetration of betamethasone 17-valerate. desonide, triamcinolone acetonide, hydrocortisone through human abdominal skin (epidermis) at room temperature. The steroids were applied in 40$ ethanol. 2 3 * Time interval Amount penetrated (mg./cm x 10J) Hours Betamethasone Desonide Triamcinolone Hydrocortisone 17-valerate acetonide + + 1 7.4 0.44 6.9 t 0.50 6.6 0.60 4.8 ± 0.50 + + + 2 1U8 0.50 10.2 ± 0.61 8.0 0.5 0 7.2 0.52 + t + + h 17.5 0.62 11.4 0.60 11.0 0.47 9.6 0.47 1 + t + 5 23.2 0.69 22.0 0.50 19.1 + 0.34 14.1 0.47 + + + 10 40.4 0.5.0 31.6 t 0.63 27.0 0.44 22.3 0.12 + + + 15 48.6 0.50 45.6 t 0.50 40.0 0.50 25.0 0.31 + + + 20 86.6 0.48 53.0 - 0.50 48.0 0.53 28.3 0.62 + + 25 65.O 0.38 60.8 * 0.50 55.2 0.60 30.0 + 0.40 + + + 30 80.0 0.40 74.3 - 0.48 68.2 0.49 38.0 0.39 * + Mean (n = 10) - standard deviation. Table 10 Cumulative penetration of betamethasone 17-valerate, desonide, triamcinolone acetonide and hydrocortisone through human abdominal skin (epidermis) at room temperature. The steroids were applied In hydrophlllc ointment base. Time interval Amount of drug penetrated (mg./cm x 10^) Hours Betamethasone Desonide Triamcinolone Hydrocortisone 17 - valerate acetonide 1 4.7 * 0.50 4.4 ± 0.33 3.8 t 0.40 2.0 * 0.63 2 7.4 1 0.31 6.7 ± 0.39 5.8 t 0.43 4.7 - 0.42 3 10.6 t 0.31 9.0 t 0.66 8.5 ± 0.44 6.2 ± 0.38 + 5 14.6 ± 0.42 0.59 12.8 t 0.50 4 0.43 13.1 - 9.3 10 25.5 1 0.60 23.0 t 0.50 21.0 - 0.50 13.4 ± 0.60 15 28.2 t 0.50 25.2 ± 0.50 24.0 * 0.50 18.0 t O.56 20 33.0 ± 0.73 31.0 - 0.56 25.5 - 0.60 22.0 ± 0.49 25 38.0 i O.56 35.0 ± 0.48 32.4 ± 0.43 25.4 t 0.50 30 50.0 ± 0.58 48.2 ± 0.61; 43.1 i 0.43 31.8 ± 0.50 Mean (n = 10) - standard deviation. 91 imum effectiveness of the drugwa:s reached at doses of 0.01$ when approximately 90$ of the sites of application vasocon- stricted. We conclude that 9 alpha-fluoro group has no effect on the potency of triamcinolone acetonide. The removal of the group leaves the physiological activity of the molecule un• impaired. Pronounced vehicle effect is noticed only at the lowest concentrations. This may be due to lack of uniform dispersion upon dilution. At higher concentrations, 0.001$ and up, vehicle and dilution effects disappear. Ointment formulations show nearly the same effectiveness as the solu• tions. It seems clear, however, that at a concentration of approximately 0.01$, desonide and triamcinolone acetonide reach the maximum in the dose-response curve. The use of higher concentrations will not cause appreciably greater numbers of persons to respond. Thus, concentrations used in practice, 0.05$ for desonide and 0.1$ for triamcinolone acetonide, represent a 50 to one hundredfold excess over the maximum response obtainable. H. Clinical Studies of Effectiveness of Desonide The clinical studies of desonide were done by one of the Table '11 Vasoconstriction produced by Triamcinolone acetonide and Desonide Concentration Number of patients with positive response Solution Cream 0.0001$ 17A3 39.5$ 6/43 14.0$ 0.001$ 27/43 62.8$ 28/43 65.0$ Desonide 0.01% 44/47 93.6$ 40/46 87.0$ 0.1% 40/46 87.0$ 35/46 76.0$ 0.0001$ 16/44 36.0$ 11/44 25.0$ 0.001$ 27/44 62.8$ 28/43 65.1$ Triamcinolone acetonide 0.01$ 44/47 93.6$ 42/47 89.4$ 0.1$ 42/48 87.5$ 38/48 79.2$ 93 VASOCONSTRICTION BIOASSAY O O m -5 -3 -2 LOG CONC (%) -1 n—-DESONIDE (SOLUTION) — " (CREAM) A —;TRIAMCINOLONE ACETONIDE (SOLUTION) -4 ED =2X10 % 50 . Pig. 20. Vasoconstriction bioassay, log concentration vs $ response 94 co-investIgators, Dr. W.D. Stewart, Head, Division of Derma• tology, Faculty of Medicine, U.B.C., Vancouver 8, B.C. The clinical effectiveness of desonide in three skin disorders, contact dermatitis, atopic dermatitis and psoriasis,, was compared to that of another potent fluorinated topical corticosteroid, betamethasone 17-valerate, using Salzburger's technique (1946). This technique involves the comparison of two drugs by use of bilaterally symmetrical lesions, randomized and double blind application and evaluation. Commercially supplied creams of desonide 0.05$ and betamethasone 17-valerate 0.1$ were used - the two concentrations most commonly prescribed in practice. Each drug was applied twice daily without occlusion. Patients were examined weekly for four weeks for anti-inflammatory and anti-pruritlc activities of each drug. Table 12, represents the results obtained with patients having contact dermatitis and Table 13,' shows the results °n of atopic dermatitis. Both the tables show percentage?of pa• tients with a better response to desonide^ and better response to betamethasone 17-valerate anH equal response to both and those in which neither was effective. Table 14, shows the comparison of desonide and beta• methasone 17-valerate in patients with psoriasis. The above Table 12 Effectiveness of desonide 0.05% vs betamethasone 17-valerate 0.1? Contact Dermatitis Total # Desonide Betamethasone Equal Neither Week Patients Superior Superior Effect Effective First 48 32$ 33$ 27$ 8 Second 44 25 36 37 2 Third 37 32 32 35 0 Fourth 29 28 38 34 0 Table 13 Effectiveness of desonide 0.05$ vs Betamethasone 17-valerate 0.1$ Atopic Dermatitis Total # Desonide Betamethasone Equal Neither Week Patients Superior Superior Effect Effective First 34 12$ 26$ 59$ 3$ Second 31 16 19 65 0 Third 27 22 19 59 0 Fourth 23 13 17 70 0 Table 14 Effectiveness of desonide 0.05$ vs betamethasone 17-valerate 0.1$ Psoriasis Total # Desonide Betamethasone Equal Neither Week Patients Superior Superior Effect Effective First 35 14$ 26^ 43$ 17$ Second 31 10 35 39 16 Third 30 10 33 34 23 Fourth 30 10 37 33 20 98 Tables 12 , 13 , Ik, , show that desonide is in the same range of effectiveness as betamethasone, although perhaps very slightly less effective in selected subjects. 99 VI SUMMARY AND CONCLUSION The vasoconstriction potency, penetration across human epidermis and the clinical effectiveness of desonide, a new non-fluorinated corticosteroid have been compared to those of two fluorinated steroids, triamcinolone acetonide and betamethasone 17-valerate. A. An attempt to establish spectrophotofluorometric method for analysis of desonide was unsuccessful. This was due to the presence ofa'l6«C hydroxy1 group in desonide, which provided unstable fluorescence. Therefore,all subsequent steroids were analyzed by a spectrophotometric method. B. Solubility of four steroidssnamely, hydrocortisone, triamcinolone acetonide, and betamethasone 17-valerate, was determined in distilled water, normal saline and k0% ethanol. The relative solubility of steroids in water and in k0% ethan• ol were of the following order: Hydrocortisone > desonide triamcinolone acetonide betamethasone 17-valerate. Octanol/water partition coefficients of the above sa-M steroids were determined at 25°± 0.1. The values obtained experimentally were compared to ether/water partition coef• ficients reported in the literature. A linear relationship was obtained when octanol/water and ether/water partition xuu coefficients were plotted vs cumulative penetration. Par• tition coefficient of the steroids followed the order in• dicated by their relative solubility. C. The penetration of hydrocortisone, triamcinolone aceto• nide, desonide, and betamethasone 17-valerate were studied using human epidermal sheets in Poulsen skin diffusion cell3- Epidermal sheets were removed by heat separation method. The intactness of epidermal sheets was checked by comparing the permeability constant of tritiated water with the re• ported literature values. The order of penetration of steroids were: Betamethasone 17-valerate > desonide > triamcinolone aceto- nice > hydrocortisone. There seems to be a linear relationship between pene• tration and partition coefficient of steroids. D. Vasoconstriction activity of desonide was compared to triamcinolone acetonide. Both desonide and triamcinolone acetonide gave coincident dose-response curves and the same o*' 6 ED5 of 2 x 10" g/ml. E. Clinical effectiveness of desonide was compared to betamethasone 17-valerate In diseases like contact derma• titis, atopic dermatitis and psoriasis. Desonide was found in the same range of effectiveness 101 as betamethasone 17-valerate although perhaps very slightly less effective in selected subjects. In conclusion, desonide has been demonstrated to have an effective degree of anti-inflammatory activity, skin pene• tration and substantial vasoconstrictive action. Desonide belongs among the more effective members of the topical anti• inflammatory steroid family. 102 VII BIBLIOGRAPHY Ainsworth, H.J.s Methods for measuring percutaneous absorption, J. Soc. Cosmet. Chem. 11:69. I960. Allenby, A.C, et all: Mechanism of action of accelerant on skin penetration, Brit. J. Derm., 82, suppl. 4:4?, 1969. Brown, T.H.: Steroids, adrenocortical, in 'Current Dermato- logic Management* ed. S. Maddin, St. Louis, The CV. Mosby Company, 1970, p. 302. Burger, A.: in "Medicinal chemistry", 3rd ed. Part 11. Wiley-intersclence, 1970, p. 892. Berliner, D.L., and Ruhmann, A.G.x The influence of di• methyl sulfoxide on fibroblastic proliferation. Ann. N.Y. Acad, sci., 141:159, 1967. Berliner, D.L., and Nabros, D.J. Jr.: Effect of cortico• steroids on fibroblast structures, in "Topics in pharmaceutical sciences," vol. 1, edited by Perlman, A., Interscience publications. New York, 1968. Bush, I.E.: Chemical and Biological factors in the activity of adrenocortical steroids, Pharmacol. Rev., 14:317. 1962. Bluefarb, A.M.: Newer topical therapy in dermatology, Int. J. Derm. 2s200* 1970. Barr, M.: Percutaneous absorption, J. Pharm. Sci., 51:395, 1962. Blank, I.H., and Scheuplein, R.J.: Transport into and within the skin, Brit. J. Derm. 81, Suppl. 4:4, 1969. Blank, I.H.: Cutaneous barrier, J. Invest. Derm. 4^:249, 1965. Blank, I.H., and Scheuplein, R.J., in "Progress in biological sciences inOrelation to dermatology," 2nd. ed. edited by A. Rook and R.H. Champion, University Press Cambridge, 1965. P. 245. Blank, I.H.: Percutaneous absorption - statement of problem and critical review of past methods, J. Soc. Cosmet. Chem., 11:59. I960. 103 Blank, I.H.: Factors which Influence the water content of the stratum corneum, J. Invest. Derm., 18:433, 1952. Barret, C.W.: Skin penetration, J. Soc. Cosmet. Chem., 20:487, 1969. Baumberger, J.P., et al* Methods for separation of epi• dermis from dermis and some physiologic and chemical properties of isolated epidermis, J. Nat. Cancer Inst., 2:413, 1942. Bernstein, I.A.: Chemical differentiation in the epidermis, J. Soc. Cosmet. Chem., 21:583, 1970. Calnan, D.D.: The significance of assay methods for the dermatologist, Brit. J. Derm. 82. Supp. 6:43, 1970. Cline, M.C, and Melman, K.L.: Plasma kinins and Cortisol: A possible explanation of the ant1-inflammatory action of Cortisol, Science, 163*1135. 1966. Coldman, M.F., Poulsen, B.J. and Higuchi, T.: Enhancement of percutaneous absorption by the use of volatilenon• volatile systems as vehicles, J. Pharm. Sci. 58:1098. 1969. Comaish, S. and Evabottom: The skin and friction: Deviation from Amontons law, and the effect of hydration and lubrication, Brit. J. Derm. 84s376, 1971. Crane, M. and Harris, J.: "Gas chromatography of steroids in biological fluids," edited by Lipsett, M., Plenum Press, New York, 1965, p. 73. Cadwallder, D.E. and Ismat Ullah: Dissolution of soluble powders under the skin conditions, 111: Transport of drug solution across screens and membrane barriers, J. Pharm. Sci. 60:1496, 1971. Dougherty, T.F., and Schneebeli, G.L.: The use of steroids as ant1-inflammatory agents, Ann. N.Y. Acad. Sci., 61:328. 1955. 104 Downes, A.M., Thomas, M.S. and Mataltsy, G.A.: Studies of epidermal water barrier, I. An improved in-vltro method for determination of the diffusion of water through skin, J. Invest. Derm., 42:230, I967. Duemllng, W.W.: New synthetic chemicals of use in finer and more efficient topical dermatologic therapy, Arch. Derm, and Syphilial, 43_:264, 1941. Dempski, R.E., Portnoff, J.B. and Wase, A.W.: In vitro re• lease and in vivo penetration studies of a topical steroid from non-aqueous vehicles, J. Pharm. Sci., J58:579, 1970. Epstein, W.L., Epstein, J.H.: Atrophic strial in patients with injured intertrigo, Arch. Derm., 8£:450, 1963. Peldmann, R.J. and Maibach, M.I.: Absorption of some organic compounds through the skin in man, J. Invest. Derm. ^4:399. 1970. Pritsch, W.F. and Stoughton, R.B.: The effect of temperature and humidity on the penetration of c1^ acetylsallcylic acid in excised human skin, J. Invest. Derm., 82:24, i960. Flfbaum, S.G. and Laden, K.: The effect of dimethylsulfoxide on percutaneous absorption: A mechanistic study, Part 1, J. Soc. Cosmet. Chem., 19_:119, 1968. Plynn, G.: Structural approach to partitioning: Estimation of steroid partition coefficients based upon molecular constitution, J. Pharm. Sci., 60:345, 1971. Frying, M. and Dougherty, J.F.: Molecular mechanisms in inflammation and stress, I. Am. Scientist, 4^:457. 1955. Fried, J. and Sabo, E.: 9<=c fluoro derivative of cortisone and hydrocortisone, J. Am. Chem. Soc. 76: 1455, 1953. Griesemer, R.D.: Biological factors affecting percutaneous absorption, J. Soc. Cosmet. Chemist, 11:80, i960. Griesemer, R.D.: Biological factors affecting percutaneous absorption, J. Soc. Cosmet. Chemist, 11:81, i960. 1U5 Grlce, K.: Tinea of hand and forearm, Betamethasone valerate atrophy, Proc. R. Soc. Med., j>2*254, 1966. Greaves, M.W. and Sondergaard, J.1 Pharmacological studies in cutaneous inflammation in man using an In vitro perfusion method, Brit. J. Derm. 82 Supp. 6:1970. Goldstein, A., Aronow, L. and Kalman, S.M.: "Principles of Drug Action," New York, Harper & Row, 1968, p. 56. Grasso, P.: Some aspects of the role of skin appendages In per• cutaneous absorption, J. Soc. Cosmet. Chem., 2£*523, 1971* Gemmel, D.H.O. and Morrison, J.C.s The percutaneous absorption of sulphanllamide, J. Pharm. Pharmacol., 10*167, 1958. Goldzieher, J.W., et alt Local action of steroids on senile human skin, Arch. Dermatol, and Syphilol., 66*304, 1952. Gilbert, D., Mier, P.D. and Jones, T.E.: An improved tech• nique for the isolation of epidermis from human skin, J. Invest. Derm. 40*165, 1963. Higuchi, T.J Physical chemical analysis of percutaneous absorption process from creams and ointments, J. Soc. Cosmet. Chem. 11*85, I960. Haleblan, J., Runkel, R., Mueller, N., Christopherson, J. and K. NG.* Steroid release from silicone elastomer contain• ing excess drug in suspension, J. Pharm. Sci. 60*541, 1971. Hlynka, J.N., Anderson, A.J. and Rledel, B.E.: Investigation of intracutaneous drug absorption II, A comparison of intracutaneous and systemic absorption as function of rest time and concentration, Can. J, Pharm. Sci. 4*92, 1969. Ive, P.A. and Mark, R.* Tinea Incognito, Brit. Med. J. 2*149, 1968. Idson, B.J.s Topical Toxicity Testing, J. Pharm. Sci. 57:1, 1968. < Idson, B.* Skin transport, Drug and Cosmetic, Ind., 108*36. 1971. Kier, L.B.s The conformation of 20-oxo-pregnane hormones from molecular orbital calculations and a consideration of the Cortisol receptor. J. Med. Chem., 11*915, 1968. xuo Keipert, J.A.: The absorption of topical corticosteroids, with particular reference to percutaneous absorption in infancy and childhood, Med. J. Aust. 1:1021, 1971. Katz, J., Shaikh, Z.I.: Percutaneous corticosteroid absorp• tion correlated to partition coefficient, J. Pharm. Sci. j&:591, 1965. Kosch, E.A.: Studies on ointments, VI. Ointments containing Chrysarobin, Arch. Dermatol, and Syphilis, 42:1, 1944. Levinson, B.B. et al: Cellular site of glucocortlcold-receptor complex formation, Science, 175:189, 1972. Lueck, L.M., Wurster, D.E. and Higuchi, T.: Investigation and development of protective ointments, I. J. Am. Pharm. Assoc. Sci. Ed., 46:694, 1957. Mantica, L., Ciceri, R., et al: Prednacinolone, a new anti• inflammatory steroid, Note 1: chemical and physical properties, Arzneim-Porsch 20:109, 1970. Mascitelll-Corlandoll, E. and Praia, A.: Prednacinolone, a new anti-inflammatory steroid, Note 2: biochemical and pharmacological properties, Arzneim-Forsch. 20:111, 1970. Menkln, V.: The biology of inflammation. Science, 123:527. 1956. McKenzie, A.W. and Stoughton, R.B.: Method for comparing the percutaneous absorption of steroids, Arch. Derm., 86:608, 1962. McKenzie, A.W.: Percutaneous absorption of steroids, J. Invest. Derm., 86:611, 1962. McKenzie, A.W. and Altklnson, R.M.: Topical activities of betamethasone esters in man, 82:74, 1964. Munro, D.D.: The relationship between percutaneous absorp• tion and stratum corneum retention, Brit. J. Derm. 81 Supp. 4:92, 1969. Medawar, P.B.: Sheets of pure epidermal epithelium from human skin, Nature, 148:783, 1941. Mars, M.J. and Voorhees, J.T.: A method for bioassay of an epidermal chalone like inhibitors, J. Invest. Dermatol., ^6:174, 1971. 10? McKenzie, A.W.: Comparison of steroids by vasoconstriction, Brit. J. Derm., £8:182, 1966. Mattingly, D.A.: Simple fluorimetrlc method for the estima• tion of free 11 hydroxycorticolds in human plasma, J. Clin. Pathol., 1^:374, 1962. McKenzie, H.M.: A spectrophotofluorometric method for the quantitative determination of hydrocortisone absorption in skin from dermatologlcal medications, M.5s.P. thesis, submitted to the University of British Columbia, Vancouver, B.C., 1967. p. 32. Martin, M.M. and Martin, A.L.A.: Simultaneous fluorometric determination of Cortisol and cortlcosterone in human plasma, J. Clin. Endoor. 28:137, 1968. Mali, J.W.H.: The transport of water through the human epi• dermis, J. Invest. Derm., 22:451, 1956. Malkinson, P.D.: The epidermis, Ed. W. Montagna and W.C. Lobitz, Jr., Academic Press, New York, Cap. 21 (1964). Marzulli, P.N.: Barrier to skin penetration, J. Invest. Derm. 22:389, 1962. Noujaim, A.A. and Jeffery, D.A.: Analysis of corticosteroids in biological samples, 1. Spectrophotometric, fluoro• metric, and gas chromatographic methods, Can. J. Pharm. j Sci., >:2, 1970. Noujaim, A.A. and Jeffery, D.A.: Analysis of corticosteroids in biological samples, 2, Radioisotopic methods, Can. J. Pharm. Sci., 6:1, 1971. Ostrenga, J., Stelnmetz, C. and Poulsen, B.: Significance of vehicle composition. I: relationship between topical vehicle composition, skin penetrability and clinical efficacy, J. Pharm. Sci. 60:1175, 1971. Ostrenga, J., Stelnmetz, C, Poulsen, B. and Yett, S.: Significance of vehicle composition II: prediction of optimal vehicle composition, J. Pharm. Sci. 60:1180, 1971. Ostrenga, J., Haleblan, J., Poulsen, B. and Ferrell.B. et alt Vehicle design for a new topical steroid, fluoclnonide, J. Invest. Derm. .56:392, 1971. 108 Phillips, B.M., Sanen, F.J., Leeling, J.L., et al.: The physical animal and human pharmacological and toxico- logical properties of desonide, a new, topically active, anti-inflammatory steroid, Toxicol. Appl. Pharmacol. (In press) 1971. Polano, M.K., Suromond, D., Lely, M.A. and Warnaar, P.: A clinical trial with hydrocortisone butyrate cream in psoriasis, Brit. J. Derm. 82:93, 1970. Poulsen, B.J.: The use of models in estimating vehicle ef• fects on the activity of topical corticosteroid formula• tion, Brit. J. Derm. 82 Supp. 6:49, 1970. Poulsen, B.J., Young, E., Coquilla, V. and Katz, M.: Effect of topical vehicle composition on the _in vitro release of fluocinolone acetonide and its acetate ester, J. Pharm. Sci. j5Z*928, 1968. Place, V.A., Viner, J. and Burdlck, K.H.: Precise evaluation of topically applied corticosteroid potency, Arch. Derm. 101:531, 1970. Reid, J. and Brookes, D.B.: Topical corticosteroids - an experimental evaluation of the vasoconstrictor test as an index of anti-inflammatory activity, Brit. J. Derm. 80:328, 1968. Robinson, S.L. and Angell, M.: Inflammation and repair, "Basic Pathology", W.B. Saunders, Co., Toronto, 1971, pp. 28-63. Rothman, S.: The mechanism of percutaneous penetration and absorption, J. Soc. Cosmet. Chem., 2:193, 1956. Roseman, T.J.: Release of steroids from silicone polymer, J. Pharm. Sci., 6l:46, 1972. Rein, H.: Experimental electroendosomotic studies on living human skin, Z. Biol. 81:125, 1924. Sneddon, I.: Adverse effect of topical fluorinated cortico• steroids in rosacea, Brit. Med. J., 1:671, 1969* Schayer, R.W.: Histamine and autonomous responses of the microcirculation, relationship to glucocorticoids action, Ann. N.Y. Acad. Sci., 116:891, 1964. 109 Stuart, D.M.: Dermatology and dermatologicals, Pharm. Index, January 1969, P« 4. Scheuplein, R.J.: Mechanism of percutaneous absorption, I. Routes of penetration and the influence of solubility, J. Invest. Derm. 4^:334, 1965. Selby, C.C.: The fine structure of human epidermis as revealed :594, b1956y th.e electron microscope, J. Soc. Cosmet. Chem., 2 Shelmlre, A.: Factors determining the skin-drug-vehicle re• lationship, Arch. Derm., 82:24, i960. Sarkany, I. and Hadgraft, J.W.: The influence of formulation on corticosteroid activity. Ibid.. 81 suppl.. 4:98, 1969. 51:702, Scheuplein1971. , R.J.: Permeability of skin, Physiol. Rev., Stoughton, R.B., Clendenning, W.E. and Kruse, D.: Percutaneous absorptio£:337, n of nicotinic and derivatives, J. Invest. Derm. 1 I960. Schutz, E.: Der Enfluss von Polyathleneglykol 400 anf die Percutane Resorption von Wirkstoffon, Exp. Pathol. Pharmakol. 2J$2:237, 1951. Stoughton, R.B. : Some In vivo and in vitro methods for measur• ing percutaneous absorption,in "Progress in biological sciences in relation to dermatology," 2nd ed., edited by A. Rook and R.H. Champion, University Press, Cambridge, 1964, p. 263. Sweat, M.: Sulfuric acid-induced fluorescence of cortico• steroids, Anal. Chem., 26:773, 1954. Shimogami15:951, A,. 1967 and ,Tsuchikura, H., et al,: Horumon to RinshoI968., through Chemical Abstract, 62:418, Solomon, L.M. et al«: Studies in the mechanism of steroid vasoconstriction, J. Invest. Derm., 4^:129, 1965. Stewart, W.D. and Runikis, J.O.: Further improvements in the dissection of metabolicall 4y activ:159,e unifor1967. m thin skin slices, J. Invest. Derm., _2 110 Schlagel, CA.: Comparative efficiency of topical anti-inflam• matory corticosteroids, J. Pharm. Sci. j>4:335. 1965. Sulzberger, M.B. et al: Methods for the rapid evaluation of the beneficial and harmful effects of agents applied to the human skin, J. Invest. Derm. 2l227» 1946. Tregear, R.T.: The structure which limits the permeability of the skin, J. Soc. Cosmet. Chem., 12:145, 1962. Tregear, R.T.: "Physical functions of the skin," Academic Press, London and New York, 1966, p. 30. Tregear, R.T.: The permeability of skin to albumin, dextrans and polyvinyl pyrolldone, J. Invest. Derm., 46:24, 1966. Treherne, J.E.: The permeability of skin to some non- electrolytes, J. Physiol. 133:171. 1956. Vickers, C.P.M.: Existence of reservoir In the stratum corneum, Arch. Derm. 88:20, 1963. Van Scot, E.J.: Mechanical separation of the epidermis from corneum, J. Invest. Derm., 18:377t 1952. Waddington, E.: Advances In the treatment of diseases of the skin, Practitioner, 201:583, 1968. Weiner, M. and Pillero, S.: Nonsteroid anti-inflammatory agents, Ann. Rev. Pharmacol. 10:171, 1970. Winkelmann, R.K.: Molecular inflammation of the skin, J. Invest. Derm. Ja£:197, 1971. Wagner, J.G.: Biopharmaceutics: Absorption aspects, J. Pharm. Sci., ^0:359, 1961. Wolf, J.: Die innere structure der zellen des stratum des- quammans des menschlichen epidermis, Z. Mikr. Anat. Forsch., 46:170, 1939. Wood, D.C.F. and Bettley, F.R.: The effect of various deter• gents on human epidermis, Brit. J. Derm., 84:321, 1971. Wolff, M.E. and Winston, H.O.s The steroid-receptor complex. Some considerations based on Sp - hybridized systems, J. Med. Chem., 2s577. 1964. APPENDIX Materials Pharmaceuticals The pharmaceuticals were used as received from the manufacturer without further purification. Hydrocortisone. U.S.P. XVIII, 11,1?,21-trihydroxy - 4 - pregnene - 3.20 - dione. Lot #17.17k, Merck Sharp and Dohme of Canada Ltd., Montreal, Canada. Mol. Wt. 362.5 Melting Point ~215-220°C (with decomposition) Solubility in water 280 ug/ml. Absorption max. 249 nm Triamcinolone Acetonide, 9- fluoro - 16 oC hydroxypred- nisolone 16,17 - acetonide. Cod #20-282, Lot #L-902. E.R. Squibb and Sons Ltd., Montreal, Canada. •CHoOH C=0 112 Mol. Wt. 434.4 Melting Point 274-278°c (with decomposition) Solubility in water 10-12 ug/ml. Absorption max. —242 nm Desonide (Tridesllon ) (Desfluorotriamcinolone acetonide), l6^?C?-hydroxyprednisolonet 16, 17 - acetonide. Code #CS-l-37. Dome Laboratories. Division of Miles Laboratory Inc., West Haven, Conn. 06516, U.S.A. CHgOH C = 0 0 Mol. Wt. >--394.5 Melting Point 274-275°C (with decomposition) Solubility in water 75.8 ug/ml. Absorption max.———242 nm Betamethasone 17-Valerate. N.F. XVII, 9 oL~fluoro - 16 o{_- methylprednlsolone - 17 - valerate. Code #12301, Lot #RL-7/ll6. Schering Corporation Ltd., Pointe Claire, Quebec. P 113 Mol. Wt. 476.6 Melting Point decomposition Solubility in water 5»5 ug/ml. Absorption max. 242 nm Tritiated Water (5 Ci/ml.) TRS - 1, catalog 70/71. Amersham/Searle, 2636 Clearbrook Drive, Arlington, Illinois. Hydrophilic Ointment, U.S.P. XVIII was prepared in this laboratory. Precautions were taken to prevent water loss during the preparation of the ointment, because loss of even small amounts of water affects the consistency of the oint• ment. The composition of the ointment is as follows: Methyl paraben 0.25 gnu Propyl paraben 0.15 gm. Sodium Lauryl Sulfate 10.0 gm. Propylene glycol 120.0 gm. Stearyl alcohol 250.0 gm. White soft paraffin 250.0 gm. Purified water 370.0 gm. To make about 1000 gm. The finished ointment was passed through the Pascall oint• ment mill to assure batch uniformity in texture and con• sistency. Steroids In Hydrophilic Ointment 1$, hydrocortisone, diesonide, triamcinolone acetonide and betamethasone 17-valerate were made in Hydrophilic oint- 114 merit base. These were used for penetration studies across the human epidermis. 0.1$ stock of the desonide and triamcinolone acetonide were also made in the same way described above for vasocon• striction studies. Steroids in 40$ Eithanol From the solubility studies of steroids in 40$ ethanol, hydrocortisone, triamcinolone acetonide, desonide and beta• methasone 17-valerate, each were weighed 14.0 mg, added 4.0 ml. of 100$ ethanol and thoroughly agitated, added distilled water slowly and made up the volume to 10 ml. The solutions were filtered through (millipore filters. Solvents and Reagents Solvents and reagents were selected for minimum im• purities. They were found to be generally of acceptable quality. Routine purification was necessary only for the ethyl alcohol. Ethyl alcohol, 100$, was purified by redistillation over 2,4 - dinitrophenylhydrazine (Sweat, 195*0 to remove alde• hydes and ketones. The ethyl alcohol containing 5 grams of 2, 4 - dinitrophenylhydrazine and 10 ml of hydrochloric acid per 1000 ml was refluxed for four to eight hours in a vacuum jacketed all-glass unlubricated packed distillation column with CORAD head. It was then distilled, the first and last 20$ being discarded, then redistilled, again discarding the first and last 20$. The alcohol was stored in 100 ml well filled aluminum foil capped bottles at 2°C to minimize re• formation of aldehydes. Sulfuric Acid (Allied Chemical of Canada, Ltd., Montreal, Quebec) was found-to vary in fluorescence impurities from lot to lot. Screening was done to eliminate unsatisfactory lots, by checking their fluorescence. Fluorescence reagent, consisting of 85 parts of sulfuric acid and 15 parts of ethyl alcohol by weight was prepared as follows: 30 grams of cold distilled ethyl alcohol was 116 weighed into a tared glass stoppered flask packed in ice. About 90 ml. of cold sulfuric acid was added slowly with constant agitation so that the solution remained cool. The solution was made up to 200 grams with cold acid. This reagent was prepared freshly just before use. Its fluorescence characteristics were reproducible from day to day so long as the same batch of acid was used. Preparation by weight was adopted because it was found that the method used by Mattingly (1962) calling for slow addition of 7 volumes of concentrated acid to 3 volumes of ethyl alcohol while cooling under the cold water tap, gave less reproduc• ible results. This was attributed to the inherent lack of accuracy in the volumetric measurement of a viscous liquid such as sulfuric acid, and to insufficient cooling. This reagent for practical purposes Is 70% acid by volume. Methylene Chloride (Matheson, Colemaniand Bell, Norwood, Ohio) was used as received from the manufacturer. This solvent is reported by the manufacturer to have a flu• orescence of 0.3 parts per billion as quinine base. 2.5 - diphenyloxazole (PPO) 1,4 -bis Q2-(5-phenyloxazole)^ benzene (P0P0P) Naphthalene, recrystallized 117 Dioxane, analytical grade Gctanol 1 (primary) Hydroxylamine hydrochloride Formula of Scintillation fluid (Downes, 1967) PPO 4.0 gm. POPOP 25.0 mg. Naphthalene 50«0 gm» Dioxane to make 1000.0 ml.