Preclinical evaluation of a new topical corticosteroid methylprednisolone aceponate

Objective : Preclinical characterization of methylprednisolone aceponate. Results : The local antiinflammatory potency of methylprednisolone aceponate was equal to the very strong glucocorticoid clobetasol 17-propionate but higher than the potency of hydrocortisone 17-butyrate after topical application in 2 animal models of inflammation. Methylprednisolone aceponate is activated enzymatically in the skin. This activation proceeds faster in inflamed tissue. In contrast to clobetasol 17-propionate, methylprednisolone aceponate was devoid of systemic effects after topical application for 3 days. Finally, whereas clobetasol 17-propionate induced marked skin atrophy, methylprednisolone aceponate induced only slight atrophogenic changes after long-term application (up to 43 days) on rat skin, comparable to the effects of hydrocortisone 17-butyrate. Conclusions : Methylprednisolone aceponate combines high local antiinflammatory potency with very low systemic side effects and only minor local atrophogenic activity. The reason for the dissociation between local antiinflammatory and atrophogenic effects is not known so far. It may be speculated that one of the reasons for the very strong local antiinflammatory activity may reside in the faster enzymatic activation in inflamed tissue. Methylprednisolone aceponate represents a new corticosteroid with which it is possible to improve the dissociation between desired antiinflammatory activity and undesired side effects of topical glucocorticoids.     

PENETRATION OF TOPICAL CORTICOSTEROIDS THROUGH HUMAN EPIDERMIS

The penetration of hydrocortisone, hydrocortisone 17-butyrate 21-propionate, betamethasone, betamethasone 17-valerate, and clobetasol 17-propionate through separated human epidermis was compared. The amount of each topical corticosteroid which was released on the skin and penetrated to saline was greater in ethanol solution than propylene glycol solution. The addition of water to the ethanol increased the release of some corticosteroids. The lipid solubility of each corticosteroid correlated well with skin penetration; however, the polarity of each corticosteroid was inversely proportional to skin penetration. The results of the blanching phenomenon caused by the application of these corticosteroids and of their skin penetration suggested to us that the local pharmacological activity of each topical corticosteroid, rather than their skin penetration, related well to their clinical effectiveness.     

Stability of retinoids in culture. All-trans-retinoic acid, 13-cis-retinoic acid and etretinate in the culture of human keratinocytes, B16 mouse melanoma cells and HeLa cells

The stability of all-trans-retinoic acid, 13-cis-retinoic acid and Ro10-9359 (etretinate) in culture was analyzed by measuring the amount of each retinoid in culture medium with HPLC. In cultures of human keratinocytes and B16 mouse melanoma cells (K440B), all-trans-retinoic acid was fairly stable, but it disappeared rapidly in cultures of HeLa cells. In general, 13-cis-retinoic acid was more unstable. When all-trans- or 13-cis-retinoic acid was used, the 13-cis or all-trans forms also appeared in the culture medium as a result of cis-trans isomerization. Human epidermal keratinocytes were able to de-esterify Ro10-9359, and Ro10-1670 was also detected in the culture medium. K440B cells had a strong capacity for de-esterifying Ro10-9359. When 10 nmole/ml Ro10-9359 was added to the culture, about 1 nmole/ml of Ro10-1670 was maintained from 24 to 96 h later. In the HeLa cells culture, no Ro10-1670 was detected after the addition of 1 nmole/ml Ro10-9359. These results indicate that, in the tissue culture system, the pharmacokinetics of each retinoid depended upon its concentration, the cell type and the cellular density.     

Study of the effects of hydrocortisone and hydrocortisone 17-butyrate ointments on plasma ACTH levels and Synacthen responses in children with eczema

This study compares the effects on the hypothalamo-pituitary adrenal (HPA) axis of two dosage schedules of hydrocortisone 17-butyrate and hydrocortisone ointments in 20 children suffering from eczema. Children with moderately extensive eczema received either 30 g of 0.1% hydrocortisone 17-butyrate or 30 g of 1% hydrocortisone ointment weekly for 4 weeks without occlusion. Children with extensive eczema received either 60 g of hydrocortisone 17-butyrate or 60 g of hydrocortisone weekly for 4 weeks. All four groups showed some clinical improvement. Although many of the children appeared to have some impairment of adrenal function prior to entering the trial, no further significant depression of the HPA axis as reflected by the plasma ACTH levels and a 30-min Synacthen test was observed during the trial. On the basis of 4 weeks' treatment, hydrocortisone 17-butyrate did not have a significantly different effect on the HPA axis in children from that of hydrocortisone.     

Screening for corticosteroid contact sensitivity

3 corticosteroids have so far been tried as markers for corticosteroid contact sensitivity: hydrocortisone, tixocortol pivalate and hydrocortisone-17-butyrate. The present study compared these steroids for screening in addition to a standard patch test series. Of 727 patients, 28 (3.9%) reacted to tixocortol pivalate and 10 (1.4%) to hydrocortisone-17-butyrate; hydrocortisone gave an allergic reaction in 2 of 521 (0.4%) patients. Serial dilutions suggested that tixocortol pivalate, not marketed in Finland, caused allergic reactions which could possibly be cross-reactions to hydrocortisone. In contrast to previously published data, frequent cross-reactions occurred with hydrocortisone-17-butyrate and tixocortol pivalate. All allergic reactions to other corticosteroids found by testing with tixocortol pivalate concurred with reactions to hydrocortisone-17-butyrate. The study suggests that the most effective choice for routine testing for corticosteroid contact sensitivity would be both tixocortol pivalate and hydrocortisone-17-butyrate.     

The hairless mouse as a model for study of local and systemic atrophogenic effects following topical application of corticosteroids.

The hairless mouse has been used as a model to distinguish between local and systemic atrophogenic effects of topical steroids. Hydrocortisone-17-butyrate, betamethasone-17-valerate, budesonide and clobetasol-17-propionate were applied topically daily for 21 days. Skinfold thickness and dermal DNA synthesis of treated and untreated skin were evaluated as parameters of local and systemic atrophogenicity. Further, body weight gain and thymus weight were assessed as markers of systemic activity. With respect to local effects, skin thickness and dermal DNA synthesis both proved to be good parameters. Of the systemic parameters, thymic involution and body weight gain paralleled quite well the skin thinning on the untreated side. The results confirmed the potency differences of the steroids. Furthermore, they emphasize the usefulness of the hairless mouse to assess the relative safety with respect to local and systemic side effects of chronically applied topical corticosteroids.     

Randomized comparison of the type 4 phosphodiesterase inhibitor cipamfylline cream,cream vehicle and hydrocortisone 17-butyrate cream for the treatment of atopic dermatitis

BACKGROUND: Therapeutic options to treat atopic dermatitis are limited. Leukocytes from atopic patients have an abnormally high activity of cyclic adenosine monophosphate (AMP)-phosphodiesterase (PDE), which can be normalized in vitro by PDE inhibitors. Cipamfylline is a new potent and selective inhibitor of PDE-4. OBJECTIVES: To compare the efficacy and safety of up to 14 days' topical treatment with cipamfylline (0.15%) cream with vehicle and with hydrocortisone 17-butyrate (0.1%) cream. PATIENTS AND METHODS: International, multicentre, prospective, randomized double-blind, left-right studies of cipamfylline vs. vehicle and cipamfylline vs. hydrocortisone 17-butyrate in adult patients with stable symmetrical atopic dermatitis on the arms. RESULTS: Both cipamfylline and hydrocortisone 17-butyrate reduced the Total Severity Score significantly (P < 0.001). The reduction with cipamfylline was significantly greater than that with vehicle (difference vehicle-cipamfylline 1.67 95% confidence interval (CI) 1.06, 2.28; P < 0.001) and was significantly less than with hydrocortisone 17-butyrate (difference hydrocortisone-cipamfylline -2.10 95% CI -2.93, -1.27; P < 0.001). Investigator and patient assessments of the overall treatment response showed a similar picture. CONCLUSIONS: Cipamfylline cream is significantly more effective than vehicle, but significantly less effective than hydrocortisone 17-butyrate cream in the treatment of atopic dermatitis.     

Discrimination of the toxic potential of chemically differing topical glucocorticoids using a neutral red release assay with human keratinocytes and fibroblasts

In inflammatory skin disease, hydrocortisone and prednisolone double esters are about equipotent to conventional medium potency topical glucocorticoids, such as betamethasone valerate. Local adverse effects, in particular skin atrophy, are a potential problem with topical glucocorticoids. Recently, cell cultures have shown promise as a means of assessing local tolerance. To investigate the toxic potential of hydrocortisone, hydrocortisone-17-butyrate, hydrocortisone aceponate, prednicarbate, triamcinolone acetonide, betamethasone valerate and desoximethasone, human keratinocytes and fibroblasts were exposed to these agents in vitro, using a modified neutral red release assay. In addition, the morphology of these cells was assessed by light microscopy. Although all the topical glucocorticoids tested proved toxic to both cell types, there were major differences between glucocorticoids in their effect on fibroblasts. Hydrocortisone and the nonhalogenated double-ester-type glucocorticoids were less toxic than the conventional medium potency topical glucocorticoids tested (betamethasone valerate and desoximethasone). In particular, hydrocortisone aceponate was less toxic than betamethasone valerate (P 0.05). In general, the effect of topical glucocorticoids on the cells, based on neutral red release, was more marked with keratinocytes than with fibroblasts. Although the ranking order with respect to the toxic potential was similar, a clear-cut difference was not observed between non-halogen a ted double-ester-type glucocorticoids and betamethasone valerate. Morphological changes due to glucocorticoid exposure followed the same pattern with both keratinocytes and fibroblasts. The neutral red release assay is able to discriminate between the cytotoxic effects of chemically differing topical glucocorticoids on human keratinocytes and fibroblasts. The present data support the hypothesis of an increase in benefit/risk ratio with the new double esters of hydrocortisone and prednisolone.     

Assessment of atrophy of human skin caused by corticosteroids using chamber occlusion and suction blister techniques

The effect of five corticosteroid ointments on epidermal thickness was studied using occlusive chamber application. Thinning of the epidermis and telangiectasia were noted 3 weeks after application of betamethasone-17-valerate (BV), budesonide (BD), clobetasol-17-propionate (CP), and fluocinolone acetonide (FA), but not of hydrocortisone (HC). Suction blisters were raised at the treated sites. Epidermal thinning was always associated with reduction in cell number. By combining different methods it seems possible to define more precisely the epidermal effects of various corticosteroid preparation.     

The chamber vasoconstrictor assay

A comparison was made, using the vasoconstrictor assay, between a chamber test and patch tests in which the patch was either covered by an aluminium foil or left unoccluded. The unoccluded patch method produced an obviously less intense degree of pallor than the other methods. With the same test substances the chamber and the aluminium paper occlusion caused equally intense vasoconstriction. The chamber proved to have the following advantages over the aluminium foil method: (1) Test substance did not leak outside the test area, and therefore the pallor was clearly outlined and easier to define. (2) The chamber requires a smaller test area of skin. Of the test substances, clobetasol-17-propionate was unquestionably the most effective in the vasoconstrictor tests. Hydrocortisone-17-butyrate and betamethasone-i7-valerate were about equally effective. The skin blanching activity of hydrocortisone was the weakest of all.     

Quantifying systemic absorption of topical hydrocortisone in erythroderma

The systemic absorption of topical hydrocortisone (HC) was quantified in seven patients with erythroderma, using the ratio of the areas under the curves for plasma concentration vs. time, following topical and intravenous administration. Over a period of 24 h. 19–93 mg of HC was absorbed systemically, corresponding to 4–19% of the total topical dose of 500 mg. Thus, topical HC therapy of erythroderma is accompanied by a pharmacologically significant systemic dose.     

Corticosteroid effect on epidermal cell size

Changes in the epidermis following application of three corticosteroids, betamethasone 17-valerate, hydrocortisone 17-butyrate, and hydrocortisone have been studied histometrically in human volunteers. The reduction in epidermal thickness observed correlated significantly with a reduction in size of the viable epidermal cells. There was no significant reduction in the number of cells constituting the viable epidermis. These findings indicate that thinning of the epidermis is a function of cell size rather than cell number. The epidermal changes developed quickly and were rapidly reversible. It is suggested that measurement of cell size may be an early and sensitive index of atrophogenicity induced by topical corticosteroids. 0.1% Hydrocortisone 17-butyrate and 0.1% betamethasone 17-valerate showed equivalent potency in causing epidermal thinning and reduction in cell size. Reduction in cell size paralleled increasing concentrations of betamethasone 17-valerate, indicating a positive dose-effect relationship.     

Effect of five anti-inflammatory steroids on collagen and glycoaminoglycan synthessis in vitro

The effect of five anti-inflammatory corticosteroids, i. e. hydrocortisone, hydrocortisone 17-butyrate, betamethasone 17-valerate, nicocortonide acetate and nicocortonide, on the synthesis of hyaluronic acid, sulphated glycosaminoglycans and collagen by cultured skin fibroblasts was studied. As inhibitors of all these parameters the steroids could be arranged in order hydrocortisone < hydrocortisone 17-butyrate < betamethasone 17-valerate, nicocortonide acetate and nicocortonide. The corticosteroid concentrations required for inhibition of hyaluronic acid were very low as compared to those required for inhibition of sulphated glycosaminoglycan and collagen synthesis.     

Corticosteroid contact allergy: an EECDRG multicentre study

This article describes the results of an EECDRG multicentre study on contact allergy to corticosteroids. A total of 7238 patients were investigated: 6238 in 13 centres in the course of 1993, and 1000 patients in 1 centre in 1993 and 1994. The 5 corticosteroids tested were budesonide 0.1% pet., betamethasone-17-valerate 1% pet., clobetasol-17-propionate 1% pet., hydrocortisone-17-butyrate 1% eth., and tixoeortol-21-pivalate 1%., pet.; 189 (2.6%) gave a positive patehtest reaction (+, ++, +++)to at least 1 of the corticosieroids. The data regarding the corticosteroid-sensitive patients, as well as the patchtest results, were recorded on a standardized form.     

Contact allergy to hydrocortisone 17-butyrate

Two female patients with stasis dermatitis developed allergic contact dermatitis to hydrocortisone 17-butyrate cream. Patch tests with hydrocortisone 17-butyrate were positive, but not with the vehicle. One patient was also allergic to two other commerical corticosteroids, but patch tests revealed positive reactions only to ingredients of the vehicles. Attention is drawn to the frequency of contact allergy to corticosteroids in patients with stasis dermatitis.     

Detection of contact hypersensitivity to corticosteroids in allergic contact dermatitis patients who do not respond to topical corticosteroids

The delayed hypersensitivity development against topical corticosteroids which are used in allergic contact dermatitis (ACD) treatment is an important clinical problem. In our study, 41 ACD patients who did not show any response to topical corticosteroid treatment were patch tested with corticosteroid series and the commercial preparations of corticosteroids and their vehicles. In corticosteroid series, there were budesonide, bethametasone-17-valerate, triamcinolone acetonide, tixocortol pivalate, alclomethasone-17-21-dipropionate, clobetasole-17-propionate, dexamethasone-21-phosphate disodium and hydrocortisone-17-butyrate. We detected positive reaction to corticosteroids in 9 of our cases (22%) (5 single and 4 multiple). The sensitivity was mostly produced by tixocortol pivalate (6 patients). This was followed by triamcinolone acetonide (2 patients) budesonide (2 patients), alclomethasone dipropionate (2 patients), dexamethasone 21 phosphate disodium (2 patients) and betamethasone-17-valerate (1 patient). As a result, it should not be forgotten that the corticosteroids used to treat ACD patients may cause ACD themselves. In ACD patients who did not respond to corticosteroid treatment, routinely applying patch test with corticosteroids should be helpful in directing the treatment.     

Oral prednisolone induced acute generalized exanthematous pustulosis due to corticosteroids of group A confirmed by epicutaneous testing and lymphocyte transformation tests

BACKGROUND: Acute generalized exanthematous pustulosis (AGEP) is a rare cutaneous eruption which is often provoked by drugs. CASE REPORT: We report 2 cases of AGEP which showed rapidly spreading pustular eruptions accompanied by malaise, fever and neutrophilia after the administration of systemic prednisolone (corticosteroid of group A, hydrocortisone type). The histological examination showing neutrophilic subcorneal spongiform pustules was consistent with the diagnosis of AGEP. In both cases the rash cleared within a week upon treatment with topical steroids (corticosteroid of group D1, betamethasonedipropionate type and corticosteroid of group D2, hydrocortisone-17-butyrate type). Three months after recovery, the sensitization to corticosteroids of group A was confirmed by epicutaneous testing and positive lymphocyte transformation tests. CONCLUSION: These cases show that systemic corticosteroids can induce AGEP and demonstrate that epicutaneous testing and lymphocyte transformation tests may be helpful in identifying the causative drug. Our data support previous reports indicating an important role for drug-specific T cells in inducing neutrophil inflammation in this disease.     

Effects of glucocorticosteroids on primary human skin fibroblasts

Summary Various glucocorticosteroids were added to logarithmically growing cultures of primary human skin fibroblasts and of mouse L929 fibroblasts. These steroids inhibited proliferation of the human fibroblasts at concentrations which fall in a range expected to occur during the topical treatment of skin disorders. In terms of the concentrations required for the inhibition hydrocortisone was least and clobetasol-17-propionate most effective. All other steroids studied (hydrocortisone-17-butyrate, triamcinolone acetonide, betamethasone-17-valerate and hydrocortisone-21-acetate) showed medium effectiveness. Fluorination as such may not enhance the inhibitory effect. The inhibition was independent of the source (baby foreskin or adult arm skin) and passage number (7th to 13th or 15th and 16th passage, respectively) of the cells. The possible relationship between the inhibition of cell proliferation by such steroids and their therapeutic effect in psoriasis and their atrophic side effects is discussed.Mouse L929 fibroblasts were affected at 10³–10⁴-fold lower steroid concentrations and the range of the effective concentrations was 10⁴–10⁵ times as wide as that for the primary human skin fibroblasts. It was concluded that these mouse fibroblasts are a poor model system for the study of in vivo effects of glucocorticosteroids in man.

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Zusammenfassung Eine Anzahl Glucocorticosteroide wurden logarithmisch wachsenden Kulturen primärer menschlicher Hautfibroblasten und L929 Mäuse-Fibroblasten hinzugefügt.Im Konzentrationsbereich, der während der lokalen Behandlung von Hauterkrankungen in der Haut zu erwarten ist, wurde die Vermehrung menschlicher Fibroblasten gehemmt. Die zur Hemmung erforderliche Konzentration war für Hydrocortison am höchsten und für Clobetasol-17-propionat am niedrigsten. Die Hemmung durch die anderen Steroide (Hydrocortison-17-butyrat, Triamcinolon-acetonid, Betamethason-17-valerat und Hydrocortison-21-acetat) erfolgte im Zwischenbereich. Die Hemmung wurde durch Fluorinierung an sich nicht gesteigert und war unabhängig von der Herkunft dieser Fibroblasten (Baby-Vorhaut oder Armhaut Erwachsenen) und von der Zahl der Passagen (bzw. 17–23 oder 15–16). Mögliche Beziehungen wurden diskutiert zwischen der Hemmung der Zellvermehrung durch diese Steroide einerseits und ihren therapeutischen Effekten bei Psoriasis anderseits, sowie ihre atrophischen Seiteneffekte.L929 Mäuse-Fibroblasten wurden schon bei 10³–10⁴fach niedrigeren Steroidkonzentrationen merklich beeinflußt und der wirksame Bereich war 10⁴–10⁵fach breiter als für primäre menschliche Hautfibroblasten. Daraus folgt, daß diese Mäuse-Fibroblasten ein ungeeignetes Modellsystem für Untersuchungen der in vivo-Effekte von Glucocorticosteroide beim Menschen bilden.

     

Cultured porcine epithelial grafts: an improved method

An improved method of in vitro cultivation of porcine keratinocytes by which keratinocyte sheets suitable for grafting can be generated rapidly is described. Epidermis from split-thickness porcine skin is enzymatically separated from dermis with 0.25% Dispase solution (37 degrees C) within 3 h, and trypsinized to a single cell suspension. Keratinocytes are grown in Dulbecco-Vogt modified Eagle medium supplemented with 20 ng/ml hydrocortisone, 100 micrograms/ml penicillin, 100 micrograms/ml streptomycin, and 20% (cells from six-month-old pigs) or 10% fetal calf serum (cells from two-month-old pigs). Freshly isolated keratinocytes are plated at a density of 1.25 X 10(6) cells/ml since their plating efficiency is about 15 times lower than that of human keratinocytes grown under comparable conditions. Primary keratinocytes plated on plastic grow to confluence faster than those plated on lethally irradiated 3T3-J2 feeder layer cells. Porcine keratinocytes grown on plastic reach senescence in the third passage but, when subsequently cultivated on a lethally irradiated 3T3-J2 feeder layer, can be passaged up to seven times. Nevertheless, plating efficiency of second-passage porcine keratinocytes is only about 5%-7%, whereas that of human newborn foreskin keratinocytes is 20%-30%. Confluent stratified primary cultures grown on plastic, or secondary cultures grown on feeder layers, are used for grafting. The sheets are detached with Dispase solution and stapled to vaseline gauze to facilitate handling. Epidermal regeneration from porcine grafts produced by this method has been demonstrated after transplantation to full-thickness wounds excised to muscle fascia in donor animals.     

Effects of human recombinant tumor necrosis factor-alpha (TNF-alpha) on the proliferative potential of human keratinocytes cultured in serum-free medium

The effects of human recombinant tumor necrosis factor-alpha (TNF-alpha) on human keratinocytes cultured in a serum-free medium were investigated. TNF-alpha markedly suppressed cell growth. The growth-inhibitory effect was reversible and cytostatic at a concentration of 1-5 U/ml, but appeared to be irreversible and cytocidal at 10 U/ml. The growth suppressive effect was more marked when TNF-alpha was added in the late growth phase or preconfluent phase than when it was added in early or mid-growth phases. No effects of TNF-alpha on cell adhesion to the substrate were observed. These results indicate that TNF-alpha is a very potent anti-proliferative agent for human keratinocytes.