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Vol. 80. No. 3 Printed in U. • . A.

Decreased Urinary Polyamines in Patients with Psoriasis Treated with Etretinate

RoY C. GREKIN, M.D., CHARLES N. ELLIS, M.D., NANCY G. GoLDSTEIN, M.P.H., NEIL A. SwANSON, M.D. ,

THOMAS F. ANDERSON, M.D. , ELIZABETH A. DUELL, PH.D., AND JOHN J _ VOORHEES, M.D.

Department of Derm.atology, University of Michigan M edical School, Ann Arbo1~ and Dermatology Service (CNE and TFA), Vetera.n8 Administration M edical Center, Ann Arbor, Michigan, U.S.A.

Oral administration of the aromatic retinoid etretinate is effective therapy for psoriasis and other epidermal hyperproliferative disorders. Since polyamine metabo­lism is known to be important in cell growth and differ­entiation, we measured urinary levels of the polyamines putrescine, spermidine, and spermine as a reflection of cutaneous polyamine metabolism in 19 psoriatic patients treated with e tretinate for 16 weeks. Using thin-layer chromatography, polyamine determinations were p er­formed on urine collected pretherapy, during therapy, and 8 weeks after therapy was concluded. Good to ex­cellent clearing of psoriasis occurred in 18 of 19 patients. All urinary polyamines showed a downward trend in the first week of therapy, prior to s ignificant clinical im­provement. At week 16 of therapy, the greatest reduction in mean urinary polyamine content occurred. Mean pu­trescine levels decreased from pretherapy to week 16 by 27 % (p < 0.001), mean spermidine values fell by 34% (p < 0.001), and mean spermine levels declined by 37% (p = 0.005). These data are consistent with the hypothesis that etretinate inhibits polyamine biosynthesis.

It is now well established tha t oral administration of the aromatic retinoid etretinate (Ro 10-9359) is a n effective pso­riasis th erapy, either alone or in combinat ion with oth er agents [1-3]. The m echanism of action of retinoids has not been establis hed; however, attention h as fo cused on polyamines a nd their possible role in the pathogenesis of psoriasis. Polyamine levels are increased in t he involved and uninvolved skin a nd in the urine of psoriasis patients [ 4,5] ; furthermore, Proctor et a l [6] a nd others [ 4,5], have demonstrated a decrease in both urina1·y and skin polya mine levels with effective treatment. Whethe r declines in polyamine values r epresent treatment­induced events or are secondary to lesional resolution produced by a n effective therapy is uncl ear.

Certa in evidence s uggests that polyamines may play a n im­portant role in the regulation of proliferation. The activity of ornithine decarboxylase (ODC), th e rate-limiting enzym e in polyamine syn thesis [7], rises prior to the increase in cellular prolifera t ion caused by various stimuli both in vitro [8] a nd in vivo [9,10). Polyamines added to cell culture systems stimulate cell growth [11], while inhibition of polyamine syn thesis by treatment with ODC inhibitors impedes cellular proliferat ion in vitro [13,14] and in the skin (15]. It has also been shown tha t retinoid therapy lowers cutaneous polyamine levels in psoriasis patients [16]. As a noninvasive procedure, we wondered whether measurement of urinary polyamine levels would refl ect

Manuscript received December 7, 1981; accepted for publication July 22, 1982.

Supported in par t by a National Institutes of Health Grant No. T32AM017197.

Reprint requests to: Dr. Roy C. Grekin, Department of Dermatology, Box 31, University of Michigan Medical School, Ann Arbor, Michigan 48109.

Abbreviations: ODC: ornithine decarboxylase

18 1

those changes known to occur in th e skin. Therefore, in 19 patients with severe psoriasis treated with etretinate, we deter­mined urinary polyamine values for putrescine, spermjdine, and spermine before and during t herapy and after th erapy had been stopped.

MATERIALS AND METHODS

Patients

Nineteen patients aged 21 to 70 years were selected for study. There were 16 men and 3 women. Patients had greater than 20% of their body surface area involved with psoriasis or had disabling psoriasis (e.g., palmoplantar psoriasis). Psoriasis types included plaque (n = 10). erythrodermic (3) , pustular (2), inverse (2) and palmoplantar (2). Ini tial treatment consisted of either etretinate or placebo in a random double­blind design. After 8 weeks, the code was broken and those patients on placebo were begun on active drug, while those patients taking etretin­ate continued to do so. All patients were treated with 1.0 mg/ kg/ day for 1 week and 0.5 mg/ kg/ day the second week. Thereafter, dosage was adjusted to mainta in maximum clinical response while keeping side effects to a degree to lerated by the patient. Etretinate treatment continued for a total of 16 weeks in aU patients. Subsequently subjects were followed for 8 weeks after therapy had been stopped. Pat1ents avoided sunlight and used only bland emollients as adjunctive therapy.

Polya.min.es

Refrigerated 24-hr collections of urine in amber jugs containing 10 ml of 12 M HCl were obtained from patients pretherapy, at weeks 1, 2, 4, 8, 12, and 16 during therapy, and weeks 4 and 8 afte r treatment had been stopped. After thorough mixing, 15-ml aliquots were removed for analysis and stored at - 70°C.

Analysis of Polyamin.es

Urine samples with added tri tiated polyamines as tracers were hy­ru-olyzed in 6 M HCl, evaporated to dryness, and resuspended in 1% perchloric acid. The polyamines were partially purified by Dowex 50 chromatography [17]. The column fractions containing the polyamines were evaporated to ru·yness and resuspended in water. The polyamines were dansylated by the method of Seiler and Wiechmann [18] with modifications [19). The dansylated amines were separated by two­dimensional thin-layer chromatography [20]. The separated com­pounds were visualized with an ultraviolet light (wavelength 365 nm), scraped from the plates, and resuspended in benzene. The concentra­tion of the polyamine derivatives was determined flu orometrica lly. Recoveries were determined by quantitating the amount of tracer in the benzene fractions.

Statistical Analysis

Statistical analysis was performed using pairwise comparisons follow­ing repeated measures analysis of variance and Student's t-test.

RESULTS

Clinical Response

Eighteen of 19 patients taking etretinate had good to excellen t clearing of psoriasis during the 16-week trial. One pat ient with plaque-type psoriasis failed to respond satisfactorily. Typical cu taneous side effects of cheilit is, peeling of the skin of palms a nd soles, and hair loss were seen, but no patient discontinued etretinate as a result.

Nine patie nts received a n 8-week placebo period, during

182 GREKIN ET AL

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FIG 1. Mea n urinar y polyamine levels a nd mean clin ica l response during the study in 19 patients with psoriasis. The mean etretinate dose is indicated by the shaded area. The vertica l line at week 8/0 indicates the conclusion of the 8-week place bo period (n = 9), and t he beginning of etretinate therapy for a ll patients. The vertical line at week 16 indicates the conclusion of etretinate t herapy; patients were monitored for an additional 8 weeks. Compared to week 0, mean polyamine levels for the 19 pa­tients were significantly decreased dur­ing etretinate t hera py: at week 1 for sper­midine, p < 0.05; at week 16 for putres­cine, p < 0.001, spermidine, p < 0.001, and spermine, p 0.005. Key: e e a ll pa tients; •- -- -• pa­tients administered placebo for 8 weeks preceding etretinate therapy (n = 9) ; e. · · · e patients receiving only etretin­ate (n = 10). Hesponse to therapy was determined at each lime point by phy­s ician ratings based on a scale of: 0 = excellent; l. = very good, 2 = good, 3 = poor, 4 = no response, 5 = worse.

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which they demonstrated no improvement in their psoriasis. Once treatment began with etretinate, their clinical course was similar to that of the group which received only etretinate therapy (Fig 1) .

Polyamine Levels

The mean 24-hr urinary levels of each polyamine decreased in the 19 patients during etretinate therapy. Decreases in all 3 polyamines occurred after 1 week of therapy prior to significant clinical response. In the first week, polyamine values declined by 21% for putrescine, 30% for spermidine, and 14% for sperm­ine; however only the change for spermidine was significant ( p < 0.05). At week 2, following a 50% decrease in the etretinate

..

16 20

dose, all polyamine levels returned toward pretreatment values. After week 2 the dosage was again increased; the subsequent mean urinary polyamine level measurements (week 4) were decreased (Fig 1, Table I). Thereafter, polyamine levels gradu­ally decreased throughout the therapy period. The decrease from pretherapy to week 16 of therapy was significant for all 3 polyamines (p :::: 0.005). The levels of all 3 polyamines rose after etretinate was discontinued, and 8 weeks after therapy had been stopped, mean polyamine values were not statistically different from pretherapy. The spermidine to spermine ratio did not vary from pretreatment levels throughout the study.

Nine patients received placebo for an 8-week period prior to their 16 weeks of etretinate therapy. Their initial mean polya-

March 1983 URINARY POLYAMINES DURING ETRETINATE THERAPY 183

TABLE I. Mean urine polyamines ±standard deviation "

Pretherapy During therapy

After therapy

"nmole/mg crea tin ine (n = 19).

Time

Week 0 Week 1 Week 2 Week 4 Week 16 Week 24

" p < 0.05 from pretherapy (week 0). ' p :::= 0.005 from pretherapy (week 0).

FIG 2. Urina ry polyamine levels in 1 patient who fai led to improve during etretinate t herapy. The dose of etretinc ate is indicated by the shaded area.

Putrescine

4.01 ± 1.81 3. 18 ± 2.27 3.66 ± 2.22 3.32 ± 1.96 2.94 ± 1.68 ' 3.65 ± 2.35

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mine levels were not statistically different from the fu·st values obtained from the group that was randomly assigned etretinate­only therapy. However, during the placebo period, polyamine levels tended to increase (Fig 1). Mea n values for each polya­mine just prior to etretinate therapy had become significantly higher (p < 0.05) in the group receiving placebo than in t he 10 patients who had only etretinate treatment (Fig 1) , which may be the result of 8 weeks of disease activity unbridled by any therapeutic measures. After the star t of etretinate therapy, the pattern of polyamine changes in the patients who had received placebo t reatment was similar to t hat of the group who took only retinoid therapy. At the end of 16 weeks of etretinate therapy, the difference in mean polyamine levels between the placebo and etretinate-only groups was not statistically signifi­cant.

In the 1 patient who failed to improve clinically, polyamine levels followed the same pattern as that of responders, decreas­ing during treatment and increasing after t herapy was discon­tinued (Fig 2).

DISCUSSION

The levels of urinary polyamines have been investigated in numerous diseases [21]. As a noninvasive procedure with min­imal patient inconvenience, we felt that monitoring polyamine changes in urine would be idea l during t herapy for benign hyperplastic conditions such as psoriasis . In this study, we have shown that urinary polyamine levels fall during the treatment of psorias is with the oral aromatic re tinoid etretinate.

Evidence consistent with a direct effect of etretinate on

Spermid ine Spermine

2.93 ± 1.35 3.21 ± 1.77 2.04 ± 0.92" 2.75 ± 1.34 2.65 ± 1.26 3.13 ± 1.4 2 2.08 ± 0.94 2.71 ± 1.08 1.93 ± 1.09 ' 2.01 ± 1.4 7'" 2.24 ± 1.86 2.54 ± 1.25

POLYAMINE LEVELS IN URINE OF NON-RESPONDER

Mean Dose of e lretinale (mg/ kg/ day)

0 0.96 0.54 0.93 1.05

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polyamine synthesis occurred during the early weeks of therapy. At week 1 we showed a downward trend (Fig 1) in a ll 3 urinary polyamine levels which preceded the onset of significant clinical improvement. Our patients received 1.0 mg/ kg/ day during week 1 and were reduced to 0.5 mg/ kg/day during week 2. The ini t ia l decrease in polyamine levels at the end of week 1 was followed at t he end of week 2 by an increase toward pretreat­ment levels in the mean values of all 3 polyamines. At the end of week 2 the dosage of etretinate was increased to a mean of 0.92 mg/ kg/day and maintained at that level for the next 2 weeks. Polyamine levels subsequently fell as measured at week 4. Thus changes in mean urinary polyamine levels appear to be closely tied to the dose of etretinate. This inverse relationship between changes in polyamine levels and etretinate dosage may have represented a dose-related effect, since apparent inhibi t ion of polyamine production by etretinate was lessened when the dose was reduced, and then restored when the dose was raised.

Further support for a direct etretinate effect on polyamine synthesis was seen in the patient whose polyamine levels de­creased despite increased disease activi ty. In this patient, changes in polyamine levels clearly were not secondary to disease improvement, and therefore we presume that these polyamine changes were due solely to etretinate administration (Fig 2) .

Evidence exists that supports the hypothesis that the reti­noids may exert their beneficial effects on psoriasis by altering the levels of polyamines in the skin [15,16,22-24). Lauharanta et al [16], using a similaT treatment protocol, achieved anala­gous results for polyamine levels in the skin of psoriasis patients.

184 GREKIN ET AL

In our study, mean urinary levels of putrescine, spermidine, and spermine declined by 27%, 34%, and 37%, respectively by week 16 of t herapy; Lauharanta et al found decreases in skin polya­mines of 72%, 66%, and 43% at remission. The lesser degrees of cha nges seen in our study may retlect "background" contribu­tion of polyamines from tissues other than skin, which would tend to obscure skin-related changes in urina ry polyamine levels. Nevertheless, urinary polyamine measurements accu­rately retlect changes occurring in cutaneous polyamine levels and are easier to obtain repeatedly.

Other investigators employing topical corticosteroids [ 4], dithranol [5,6), or specific inhibitors of ODC [25) have demon­strated decreases of either ODC activity or po lyamine synthesis or both in skin and urine in association with improved psoriasis. Several investigative groups [15,22- 24) have suggested that the t herapeutic efficacy of retinoids in hyperproliferative skin dis­orders may result from inhibition of ODC. Although we did not measure ODC activity per se, we did demonstrate alterations in the products of that enzyme, namely polyamine levels, which varied with the retinoid dose. Urinary polyamine levels declined in our study prior to clinical improvement a nd then remained at reduced levels throughout the period of etretinate therapy, further supporting the hypothesis that retinoids directly mod­ulate polyamine synthesis. It is likely that other compounds possessing the ability to inhibit the synt hesis of polyamines wil l be effective in the treatment of psoriasis. We believe that a continued search for and investigat ion of such agents are war­ranted.

We thank Lenore Rhodes for technical assistance and Mary Anne Jordan for preparing the figures. Hoffmann-LaRoche, Inc., Nutley, New J ersey supplied the etretinate.

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Voorhees JJ: Glucocorticoid inhibits elevated polyamine biosyn­thesis in psoriasis . J Invest Dermatol 71:177-181, 1978

5. Bohlen P, Grove J, Beya MF, Koch-WeserJ , Henry MH, G•·osshans E: Skin polyamine levels in psoriasis: the effect of di thranol therapy. Eur J Clin Invest 8:215-218, 1978

6. Proctor MS, Wilkinson Dl, Orenberg EK, Farber EM: Lowered cutaneous a nd urinary levels of polyamines with clinical improve­ment in treated psoriasis. Arch Dermatol 115:945-949, 1979

Vol. 80, No. 3

7. R ussell DH, Durie GM: Polyamines as Biochemical Markers of Normal and Malignant Growth. New York, Raven Press, 1978 pp 1- 13 '

8. Paranjpe MS, Delar co JE, T odaro GJ : Retino ids block orni thine decarboxylase induction in ce lls treated with the tumor promoter TPA or the peptide growth hormones, EGF and SGF. B ioch em Biophys Res Commun 94:586-591, 1980

9. Verma AK, Lowe NJ, Boutwell RK: Induction of mouse epiderma l ornith ine decarboxylase activity and DNA synthesis by ultravi­olet light. Cancer Res 39:1035-1040, 1979

10. Morrison DM, Goldsmith LA: Ornithine decarboxylase in rat s kin . J Invest Dermatol 70:309-3 13, 1978

11. Stoner CD, Ha rris CC, Myers GA, Trump BG, Connor RD: Pu­trescine stimulates growth of human bronchial epithelial ce lls in primary cul ture. In Vitro 16:399-406, 1980

12. Mamont PS, Boh len P , McCann PP, Bey P, Schuber F, Tardif C: a -Methyl ornithine, a potent competit ive inhibi to r of orni thine decarboxylase, blocks proliferation of rat hepatoma ce lls in cul ­ture. Proc Nat! Acad Sci USA 73: 1626-1630, 1976

13. Haddox MK, Scott KFF, Russell DH: Retinol inhibi tion of o rni ­thine decarboxylase induction and G, progression in chi nese hamster ovary ce lls. Cancer Res 39:4930-4938, 1979

14 . Haddock DH, Haddox MK: Antiproliferative effects of re tino ids related to the cell cycle-specific inhibition of orni thine decarbox­ylase. Ann NY Acad Sci 359:281-297, 1981

15. Lowe NJ , Kaplan R, Breeding J : Etretinate treatment for psoriasis inhibi ts epidermal ornithine decarboxylase. J Am Acad Dermatol 6:697- 698, 1982

16. Lauharanta J, Kousa M, Kapyaho K, Linnamaa K, Mustakallio K : Reduction of increased polyamine levels in psoriatic lesions by retinoid a nd PUVA t reatments. Br J Dermatol 105:267-272, 198 1

17. Inoue H , Mizutani A: A new method for isolation of polyam ines from animal tissues. Anal Biochem 56:408-416, 1973

18. Seiler N, Wiechmann M: Die Mikrobestimmung von Spermin und Spermidin a ls 1-dimethylamino-naph tha lin-5-sulfonsaure- Deri­vate. Z Physiol Chem 348:1285- 1290, 1967

19. Vandemark FL, Schmidt GJ, S lavin W: Determination of polya­mines by liquid chromatography a nd pre-column la belling for flu orescence detection. J Chromatogr Sci 16:4 66-469, 1978

20. Abdei-Monem M, Ohno K, Newton NE, Weeks C: Thin layer chromatography and high pressure liquid chromatography of the dansyl polyamines, Advances in Polyamine Research, vol 2. Edited by R Campbell , D Morris, D Bartos, G Daves, F Bartos. New York, Raven Press, 1978, pp 37-49

21. Russell DH, Durie GM: Polyamines as B iochemical Markers of Normal and Malignant Growth. New York, Raven Press, 1978, pp 139- 155

22. Voorhees JJ: Polya mines and psoriasis. Arch Dermatol 115:943-944, 1979

23. Haddox MK, Russell DH: Cell cycle-specific locus of vitamin A inhibi tion of growth. Cancer Res 39:2476-2480, 1979

24. Verma AK, Rice HM, Shapas BG, Boutwell RK: Inhibi tion of 12-0-tetra-decanoylphorbol- 13-acetate-induced orni thine decm·box­ylase activity in mouse epidermis by vita min A analogs [reti­noids]. Cancer Res 38:793-801, 1978

25. Grosshans E, Henry M, T ell G, Schechter PJ, Bohlen P, Grove J , Koch-Weser J: Les polyamines dans le psoriasis. Ann D ermatol Venereol 107:377-387, 1980

The Pennsylvania Academy of Dermatology, In c. (A nnua l Meeting) a nd the Postgraduate Dermatology Semin ar of the Toronto Academy of Medicin e wi ll be a conjo in t mee ting held at the Hote l Westin, Toronto, Canada, October 28-30, 1983. For information: Maurice A. Thew, M.D. , Secreta ry P.A.D., #19 The Commons, 3516 Silverside Hoad, Wilm in'gto n, Delaware 19810.

The 5th World Congress ofCtyosurgety wiU take place at the P hilippine In ternationa l Convent ion Center, Ma nill a, P hilippines on November 17-19, 1983. Abstracts to: Dr. Sajio S umida, The National Fukuoka Central Hospita l 2-2, Jona i, Chuoku, Fukuoka 810, Japan, for receipt no late r than M ay 31, 1983. For information abo ut registration fees a nd program: Dr. Samuel M. T a nchoco, S ui te 31, Lagasp i Towers, 300 Roxas Boulevard, Ma nill a, P hilippines.