Indian Journal of Biochemistry & Biophysics Vol. 39, February 2002, pp. 22-27

Constant variation in structure and function of geometrical isomers of acitretin under natural light

Akira Murayamat, Takakazu Suzukit, Minoru Iwamoto:!: and Sridhar Rao Kunchala*

tlnstitute for Medicine and Human Science, 2-27-18-701 Honkomagome, Bunkyo-ku, Tokyo 113-002 1 and Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Science,

3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan :j:T. Hasegawa Co. L TO. Itakura Factory, Itakura-cho, Gunma-ken, 374-0131, Japan

*Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007 (AP), India

Received 5 November 2001; revised and accepted 20 December 2001

Acitretin, a beneficial retinoid, was shown to undergo constant structural interconversions among its geometrical iso­mers (all-trans-acitretin, 9-cis-acitretin, 13-cis-acitretin, 9, 13-di-cis-acitretin, etc.) by photoisomerization under natural light. The photoisomerization was zero order reaction with an apparent velocity of 4x 10.7 Mlmin under illumination by white Ouorescent lamps (I, 200 Ix). An equilibrium mixture of the geometrical isomers (all-trans-acitretin 20%, 9-cis­acitretin 15%, 13-cis-acitretin 30%, 9, 13-di-cis-acitretin 15%, and unidentified compounds 20%) was formed at around 30 min. Equilibrium mixtures with similar composition were obtained by photoisomerization reactions starting from other geometrical isomers. Geometrical isomers of acitretin thus formed, showed different effects to induce differe;l tiation of hu­man acute promyelocytic leukemia cells (HL-60 cells) : activity of all-trans-acitretin (E050, 3.2x 1O.6M), 9-cis-acitretin (ED50, 2.3x 1O.5M), 13-cis-acitretin (ED50, 1.1 x 10.5 M), 9, 13-di-cis-acitretin (E050, 2.6x 10·6M). 9-cis-Acitretin acted syner­gistically with all-trans-acitretin, 13-cis-acitretin and 9, 13-di-cis-acitretin on HL-60 cells. On the other side, all-trans­acitretin, 13-cis-acitretin and 9, 13-di-cis-acitretin acted additively. Geometrical isomers of acitretin showed different effects on differentiation of human epidermal keratinocytes; expression of keratinocyte differentiation markers, keratin I and kera­tin 10, were suppressed more strongly by 9-cis-acitretin and 13-cis-acitretin as compared to all-trans-acitretin or 9, 13-di­cis-acitretin.

We have recently reported that the geometrical isomers of retinoic acid undergo constant structural interconversions by photoisomerization reaction under natural light (velocity of the photoisomeriza­tion under 1, 200 Ix white fluorescent light >8x 10.7 Mlmin)I.3. The geometrical isomers of retinoic acid differed in their side chain configurations and possessed different abilities to induce differentiation of human acute promyelocytic cells HL-60 cells or normal human epidermal keratinocytes '·

3. The study

of variation in structure and function of similar biosignal molecules under natural light is a pre­requisite for correct understanding of their mechanism of action. Acitretin is widely used as a therapeutic agent in the treatment of a variety of dermatological diseases like psoriasis, icthyosis, acne etc.4

.6

. In this paper, we describe the photoisomerization of acitretin under natural light and its biological effects.

* Author for correspondcncc E-mail: srecdhar@gene.ccmbindia.org Fax: 040-7171195

Materials and Methods To avoid problems associated with photoisomeri­

zation of acitretin, all experiments were carried out under FLR40S Y -FIM coloured fluorescent lamps (Matsushita Electric Industrial Co., Osaka, Japan), unless otherwise stated.

High-performance liquid chromatography (HPLC) was carried out on a TSKgel ODS-80Ts column (Toso Co., Tokyo, Japan). The mobile phase consisted of a mixture of CH3CN/O.l % CH3COONHJCH3COOH (700:300:4) as described earlier7

• Assignment of the ' peaks was carried out by running authentic samples of the geometrical isomers of acitretin.

IH-NMR spectra of geometrical isomers of aci­tretin were measured with JEOL EX-400 spectrome­ter in CDCb with tetramethylsilane as an internal standard. Acitretin numbering conforming to IUPAC norms was used for NMR analysis. High-resolution MS analyses were conducted on a JEOL OX-303 high-resolution mass spectrometer.

Monoclonal antibodies for human keratins K I and for KIO were purchased from Progen Biotechnik

MURA Y AMA et at.: CONSTANT VARIATION IN STRUCTURE AND FUNCTION OF ACITRETIN 23

OH POCb/ ~ ~

~ M92S0'~ DMF )§eCHO KH/THF ~ KOH/Meo~ ' -.-: o -+ 0 -+ 0 ~ 0 Mg2S0. 0 NaOH MeO .-vCOOMe MeO COOMe MeO COOMe

r

silica gel I rCOOMe ~ MnO. CHO chromatography ~ 0 -.-: ~ ~ )9l... ~ -.-: OH ~ 0 -.-: -.-:

MeO vltrlde MeO MeO

rCHOKH/THF. ~ ~ ~COOH MeO )....,COOMeMeO COOH MeO

'ii; 13-cis-acitretin all-trans- acltretin

~ MeO CHO

KH /THF

;l,..COOMe 'ii; ~h~ 0-'-: -+ 0-'-:

MeO MeO -.-: COOH

9,13-di-cls-acitretin 9-cls-acitretin COOH

Fig. I-Synthesis of geometrical isomers of acitretin

GMBH (Heidelberg, Germany) and ICN Pharmaceu­ticals Inc. (Ohio, USA) respectively . Bioassays in­cluding NBT-reduction assay for HL-60 differentia­tion and Westet:n blot analysis for keratinocytes dif­ferentiation markers were carried out as described previousl/.

Results all-trans-Acitretin was synthesized as reporteds.

Geometrical isomers of all-trans-acitretin (9-cis­acitretin, 13-cis-acitretin, 9, 13-di-cis-acitretin) were synthesized via similar procedure shown in Fig. 1. Spectral data of synthesized geometrical isomers of acitretin are as follows:

(2£, 4£, 6£, 8E)-9-(4-methoxy-2, 3, 6-trimethyl­phenyl)-3, 7-dimethyl-2, 4, 6, 8-nonatetraenoic acid (all-trans-acitretin). UV Amax (EtOH) 357 nm (£ 41, 500). HRMS calculated for C21H260 3 326.1875 ; Found, 326.1879. IH-NMR (CDCb): =2.12-2.39 (ISH, m: Ar (aromatic) CrMe, ArCrMe, ArC6-Me, C3-Me, CrMe), 3.82 (3H, s: ArC4-OMe), 5.82 (lH, s: C2-H), 6.21 (lH, d J=12Hz: C4-H or C6-H), 6.26 (lH, d J=17Hz: Cs-H), 6.35 (lH, d J=15Hz: C4-H or C6-H), 6.61 (lH, s: ArCs-H), 6.71 (lH, d J-17Hz: C9-H), 7.08 (lH, dd J=12Hz and 15Hz: Cs-H).

(2£, 4£, 62, 8£)-9-(4-methoxy-2, 3, 6-trimethyl­phenyl)-3, 7-dimethyl-2, 4, 6, 8-nonatetraenoic acid

(9-cis-acitretin). UV Amax (EtOH) 349 nm (£ 36,500), HRMS calculated for C21 H260) 326.1875; Found, 326.1883. IH-NMR (CDCb+DMSO-d6): 0=2.11-2.31 (l5H, m: ArCrMe, ArC)-Me, ArC6-Me, C)-Me, Cr Me), 3.80 (3H, s: ArC4-OMe), 5.74 (lH, s: Cz-H), 6.14 (lH, d J=IIHz: C6-H), 6.27 (lH, d J=lSHz: C4-

H), 6.62 (lH, s: ArCs-H) 6.68 (lH, d J=16Hz: CsH or C9-H), 6.80 (lH, d J=16Hz: Cs-H or C9-H), 7.05 (lH, dd J=11Hz and 15Hz: Cs-H).

(22, 4£, 6£, 8E)-9-(4-methoxy-2, 3, 6-trimethyl­phenyl)-3, 7-dimethyl-2, 4, 6, 8-nonatetraenoic acid (I3-cis-acitretin). UV Amax (EtOH) 359 nm (£ 40,800) . HRMS calculated for C21 H260 ) 326.1875; Found, 326.1990. IH-NMR (CDCb): 0=2.11-2.30 (I5H, m: ArCrMe, ArC)-Me, ArC6-Me, C)-Me, CrMe), 3.82 (3H, s: ArC4-OMe), 5.69 (lH, s: C2-H), 6.28 (lH, d J=16Hz: Cs-H), 6.32 (IH, d J=12Hz: C6-H), 6.60 (lH, s: ArCs-H), 6.70 (IH, d J=16Hz: C9-H), 7.06 (IH, dd J=12Hz and 15Hz: Cs-H), 7.77 (lH, d J=15Hz: C4-H).

(22, 4£, 62, 8E)-9-(4-methoxy-2, 3, 6-trimethyl­phenyl)-3, 7-dimethyl-2, 4, 6, 8-nonatetraenoic acid (9, 13-di-cis-acitretin). UV Amax (EtOH) 351 nm (£ 39,300). HRMS calculated for C21H260 ) 326.1875 ; Found, 326.1889. IH-NMR (CDCI) : 0=2.04-2.32 (I5H, m: ArCrMe, ArC)-Me, ArC6-Me, C)-Me, Cr Me), 3.83 (3H, s: ArC4-OMe), 5.67 (lH, s: Cr H), 6.26 (lH, d J=11 Hz: C6-H), 6.63 (lH, s: ArCs-H),

24 INDI AN J. BIoeHEM. BIOPHYS., VOL. 39, FEBRUARY 2002

E C

0 to C')

..... ctS 0) u C ctS .0 ~

° (f) .0 <l: 0 15 20 0 15 20 0 15

Retention time ( min) 20 0 15 20

Fig. 2-HPLe analysis of photoisomerization of solutions of all-trans-acitretin illuminated by white fluorescent light [Ethanolic solutions of all-trans-acitretin in quartz cuvette (lOxSx4Smm) were ill uminated by white fluorescent lamps (1,200 Ix) for IG hr by coloured fluo­rescent lamps (FLR40S Y -FIM, 120 Ix) at 1O-5M (A); or by white fluorescent lamps (1,200 Ix) for 30 min at 1O-5M (B); IO-4M (C); or 10-3M (D); and analyzed for photoisomerization by HPLC. Shaded peaks represent starting all-trans-acitretin and unshaded peaks represe nt photoisomerized acitretins. PI, P2, unidentified; P3, 9, 13-di-cis-acitretin; P4, all-trans-acitret in; PS, 13-cis-acitretin; P6, 9-cis-acitretin]

~ 100 .--- ---, (f) '­

'+- 0)

°E C o .Q(f) ...... -'iii -o ~ 0... .-E ~ 0 0)

o §

50

o 10 300 10 30 0 10 300 10 30 0) O'l Time illuminated ( min )

Fig. 3-Time course of photoisomerization of solutions of geometrical isomers of acitretin under white fluorescent light [Ethanolic solu­tions (10-5 M) of all-trans-acitretin (A); 9-cis-acitretin (B); 13-cis-acitretin (e); and 9, 13-di-cis-acitretin (D) in quartz cuvettes were illu­minated by white fluorescent lamps (1200 Ix) and then analyzed for photo-isomerization by HPLC. 0, all-trans-acitretin; .A. , 9-cis­acitretin ; e, 13-cis-acitretin ; ,9, 13-di-cis-acitretin; . and are unidentified compounds]

6.71 (tH, d J=17Hz: Cg-H or C9-H), 6.80 (tH, d ]=17Hz: Cg-H or C9-H), 7.10 (1 H, dd J=llHz and 16Hz: C5-H), 7.71 (lH, d ]=16Hz: C4-H) .

Results of the analysis of photoisomerization of solutions of all-trans-acitretin with white fluorescent lamps (FLl5BA-37K, 1, 200 Ix) or coloured fluores­cent lamps (FLR40S Y-F/M, 120 Ix) at different con­centrations (l0-3M, 1O-4M, 1O-5M) are shown in Fig. 2. Photoisomerization of all-trans-acitretin was not de­tected under coloured fluorescent lamps exclud ing light under 500 nm even after 10 hr (Fig. 2A). How­ever, under white fluorescent lamps, all-trans-acitretin underwent photoisomerization to considerable extent. Equilibrium state of photoisomerization (100% pho­toisomerization) was reached for 10-5 M after 30 min (Fig. 2B). Degree of photoisomerization reached around 80% and 15%, respectively, for solutions of 1O-4M and 1O-3M after 30 min (Fig. 2C and 20). These

results supported that photoisomerization is a zero order reaction and that degree of photoisomerization of all-trans-acitretin is inversely proportional to con­centration at concentrations from 1O-3M to 1O-5M un­der the experimental condition .

The time course of photoisomerization of 1O-5M solutions of geometrical isomers of acitretin under white fluorescent lamps is shown in Fig. 3. The ap­parent velocity of photoisomerization reaction of all­trans-acitretin was approximately 4xlO-7Mlmin (Fig. 3A). The equilibrium mixture of geometrical isomers of acitretin consisting of all-trans-acitretin (20%), 9-cis-acitretin (15 %), 13-cis-acitretin (30%), 9, 13-di-cis-acitretin (15%) and un identified com­pounds (20%) was formed by about 30 min (Fig. 3A). Other geometrical isomers of acitretin, viz 9-cis­acitretin, 13-cis-acitretin and 9, 13-di-cis-acitretin also undergo similar photoisomerization reaction (Fig. 3B,

MURA YAM A et al.: CONSTANT VARIATION IN STRUCTURE AND FUNCTION OF ACITRETIN 25

~~COOH

all-trans-acitretin

COOH 9-cis- acitretin

~ COOH

3-cis-acitretin

*\~ Velocity of ptlotoisomerization " COOH under white fluorescent light

(1,200 Ix)

9,13-di-cis-acitretin 4x1O -7 M/min

Fig. 4-Constant variation of structure among the geometrical isomers of acitretins under natural light

X10-6 M

........ 0 o L()

o -W1 0 .......... c c o 0 :;::;~ () CO ::J~ "0 C 2 C Q)

- L-Q)

~ "0 o 10 20 30

Time illuminated ( min)

Fig. 5-Biological activity of solutions of geometrical isomers of acitretin to induce differentiation of HL-60 cells depending on illumination by white fluorescent light [Ethanolic sol utions (I0·sM) of all-trans-acitretin (0); 9-cis-acitretin (M; and I3-cis­acitretin (e) in quartz cuvettes were illuminated by fluorescent lamps (1200 Ix) and analyzed for activity to induce differentiation of HL-60 cell s]

3C, 3D). These results showed that acitretin con­stantly undergoes structural variation among multiple geometrical isomers by photoisomerization under natural light (Fig. 4).

Ability of solutions (lO-5M) of geometrical isomers of acitretin to induce differentiation of HL-60 cells into granulocytes varied depending on illumination by white fluorescen t light (Fig. 5) . Activities of the solu-

Table I-Biological activity of geometrical isomers of acitretin to induce differentiation of HL-60 cells

Acitretin

all-trans-Acitretin

9-cis-Acitretin

13-cis-Acitretin

9, 13-di-cis-Acitretin

Induction of differentiation

(EDso, l1M)

0.32

2.3

1.1

2.6

tions of all-trans-acitretin and 13-cis-acitretin in­creased slightly, while activity of solution of 9-cis­acitretin was increased to 1.6-fold by illumination.

Activities of the geometrical isomers of acitretin to induce differentiation of HL-60 cells are summarized in Table I. all-trans-Acitretin showed approximately 3-fold higher activity than 13-cis-acitretin, and 7-fold higher activity than 9-cis-acitretin or 9, J 3-di-cis­acitretin. These results showed that geometrical iso­mers of acitretin formed by photoisomerization under natural light showed remarkably different biological activity on HL-60 cells.

Combined effects of geometrical isomers on their ability to induce differentiation of HL-60 cells are summarized in Table 2. 9-cis-Acitretin acted syner­gistically with other geometrical isomers, i.e. all-

26 INDIAN J. BIOCHEM. BIOPHYS., VOL. 39, FEBRUARY 2002

Table 2- Combined effects of geometrical isomers of acitretin on induction of differentiation of HL-60 cells

all-trans- 9-cis- 13-cis- 9, 13-di-cis- Induction of Acitretin Acitretin Acitretin Acitretin differcntia-

ti on

(0. 1 )lM) (0.7 )lM) (0.3 )lM) (0.7 )lM) (%)

+ 15.5

+ 14.4

+ 11 .7

+ 10.8

+ + 46.3

+ + 29 .1

+ + 28.2

+ + 50.5

+ + 46.6

+ + 20.9

Numeral s indicate the rate of differentiation o f HL-60 cells evalu­ated by NBT-reduction activity

Table 3-Biological activities of geometrical isomers of acitretin to induce synthesis of keratin K I and keratin K 10 of human

epidermal kcratinocyte

Acitrctin KI K IO (%) (%)

all-t ralls-Acitretin 60 53

9-cis-Acitretin 23 25

13-cis-Acitn:tin 17 21

9, 13-di-cis-Acitretin 71 76

Control 100 100

trans-acitretin, 13-cis-acitretin and 9-13-di-cis-aci t­retin. However, all-trans-acitretin, 13-cis-acitretin and 9-13-di-cis-acitretin acted addi ti vely. These results showed that geometrical isomers mutually exerted complicated combined effects on HL-60 cells.

We also investigated the effect of the geometrical isomers on differentiation of normal human epidermal keratinocytes, the target cells of acitretin in therapeu-

tic use. The isomers of acitretin showed different ac­tivities to alter expression of differentiation markers of keratinocytes: 9-cis-acitretin and 13-cis-acitretin showed stronger activity to inhibit expression of K1 and K 10 than all-trans-acitretin or l 3-di-cis-acitretin (Table 3).

Above results indicated that multiple geometrical isomers of acitretin formed by photoisomerization under natural light acted quite differwtly on different cel! types.

Discussion The structure and function of acitretin constantly

varied among its multiple geometrical isomers by photoisomerization reaction under natural light. The velocity of photoisomerization of acitretin (4x 10' 7M/min) under natural light was approximately 1/2 of the velocity of retinoic acid reported previously'. Velocity of photoisomerization is constant under constant condition of illumination (zero order reac­tion). It is assumed that the effect of photoisomeri­zation is dependent on concentration. At concentra­tions of normal organic synthesis of acitretin, effect of photoisomerization is assumed to be low, but is expected to be noticeable at lower concentrations. When concentration of acitretin was in the range of 10-3 to 10.5 M, relative composition of geometrical isomers of acitretin changed rapid ly as the result of photoisomerization, and observed degree of photo­isomerization was inversely proportional to concen­tration. The results presented in this report indicated that velocity of photoisomerization of acitretin under natural light is physiologically quite high .

We showed previously that photoisomerization of retinoic acid was triggered by the absorbed light', and that velocity of photoisomerization of retinoic acid analogs decreased drastically with a change of their side chain conjugation system2

• The side chain struc­ture with tetraenoic carboxyl conjugation system is assumed to be required for responding to natural light causing the unique photoisomerization reactions of acitretin and retinoic acid. Supporting the assumption, all-trans-acitretin showed Amax at 357 nm (£ 41,500), and all-trans-retinoic acid showed "'max at 355 nm (£ 45,300).

Therapeutic efficacy of acitretin is assumed to be greatly influenced by photoisomerization under natu­ral light, since geometrical isomers of acitretin formed by photoisomerization possessed remarkably different biological activity, and exhibited complicated mutual cooperativity.

MURA Y AMA et al.: CONSTANT VARIATION IN STRUCTURE AND FUNCTION OF ACITRETIN 27

Acknowledgement We thank Dr Hidekazu Oyamada of Souzoukagaku

Institute Co. Ltd., Nihonbashi, Tokyo for measure­ments of NMR spectra and for the assignments of sig­nals. We also thank Dr Masanao Matsui for valuable discussions. One of the authors (KSR) is thankful to the Tokyo Municipal Government for providing vis­iting scientist fellowship to undertake this research work.

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minol43 , 167-176

2 Suzuki T, Sridhar Rao K, Matsui M & Murayama A ( 1998) J Nutr Sci Vitaminol 44, 729-736

3 Sridhar Rao K, Suzuki T & Murayama A (2000) Indian J Biochem Biophys 37, 71-76

4 Geiger J & Czarnetzki B (1988) Dermatologica 176. 182-190.

5 Blanchet-Bardon C, Nazzaro V, Rognin C, Geiger J M & Puissant A (1991) J Am Acad Dermatol 24. 982-986

6 Steijlen P M, van Dooren-Greebe R J & van de Kerkhof P C M (1994) Brit J Dermatoll39, 211-214

7 Decker M A & Zimmerman C L (1995) J Chromatogr B 667,105-113

8 Bollag W, Ruegg R & Ryser G (1974) German Patent 2, 414 &619