Biological activity and enzymatic hydrolysis of retinoates in vitro

EUROPEAN

ELSEVIER

European Journal of Pharmaceutical Sciences 3 (1995) 71-76

*JOURNAL

OF

PHARMACE[TICAL SCIESCES

Biological activity and enzymatic hydrolysis of retinoates in vitro M. Salo a,*, j. Kiviranta a, V. Knuutila b, L. Kangas b, H. Vuorela a "Department of Pharmacy, University of Helsinki, P.O. Box 15, FIN-O0014, Helsinki, Finland bOrion-Farmos Pharmaceuticals, Turku, Finland Received 23 March 1994; revised 28 October 1994

Abstract

The enzymatic hydrolysis and biological activity of a series of retinoates, esters of all-trans retinoic acid, were studied. Hydrolysis was conducted in pig liver esterase in aqueous solution. Toxicity was measured by brine shrimp (Artemia salina) bioassay and the anticancer effect by cell culture assay with human cancer ovarii cells and potato disc assay. Retinoates were effective in both tests, but their activities were lower than those of retinoic acid. The effects were related to the rate of hydrolysis. The reaction rates and the biological activities correlated with the molecular size and the retention factor measured by reversed-phase liquid chromatography. Keywords: Biological assay; Hydrolysis; Retinoids; Structure-activity relationship

I. Introduction All-trans retinoic acid (tretinoin, RA) is a major oxidative metabolite of retinol (vitamin A, ROH), and can substitute it in growth promotion and epithelial differentiationand maintenance (Arens and Van Dorp, 1946). The biological significance of RA in the differentiation and maintenance of epithelial tissue has lead to its therapeutic, topical use in skin disorders, such as the treatment of acne (Kligman et al., 1969). RA is effective against psoriasis (Frost and Weinstein, 1969). RA has been effective in inhibiting the growth of different types of cancer cell in vitro (Newton et al., 1980) and also in clinical studies with patients suffering from acute promyelocytic leukemia (Huang et al., 1990), but the use of RA is limited by its narrow therapeutic ratio. A search for an effective retinoid with an improved therapeutic ratio has produced over 1500 re* Corresponding author. Tel. ( + 358-0) 19 13 231. Fax ( + 358-0) 19 12 786. 0928-0987/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved S S D I 0928-0987(94)00076-X

tinoids for pharmacologic studies as recently reviewed by Tallman and Wiernik (1992). The pharmacological properties of retinoids have been measured by cell culture (Sporn et al., 1976; Shudo and Kagechika, 1993) or organ culture (Newton et al., 1980) assays in vitro. The Artemia salina bioassay has been used in the toxicity study of cyanobacteria (Kiviranta et al., 1991), drug and chemical compounds (Lewan et al., 1992; Solis et al., 1993) and extracts of some Ficus species (Mousa et al., 1994). The antitumor effect of the Ficus extracts has also been studied by potato disc assay (Mousa et al., 1994). The activity of cytotoxic drugs has been evaluated in vitro by measuring the bioluminescence of cellular ATP (Kangas et al., 1984; Minn et al., 1991). Pharmacological screening of a large number of compounds involves a considerable amount of laborious and time-consuming research. Quantitative structure-activity or -property relationship (QSAR or QSPR) studies provide a means for the systematic investigation of those characteris-

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M. Salo et al. / European Journal of Pharmaceutical Sciences 3 (1995) 71-76

tics of molecular structure which affect the pharmacological activity or other properties, such as the pharmacokinetics, solubility and reversedphase retention, of the compound. The retention in reversed-phase liquid chromatography (RPLC) is used to estimate relative biological activities within a series of compounds (Kaliszan, 1993a). Esterification of RA alters its biological effects, both the activity and the toxicity. The biological effects of methyl and ethyl retinoates have been reported in the literature (Sporn et al., 1976; Newton et al., 1980; Strickland and Sawey, 1980), but no data on other esters of RA were found. The aims of the investigation were to study the enzymatic degradation and inhibition of tumor cell growth by a congeneric series of retinoates in vitro.

H CH3 CH2CH3 (CH=)=CH3

RA MeR EtR PrR

(CH2)3CH3

BuR

0.541 0.915 1.000 1.119 1.234

(CH2)4CH3 PeR CH(CH=)= IprR CH2C(CH3)3 TbucR

1.357 1.073 1.285

CsH11

ChR

1.386

ROH

0.533

~,,,~CH2OH

Fig. 1. Structures, abbreviations and the retention factor values (log k measured on an octadecyl stationary phase with 86% aqueous methanol as the mobile phase) of the retinoids in the study.

2. Experimental procedures All-trans RA and retinol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Retinoates were synthesized at the Department of Pharmacy, University of Helsinki (Salo and Halmekoski, 1990). The series included five straight-chain esters from methyl to pentyl, isopropyl, t-butylcarbinyl and cyclohexyl esters. The structures and abbreviations used are given in Fig. 1. Retention factors had been measured previously on an octadecyl stationary phase with 86% aqueous methanol as a mobile phase at 40°C (Salo et al., 1993) (Fig. 1).

2.1. Enzymatic hydrolysis Pig liver esterase suspended in 3.2 M ammonium sulphate solution (pH 8) was purchased from Sigma Chemic~al Co. (St.Louis, MO, USA). The hydrolyses were conducted at 37°C in solutions containing esterase solution diluted with phosphate buffer (pH 7.4) to which the ethanolic ester solution was added to give final concentrations of 15 /zM of the ester and 580 IU/ml esterase (1 IU hydrolyzes 1 ~mol of ethyl butyrate per min at pH 8.0 at 25°C) in 1 ml. Samples (0.1 ml) were taken at 1, 2, 4 and 6 hours (at 0.5 h for MeR, EtR and IprR), diluted with 0.9 ml methanol, shaken and centrifuged for

20 min (3000 RPM). After filtration the compounds were chromatographed on a Lichrospher RP-8 column (125 x 4 mm, 5/zm) equipped with a 10 mm guard column (Lichrospher RP-18, 5 /zm) (E. Merck, Darmstadt, Germany) with a mobile phase consisting of methanol w 0.05% acetic acid 90:10 at 40°C, detection at 350 nm. The reactions were monitored for 6 h. The hydrolysis rate constant was calculated using the ester concentration, k~(obs), and retinoic acid concentration, k2(obs), in order to monitor the possible non-hydrolytic degradation of the esters. The rate constants were calculated with concentrations taken from the linear part of the concentration curve, before other degradation reactions were observed.

2.2. Assay of toxicity by Artemia salina The assay was performed by the method described by Kiviranta et al., 1991. The larvae of brine shrimp (Artemia salina) were bred from dry eggs (Sera, Heidelberg, Germany) in saline water (Artemia medium). The retinoids were dissolved in dimethylsulfoxide (DMSO) (10 mg/ml) and diluted with Artemia medium to give solutions containing 100 /zg/ml retinoid in Artemia medium and 1% DMSO. Nunclon flat bottomed spot plates, 400/zl in size, were used as test vials.

M. Salo et al. / European Journal o f Pharmaceutical Sciences 3 (1995) 71-76

50 /zl of Artemia medium containing 10-40 one day old larvae were pipetted into each well. Test solutions were added to give final concentrations of 2, 4, 7, 14, 43 and 83 p,g/ml. Enough medium containing 1% DMSO was added to give a final volume of 350/.tl. The plates were incubated at 22°C in the dark. After 48 h, the number of dead or atypically moving larvae was counted under a stereoscopic microscope. The living larvae were killed by pipetting 50/zl of M e O H into the wells. The total number of larvae was counted on the next day. Saline water containing 1% DMSO was used as control. ECs0 (concentration that affects half of the larvae) was calculated by probit analysis (Finney, 1963).

2.3. Potato disc bioassay Agrobacterium tumefaciens (strain H A M B I 1217), which carries the Ti (tumor inducing) plasmid, was cultured as described by McLaughlin (1991). The samples were prepared by dissolving 4.0 mg of compounds in 1 ml of DMSO. The samples and potato tubers were processed according to McLaughlin (1991). Controls were prepared with pure DMSO. The assay was made with 12 replicates of each compound. The Petri dishes were kept in the dark at 22°C for 14 days. The number of tumors was counted at the end of the incubation period. The differences in the tumor numbers for the controls and t h e retinoates were tested statistically by the paired

73

t-test. Combined results from two separate assays were used in the calculations.

2.4. Cell culture bioassay Retinoates were incubated in solutions containing HOV-030 cells (human cancer ovarii) in RPMI-1640 medium, to which 5% of fetal calf serum was added. The cell line was established from a fresh tumor sample as described by Minn et al. (1991). The compounds were dissolved in DMSO. Concentrations of !, 10 and 100 /zM were tested in triplicate together with a control. The vials were kept in the dark at ambient temperature. The amount of living cells was measured with a luminometer as the amount of ATP after 3 and 5 days of incubation as described by Kangas et al. (1984). IC50 values (concentration needed to inhibit 50% growth) were calculated by regression analysis.

3. Results and discussion

The rate constants of hydrolysis, k~(obs) and k2(obs ), are presented in Table 1. Even though the hydrolysis rates were calculated from the linear part of.the concentration curve, it can be deducted from the rate constants that retinoic acid is degraded in the reaction solutions. The rate of R A degradation is probably slower than

Table 1 Observed rate constants for hydrolysis, k~(obs) and k2(obs ) ( h - l ) , toxicity as ECso in the A r t e m i a salina assay ( - , not determined), and cell growth inhibition as the IC5o in the cell culture assay

MeR EtR PrR BuR PeR IprR TbucR ChR RA ROH

k~(obs) --

k2(obs )

ECso x 10 -6 M

ICso × 10 -6 M

1.86 1.32 0.48 0.37 0.18 1.08 0.33 0.09 -

1.50 1.39 0.25 0.19 0.01 0.28 0.05 -

25 (22-32) 40 (34-49) 73 (56-99) 138 (110-177) 297 (>216) non-toxic 10 (3-13) 420 (>280)

40 (25-65) 47 (39-55) 50 (41-59) 46 (38-55) 53 (50-57) 47 (37-59) 31 (5-92) 49 (44-76)

95% Confidence limits in parentheses (abbreviations of retinoids as in Fig. 1).

M. Salo et al. / European Journal o f Pharmaceutical Sciences 3 (1995) 71-76

74

the hydrolysis rate of the smaller esters. W h e n the hydrolysis rate approaches the degradation rate of retinoic acid, the concentration of the acid due to hydrolysis cannot be measured. The rate constant calculated using the concentration of the acid (k 2(obs)) describes the rate of hydrolysis and acid degradation combined in one constant value, and cannot be used as a measure of the hydrolysis rate. The rate constants calculated by the two m e t h o d s differ considerably when the degradation of retinoic acid is significant. The activity of the e n z y m e was not m o n i t o r e d during the experiments. A possible decrease in enzymatic activity affects especially the results obtained for the m o r e slowly hydrolyzed esters. The hydrolysis rate was not studied as a function of substrate concentration, and the e n z y m e kinetic constants were not m e a s u r e d . The concentration range available for the experiments was limited by the low water solubility of the retinoids. The relative reaction rates of the esters were comp a r e d with each other. At the high enzyme c o n c e n t r a t i o n used the half-lifes, calculated using k l ( o b s ), ranged b e t w e e n 0.37 and 7.7 h. The rate of hydrolysis was highly d e p e n d e n t on the alcohol m o i e t y of the ester. The rate decreased very rapidly w h e n the chain length was increased. T h e toxicity to Artemia salina (as ECs0 ) and inhibitory effect in cancer cell culture (as IC50 ) are presented in Table 1. IC50 values were calculated using nine data points measured at t h r e e concentrations. 95% confidence limits were

calculated and are p r e s e n t e d in Table 1. The results from the potato disc assay are p r e s e n t e d in Table 2. Retinoates were m o r e toxic to Artemia salina larvae than R O H but less toxic than R A . The p o t a t o disc assay indicated a similar trend in the activity of the c o m p o u n d s as the Artemia salina bioassay. The potato disc assay is associated with high variation, and thus the assay can be r e g a r d e d as an activity indicating assay. The inhibitory effect of retinoates on cell growth in the l e u k e m i a cell culture was smaller than that of R A , and about the same magnitude as the effect of R O H . Smaller activities than that of R A have b e e n previously r e p o r t e d for M e R and E t R (Sporn et al., 1976; N e w t o n et al., 1980). A n effective retinoid has to have either a free carboxyl group or a group which can be oxidized to a carboxyl group, and the toxicity and antitumor effects are related (Tallman and Wiernik 1992). The toxicity and the inhibitory effects are possibly related to the rate of hydrolysis of retinoates: an ester which hydrolyzes faster is also m o r e toxic and effective (Fig. 2). The rates of enzymatic hydrolysis of carboxylic acid esters in plasma (Nielsen and Bundgaard, 1987) and the oxidation reaction of simple c o m p o u n d s with a l c o h o l - d e h y d r o g e n a s e (Hansch et al., 1972) increase as the h y d r o p h o b i c nature of the compounds increases (Hansch et al., 1972; Nielsen and B u n d g a a r d , 1987). H o w e v e r , after the hydrophobicity has r e a c h e d an o p t i m u m value the activity decreases (Hansch et al., 1972). The

Table 2 Effect of retinoates on crown gall tumor growth in the potato disc assay x. . . . Control RA ROH MeR EtR PrR BuR PeR IprR

18 12 11 13 12 12 15 20 15

--"

S.D.

n

% change

Significance level

11 10 10 8 8 7 8 7 9

26 25 22 24 23 28 28 28 21

-33 -39 -28 -33 -33 - 17 + 11 - 17

** * *

x . . . . = the average number of tumors per potato disc; mean values were compared with the paired t-test, significance level: ***P<0.1; **0.1 < P < 1.0; *l.0
M. Salo et al. / European Journal of Pharmaceutical Sciences 3 (1995) 71-76

•

"

'

,

.

.

.

75

T

. . ,

EC50

IC50

k I (obs)h -1

(pM)

2

@

200

I00 0.5 50

I

I

0.3

II

0.2 30 I

0.2

I

•

•

•

i

i

,

0.5 1 k 1 (obs)(h - 1 )

I

I

,9

2

Fig. 2. Effect of the rate of hydrolysis, k](obs) (h -1) on the toxicity (Artemia salina bioassay, @, ECso x 10 -6 M) and inhibitory effect on cancer cell growth (cell culture assay, I , IC~0 x 10 -6 M) of retinoates plotted on logarithmic scale.

I

I

I

I

1 1

1,2

I ,3

1,4

i

Iogk

EC50 (~al)

IC50 (i.tM)

•

b

J

200 Q

retinoids are highly hydrophobic compounds, the reaction rates of which probably become smaller when the size of the alcohol moiety of the ester increased. The observed hydrolysis rate constant and the biological effects of retinoids correlated well with the retention in RPLC (Fig. 3a and 3b, respectively). The retention of a homologous series is governed by the hydrophobicity and the size of the molecules (Kaliszan, 1993a), which are probably also the factors affecting the reaction rate. As demonstrated in this work, RPLC can be used in predicting the biological activities of congeneric compounds. Retention data are useful in QSAR and QSPR studies of homologous series of compounds, especially when the activities are dependent on non-specific properties (Kaliszan, 1993b). In conclusion, retinoates were degraded by enzymatic hydrolysis, the rate of which diminished strongly with increasing molecular size. Retinoates exhibited similar cytotoxic activity in vitro as retinoic acid. The activity of the esters was lower than that of the acid, but at least the same as that of retinol. The biological effects were related to the rate of enzymatic hydrolysis

I00

•

50 •

•

I

I

30 I

,9

I

I

I

!

1,1 ,2 Iogk

I

I

I ,3

I ,4

Fig. 3. Dependence of the hydrolysis rate and biological activities on the retention in RPLC as log k (octadecyl stationary phase, 86% aqueous methanol mobile phase) on a semilogarithmic scale: (a) rate of hydrolysis, k](obs) (h-l); (b) toxicity (Artemia salina bioassay, 0 , ECs0 × 10 -6 M) and inhibitory effect (cell culture assay, I , IC50 × 10 -6 M).

of the esters. The results give rise to further pharmacokinetic and pharmacodynamic studies.

Acknowledgements The authors wish to thank Dr. Jyrki Taskinen (Orion-Farmos Pharmaceuticals, Espoo, Finland) for fruitful discussions.

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M. Salo et al. / European Journal of Pharmaceutical Sciences 3 (1995) 71-76

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