Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp. 216-226

Formation of site and conformation specific antibodies to centchroman, a new non-steroidal contraceptive drug

Apurva K Srivastava* and P K Grover

Pharmacokinetics and Metabolism Division, Central Drug Research Institute, Lucknow 226 00 I, Indi a

Received 14 September 1998; revised 28 January 2000

Centchroman [ Ormeloxi fene, 3 ,4-trans-2, 2-di meth yl-3-phenyl-4-(p-pyrrolidino ethox y)phenyl-7 -methoxy-chroman (2a)] is a new non steroidal oral contraceptive for women . In an attempt to develop sensitive immunoassay to measure centchroman concentration in biological fluids, we describe production of antibodies against four hapten deri vatives of centchroman , and evaluation of e fli cacy of these antibodies in developing sensitive and specific immunoassay of centchroman. Antigens were made by coupling four hapten derivatives, 3,4-trans-2,2-dimethyl-3-phenyl-4-(p­pyrrolidinocthoxy)phenyl-7-carboxymcthoxyl-chroman (2c), 3,4-trans-2,2-dimethyl-3-phenyl-4-(p-succinoylethoxy)phenyl-7- methox ychroman (3a), 3, 4-t rans-2,2-di methyl-3-(m-hydroxyl)phenyl-4-(p-pyrro-1 idinoethox y)phenyl-7 -methoxy­chroman (Sa), and 3,4-trans-2,2-d imethyl-3 -phenyl-4-(3' -amino,4' -pyrrolidinoethoxy)phenyl-7-methoxy-chroman (6a), to bovine serum albumin (BSA). Three enzyme tracers, made by coupling centchroman derivatives (2c, 3a, and Sa) to peroxidase (HRP), were utilized to monitor four antisera. Four homologous and heterologous enzyme immunoass:ty formats were developed and speciticity of each antiserum was analyzed against anticipated metabolite of centchroman. An tiserum raised against centchroman derivati ve (6a) was found to be most specific, showing <4% crossreactivity with a putative metabolite, 7-desmethyl centchroman (2b). This antiserum, when used in solid phase EIA format, exhibited sensi tivity of 2SO pg/ml , total assay variance of CY < 14%, and analyti cal recoveries between 82-106%. Comparison of the EIA with a RIA, which was developed by using same antibody, showed a close correlation (r=0.9, n=40). Specificity of all an tisera, as determined by the respect ive enzyme immunoassays, followed an uncharacteristic pattern of crossreactivity towards cis centchroman. Especially, antiserum gener::tted against an immunogen (Sb) showed high crossreaction with cis centchroman. Evidence provided in th is study showed that immunogen (Sb) produced a sub population of antibodies to a flipped conformation of the drug, which conformationally looks similar to cis centchroman. These conformation specific antibodies accounted for high crossreactivity towards cis centchroman. In conclusion, we describe the formation of conformation specific antibodies and the significance of site of attachment of centchroman derivative to carrier protein on the specificity of antibodies towards anticipated metabolites. Furthermore, by using antiserum generated in thi s study, sensitive immunoassays were developed and validated for the measurement of centchroman in serum.

Centchroman 1 [Ormeloxifene, 3,4-trans-2,2-dimethyl-3-phenyl-4-(p-pyrrolidino ethoxy) phenyl-7-methoxy­chroman (2a)] ts a new non steroidal, ora l contraceptive for women. This drug is currently marketed in India. In clinical practice, centchroman is recommended 30 mg biweekly for 12-weeks followed by 30 mg once a week. Its pharmacokinetic studies in the normal female human volunteers showed the biological half-life as seven days2

. There was no evidence of any metabolites in this study. However, in a recently published3 study, presence of 7-

*Author for correspondence at the following address: Musculoskeletal Di sease Center & Mineral Metabo lism Divi sion , Jerry L Pettis VA Med ical Center, 1120 I Benton Street , Loma Linda, CA 923S7. Tel. 909-82S-7084- ext. 1827; Fax: 909-796-1680; E-mail: apurva.srivastava @mcd. va.gov

desmethyl centchroman is indicated based upon HPLC data. T-his however needs confirmation. The HPLC assa/ used for the pharmacokinetic study had a sensitivity of 2 ng/ml requiring large amount of serum. To improve the sensitivity of detection and to simplify the measurement of the drug in human serum, development of immunoassay of centchroman was initiated.

As most of the drugs are non-immunogenic, they must be covalently coupled to large protein molecules to produce antibodies. The site of the drug molecule used for such linkage is known to influence the specificity 14

·15 of antiserum. Although a non-specific

antiserum has its own advantages, such as , an antiserum with high crossreactivity with metabolites of a compound can be used to quant itate all the crossreacting compounds, using same an tibody if the

SRIVASTAVA & GROVER: FORMATION OF ANTIBODIES TO CENTCHROMAN 217

metabolites can be separated by chromatographic techniques and assayed4 separately. However, the study was aimed at developing specific antibodies, which can permit use of straight serum in the assay without any cumbersome sample preparation and chromatographic purification 11 steps. In a new compound like centchroman, it is difficult to predict the response of a particular immunogen in terms of crossreacttvtty of the antibodies to possible metabolites. Therefore, antibodies were generated against conjugates made at four different sites of the drug molecul e. Three of the haptenic sites used for conjugation to carrier protein in volved positions that were farth est from the ring-A of centchroman. The rationale was to expose ring-A for antibody production, thus minimizing the crossreactivity of the antibodies with 7-desmethyl centchroman (DMC), a putative metabolite of centchroman. The fourth conjugate was involved in linkage to carrier protein at 7-position of centchroman and it was anticipated that this will produce antibodies that will show hi gh cross reactivity with DMC, thus enabling us to develop an immunoassay spec ifi c for the measurement of DMC in serum. Earlier we reported5

·6 synthes is of hapten

derivatives of centchroman , and BSA conjugates : I ) 3, 4-trans-2, 2-dimethyl-3-phenyl-4-(p-pyrrol idinoeth­oxy)phenyl-7-carboxymethoxylchroman-BSA (2d); 2) 3, 4-trans-2, 2-d i methyl-3-pheny 1-4-(p-succ i noy leth­oxy)phenyl-7-methoxychroman-BSA (3b); 3) 3,4-trans-2, 2-d i methyl-3-(m-hydrox y I )phenyl-4-(p-pyrro­!idinoethoxy)phenyl-7 -methoxychroman-BSA (5b) (linkage through methylene at para posit ion by Mannich condensation)]; and 4) 3, 4-trans-2, 2-dimethyl-3-pheny l-4-(3' -amino,4' -pyrro lid inoethox y) phenyl-7-methoxychroman-BSA (6b) (linkage via glutaraldehyde). In this study, we describe production of antibodies against these conjugates and charac­terization of antisera using three horse radi sh peroxidase (HRP) tracers . We used three different enzyme tracers because it has been reported that sensitivity and in some cases specifici t/ 2

·15 of the

immunoassays are influenced by site of conjugation of hapten derivative to enzyme tracer. Synthesis of two hapten derivatives used for making HRP conjugates, 3, 4-trans-2, 2-dimethyl-3-phenyl-4-(p­pyrrolidinoethoxy) phenyl-7-carboxymethoxy-chro­man (2c), and 3,4-trans-2, 2-dimethyl-3-phenyl-4-(p­succinoylethoxy) phenyl-7-methoxychroman (3a) have been reported5

·6 earlier. Synthesis of the third

hapten derivati ve, 3, 4-trans-2, 2-dimethyl-3-(m-carb-

oxymethoxyl) phenyl-4-(p-pyrrolidinoethoxy) phenyl-7-methoxychroman (Se), and HRP conjugate (5c) is described in this study.

A low complexity, double-antibody (liquid-phase) based enzyme immunoassay format was used for initial evaluation of all four antibodies with the three enzyme tracers (2e, 3c, and 5c). Only those assays, which produced high signal and low background noise were followed for further evaluation of sensitivity and specificity. Once an antibody and enzyme tracer producing the des ired specificity was identifi ed by liquid phase EIA format , more robust solid phase EIA and RIA were developed. In the absence of human control serum, analytical performances of these immunoassays were evaluated by using rat serum supplemented with known amounts of Centchroman .'

Materials and methods

Materials

All organic solvents were of laboratory grade and were purified before use. Sephadex G 25, bovine serum albumin (BSA), horseradish peroxidase (HRP) (E.C. I.ll.l.7, Type VI), Freund's complete adjuvant (FCA) and a-phenylenediamine tablets (30 mg) were all obtained from Sigma Chemical Co. St. Louis. MO, USA. Tri-n-butyl amine, isobutyl chloroformate were from Jansson Chemical Co., USA. Monosodium and disodium phosphate and ammonium su lphate were of analytical grade from Qualigens, India. Radioiodine Nal 125 (carrier free) was purchased from Bhabha Atomic Research Centre (BARC) Mumbai, India. Polystyrene tubes (RIA tube Cat#8500 I 0) were from Tarsons, India. Antirabbit gamma globulin antiserum was generated in goats in our laboratory.

Instruments Melting points were determined on an electrically

heated block and are uncorrected. Ultraviolet and visible spectra were recorded on Kontron Uvicon 810 spectrophotometer, infrared (TR) spectra were recorded on Pye Unicham (wave numbers are given in em -1

), PMR spectra were recorded on R-32 Brucker or CFT-20 Varian NMR spectrometer using TMS as internal standard [chemical shifts (8) are given in ppm] and mass spectrum on a Joel JMS-D-300 instrument (m/z values are given agai nst their relative intensities).

218 INDIAN J BIOCHEM BIOPHYS, VOL. 37, AUGUST 2000

Synthesis of hapten, BSA conjugates, HRP and radio labeled tracers

Structures of immunogen (2d, 3b, Sb, and 6b), and enzyme tracers (2e, 3c, and 5c) are shown in Fig. I. Purity of all derivatives synthesized in this study was based on TLC (precoated sil ica gel-60, 5 em x 20 em from E. Merck, Germany) and spectral investigation. Synthesis of BSA conjugates have been described in

d o 56 h o d prece tng · papers, enzyme tracers were synt es1ze by mixed anhydride7 method.

1

H3C ' O

H3C '0

3 a:R=OH b:R=BSA

c:R=:~o

6 5 a:R1=H;R2=0H;

b:R1=CH2NH-BSA; R2=0H

c:R1=H; R2=0CH2CONH-HRP

d:R1=0H;R2=125

1

e:R1=0CH2COOH

f :R1=H;R2=0CH2COOEt

Synthesis of 3, 4-trans-2, 2-dimethyl-3-(m-ethoxycar­bonylmethoxy)phenyl-4-(p-pyrrolidino-ethoxy) phenyl-7-methoxychroman (51)

To a solution of 3,4-trans-2, 2-dimethyl-3-(m-hy­droxy )pheny 1-4-(p-pyrrol idinoethox y )phenyl-7 -meth­oxychroman (Sa) (0.845 mM) in dry dimethyl su lfoxide (5 ml) was added sodium hydride ( 1.0 mM) and reaction mixture was stirred for 30 min . Ethyl bromo acetate (0. 11 ml, I mM) was added and stirring continued for another 18-20 hrs, the reaction

R '0

H3C ' 0

H3C ' 0

2 a: R=Me; b:R=H

c: R=CH2COOH

d: R=CH2CONH-BSA

e: R=CH2CONH-HRP

OH

6 0

6 a:R= NH 2

b:R= N=CH-(CH2) 2 -CH=N-BSA

Fig. !-Structure of hapten derivatives. imrnunogens. enzyme tracers. and radioactive tracer of centchroman.

SRIVASTAVA & GROVER: FORMATION OF ANTIBODIES TO CENTCHROMAN 219

mixture was poured on to ice (I 00 ml) and extracted with ethyl acetate. Organic layer was separated, washed with water and saturated saline solution, and dried over sodium sulfate. Ethyl acetate was removed under reduced pressure and the product (Sf) was purified by preparative thin layer chromatography (silica gel). Yie ld, 4SO mg; m.p . SO-SS°C; IR, 1740 (C=O of carboxyl), 1610, IS80; PMR (CDCI 3), I .23 (m, 6H, CH3, CH2CH,), 1.36 (s, 3H,CH3) 2.03 (m, 4H,CH2CH2), 3.19 (m, 6H,N (C!:hh), 3.7S (s,3H,OCH3), 4.2(q,2H,CH2CH}), 4 .S4 (s, 2H,OCH2), 6.3-7.2(m,IIH, Ar-H); Chemical Ionization Mass : S61 ( 16) (M), S62 (7) (M+ +I), S63 (2) (M+ +2) , 228(12), 229(6), 230 (3), 98(40), 84(100).

Synthesis of 3,4-trans-2,2-dimethyl-3-(m-carboxy­methoxy)pheJty !-4-(p -pyrrolidinoethoxy)phenyl-7-methoxychroman (5e)

A solution of ester derivative (Sf) (60 mg, 0.106

mM) in methanol (4 ml), IN sodium hydroxide (0.0 16

ml , O.IS9 mM) was stirred for three hours at room

temperature. Methanol was removed under reduced

pressure, ice added and solution was made neutra l

(pH 7.0). Solid was filtered, washed with water and

dried . Yield, 46 mg; m.p. 136-40°C; IR, 2960, 1600,

IS80; PMR (CDCb), l.l9 (s , 3H,CH3), 4.4 (s ,

2H,OCH2); Chemical Ionization Mass : 487 (S) (M+­

C02), 488 (2) (M+ +I -C02), 227(6), 97(20), 84( I 00).

Synthesis of HRP conjugates (2e , 3c, and 5c) To a cool ( -IS0 C) mixture of 2c (9.29 mg, 0.0 17S

mM) and tri-n-butylamine (4 .8 ).ll , 0 .2 1 mM) in dry

DMF (400 ).1.1) was added isobutyl chloroformate

(2.33 ).ll, 0.017 rn:\1) under stirring and the reaction

was allowed to proceed for 30 min . The mixed

anhydride was added to a solution of HRP (I 0 mg,

0.0002S mM) dissolved in a mixture of water:DMF

(600 ).1.1 :200 f-ll) and 0.1 N sodium hydroxide (0.0 I

ml) with constant stirring for 4 hrs at -IS0 C. After

completion of reaction the conjugate was dialyzed

against phosphate buffer (0.0 I M, pH 7 .0) for 48 hrs

and then purified on Sephadex G-2S column

(I cmxSO em) . The fractions with maximum

immunoreactivity were aliquoted , freeze-dried and

stored at 4°C until further use.

The other HRP conjugates (3c and Sc) were

synthesized by the same procedure used for making

2e.

Preparation of 3,4-trans-2,2-dimethyl-3 -(m-hydro.xy) phenyl-4-( p-py rroidino-e thoxy )phenyl-7 -methoxy­chroman-1251 tracer ( 5d)

A solution of 3, 4-trans-2, 2-dimethyl-3-(m-hydr­oxy)phenyl-4-(p-pyrrol idinoethoxy) phenyl-7 -meth­

oxychroman (Sa) ( 1.0 J-lg, dissol ved in 0.02S ml 0.2S moll! sodium phosphate buffer, pH 7 .8) was mixed with 0.020 m1 of Na 125 I (I mci , 0.2S moll! phosphate buffer, pH 7.8) . Iodination was initiated by addition

of 20 ~Lg of chloramine-T (0.010 ml of 0 .2S sodium phosphate buffer, PB) to the above solution, with continuous stirring. The reaction was stopped after

I 0 min by addition of 20 ).lg of sodium metabi sulfite in 0.020 ml of 0.2S M PB. The reaction mixture was diluted by addition of 0.2 ml of water and extracted

with 3x0.2 ml of ethyl acetate. The ethyl acetate layer was separated , dried over anhydrous sodium sulfate, concentrated under stream of nitrogen, and purified by thin layer chromatography (precoated silica gel, 60x20 mm). The thin layer plate (TLC) was developed in chloroform: methanol: ammonia (9:0.9S:O.OS, rf-0 .7) solvent system. A band corresponding to 3,4-trans-2 , 2-dimethyl-3-(m-hydr­oxyl)phenyl-4-(p-pyrrolidinoethoxy) phenyl-7 -meth­oxychroman (Sa) was scraped from TLC plate, eluted with ethyl acetate, ethyl acetate removed under nitrogen stream and tracer was redissolved in ethanol and kept at -20"C in ethanol so lution until further use.

Antiserum Antibodies were raised in female white Belgian

rabbits. For each immunogen a group of three rabbits were immunized by intradermal multisite injections8

with approximately I mg of conjugate (equivalent to 40 ).lg of drug) emulsified in Freund ' s complete adjuvant (FCA). After 4S days, each rabbit received an intramuscular booster of I mg immunogen in FCA. Further boosters were given at monthly intervals (in incomplete Freund 's adjuvant) and bleedings were collected from central ear artery IS days after each booster injection . Serum after third bleeding was used for titer and specificity studies .

For simplicity, antisera raised against immunogens 3b, 6b, and Sb were labeled as AS-I, AS-2, and AS-3, respectively. Titer of antiserum generated against immunogen 2d was extremely low with all three­enzyme tracers used in this study, consequently, it was excluded from further analysis.

For the development of solid phase assay, antiserum AS-2 was further purified by affinity

220 INDIAN J BIOCHEM BIOPHYS, VOL. 37, AUGUST 2000

chromatography. The gamma globulin fraction of antiserum AS-2 was precipitated at room temperature by addition of 33% ammonium sulfate (w/v), the precipitate was dissolved in equal volume of sodium chloride (I 54 mmol/1) and the process was repeated once more. The dissolved precipitate was dialyzed against 0.0 I mol/1 phosphate buffer, pH 7 .0 for 24 hrs at 4°C. The dialysate containing JgG fraction was incubated for I hour with DEAE-cellulose at room temperature (DEAE-ce llulose previously washed and equilibrated with 0.0 I mol/1 phosphate buffer). The unabsorbed fraction was passed through a Sepharose-4B-BSA column, prepared by known methods3 to absorb out anti-BSA antibodies.

Liquid phase enzyme immunoassay (E!A) Liquid phase EIA was performed in 12x75 mm

glass tubes. Different concentration (0.2 ml) of centchroman standards (31 .25 pg to I 000 pg/ml solution prepared in 0.1 M phosphate buffer containing 0 .9 M sodium chloride and 0.5% BSA, pH 7.0, PBS/BSA) , 0.1 ml of HRP conjugate (diluted in PBS/BSA) and 0.1 ml antiserum (appropriately diluted in PBS/BSA) were mixed and incubated overnight at 4"C. Goat antiserum against rabbit gamma globulins (0.075 ml diluted I : I with PBS/BSA) and normal rabbit serum (0.025 ml of I :4 diluted with 0.1 M PBSIBSA) were added and after a brief mixing incubated overnight at 4°C. After completion of the second incubation period, 2.0 rnl of PBS/BSA was added to each tube and centrifuged at 3000g at 4°C for 30 min . The supernatant was discarded, precipitate washed with I ml PBS/BSA and finally suspended in 0.4 ml PBS/BSA. Freshly prepared substrate buffer (2 rnl per tube, prepared by dissolving 0.06 g of a-phenylenedi amine, 0.025 ml of hydrogen peroxide (30%) and 3.3 g ammonium sulfate in 100 ml 0 . 1 M phosphate buffer, pH 7.0) was added to each tube and incubated for 4 hours at 4°C. Reaction was terminated by addition of 6 M sulfuric acid (0.6 ml) and absorbance measured at 492 nm.

Solid phase enzyme immunoassay ( EIA ) Polystyrene tubes ( 12x55 mm) were coated with

affinity-purified anti-centchroman IgG fraction of rabbit antiserum AS-2 . Antibody solution 400 J.ll (0.5 J.lg/tube in 0.1 rnol/1 carbonate buffer, pH 9.5) was dispensed in each tube and incubated overnight at 4°C. Unabsorbed antibodies were washed off with distilled water containing 0.05 % Tween-20. The

remaining protein binding sites were blocked by coating with 0.5 ml of BSA, I 0 g/1 in 0 .0 1-mol/1 phosphate buffer, for one hour at 3rc. Assay tubes were then washed three times with distilled water and used immediately.

The EIA was initiated by addition of 0.1 ml of

centchrornan standards (9 .32-300 pg/tube in 0.0 I mol/1 phosphate buffer containing 0.9% sodium chloride and 0.5 % BSA, assay buffer) or serum extracts (0.05 ml of rat serum spiked with centchrornan was diluted to 0.1 ml with distilled water, extracted with 3 ml of ether, and ether extracts were reconstituted in 0 .25 ml of assay buffer), and 0.2 ml of enzyme label 3c (diluted to I :200,000 with assay buffer) were added to the antibody coated tubes in triplicate and incubated overnight at 4°C. The contents of the tubes were decanted and tubes were washed thoroughly with distilled water. HRP activity was measured by the method described for liquid phase EIA after one-hour incubation with substrate at room temperature.

Radioimmunoassay (RIA) Pooled normal rat serum spiked with centchroman

was used for preparing the serum standards ranging from 625 pg/ml to 20 ng/ml. 0.05 ml of serum standard or unknown sample was diluted with 0.95 ml of 0.0 I mol phosphate buffer conta ining 0.89% sodium chloride and 0.5% BSA (assay buffer) and heat inactivated at 60°C for 30 min (extraction of serum samples was not required) . After cooling 0.2 ml of this diluted serum was used for measuring centchroman concentration. The assay was performed in glass tubes ( 12x55 mm) to wh ich 0.2 ml of different concentrations of centchroman standard, 0 .1 ml of purified anti-centchroman IgG fraction (AS-2, diluted I :2000 in assay buffer), and 0 .1 ml of centchroman tracer (5d) (reconstituted in assay buffer) were added and incubated overnight at 4°C. Normal rabbit serum 0.025 ml (diluted 1:4 in assay buffer) and goat anti rabbit gamma globulin antiserum 0 .075 ml (diluted I :2 in assay buffer) were added to all the tubes and further incubated for 16-20 hrs at 4°C. After addition of I ml of assay buffer, all the tubes were centrifuged at 3000 g for 15 min at 4°C. Supernatant was discarded and precipitate counted for a minimum of l min in a Phillips Raytest y-counter.

SRIVASTAVA & GROVER: FORMATION OF ANTIBODIES TO CENTCHROMAN 221

Table !-Binding characteristics of liquid-phase EIA formats

Antiserum Label Standard curve range Sensitivity Affinity (Titer) (Dilution) (pg/tube) (pg/tube) constant

AS-I 3c 31.25-1000 31.25 2.4 X 109 UM

(I :2400) ( I: 100000)

AS-2 3c 31.25-1000 31.25 2.4 X 109 UM

(I :800) ( I : 100000)

AS-3 Sc 62.5-1000 62.5 2.4 X 109 UM

(I: 1600) (I :200000)

AS-3 2e 31 .25-500 31.25 2.4 X 109 UM

(I :4000) (I: 100000)

Table 2-Crossreactivity of three antisera against centchroman determined by liquid-phase EIA format

Crossreacting compounds

Centchroman (2a)

Cis centchroman (I)

7-Desmethyl centchroman (2b)

3,4-trans-2,2-dimethyl-3- phenyl-4-(p-hydroxyl)phenyl-7-methoxychroman (4)

Results Liquid phase assay

AS-I & Label 3c

100

<1.0

9.1

52.8

Binding characteristics of all the antibodies used in the liquid phase assays are summarized in Table I. Affinities of antibodies were calculated by plotting the Scatchard plot. Calibration curves of the liquid phase EIA are shown in Fig. 2. Table 2 summarizes the specificity of all the antisera evaluated with one possible metabolite, DMC and three other related compounds, cis centchroman (I), and 3, 4-trans-2, 2-dimethyl-3-phenyl-4-(p-hydroxyl) phenyl-7-methoxy­chroman (4) . Crossreactivity was calculated by com­paring 50% inhibition of labeled centchroman by crossreacting compound [% crossreactivity=(con­centration of centchroman at 50% inhibition of binding at zero concentration of centchroman/concen­tration of crossreacting compound at 50% inhibition of binding at zero concentration of centchroman) x 100] according to the method described earlier9

(Table 2). Crossreactivity of centchroman was arbitrarily set at I 00 per cent.

Solid phase EIA

Limit of detection

The solid phase EIA was developed by usmg

purified antiserum AS-2, and HRP tracer 3c. The

smallest amount of centchroman concentration that

Percentage crossreactivity AS-2 & Label 3c AS-3 & Label 5c AS-3 & Label 2e

100 100 100

<1.0 46.8 51

6.9 90 178

13.4 <1.0 <I~

1.25

1.00

0.75 c 0

0.50

0.25

0.00 10 100 1000

Centchrornan concentration (pg/ml)

Fig. 2-Standard curves of liquid-phase enzyme immunoassays [(a), 0-0 antiserum AS-1 and enzyme label 3c; (b), *-* antiserum AS-2 and enzyme label 3c; (c), • - • antiserum AS-3 and enzyme label Sc; and (d), 0--0 antiserum AS-3 and enzyme label 2e]

can be distinguished from 0' dose by more than 2SD

values was <10 pg/tube, and the standard curve range

was set at 9.3-300 pg/tube. Taking in account the

extraction procedure, the sensitivity of the EIA was

250 pg/ml of serum. The 50% intercept of the

standard curve was at 75 pg/tube. A graphical

representation of standard curve (B/Bo vs log

concentration) obtained by solid phase assay is

illustrated in Fig. 3a.

222 INDIAN J BIOCHEM BIOPHYS, VOL. 37, AUGUST 2000

Specificity Table 3 li sts crossreactivity of AS-2 in solid phase

EIA format. The specific ity was determi ned by the method described for liquid phase ErA. Crossreactivity with centchroman was arbitrarily set as 100%. Each value represents mean of two measurements.

1 00,-----------------------------------~

0 0 ~

X 0

[lJ

80

60

40

20

0 1

[ij 100

75

50

25

(A)

o (B)

10 100 1000

Centchroman concentration (pg/tube)

10 1 0 1000

Centchroman concentration (pg/ml)

Fig. 3-Calibration curves of (A): solid-phase EIA usi ng antiserum AS-2 and enzyme label 3c and (B) RI A that utilizes anti serum AS-2 and radioiodi nated centchroman derivative Sd . [Error bars represent standard error of mean at each concentration (n=5) . In y-axis, ' B' represents binding of the enzyme or radio labeled tracer in the presence of ccntchroman, and Bo represents binding at zero concentration of ccntchroman]

Recoveries The percentage recoveries were calcul ated from

centchroman sp iked rat serum at three different concentrations: 600 (low) , 1200 (medium) and 2400 (high) pg/ml. Mean recoveries ranged between 83-93% (Table 4) .

In tra- and inter-assay precision Three spiked serum pools were used to study the

with in and between assay coeffic ien t of variance (CV) . CV ranged between 6-1 4% (Table 5).

Centchroman spiked serum sample was diluted 2 to 8 folds and stud ied along with zero standard . The diluti on curve and standard curve were parall el to each other (data not shown).

Recoveries in the presence afmzticipated metabolites Pooled rat serum was supplemented with

centchroman ( 1200 pg/ml), and its putative meta­bolites 7-desmethylcentchroman (2b) ( 1200 pg/ml) and 3, 4-trans-2,2-d imethyl-3-phenyl-4-(p-hydroxyl) phenyl-7-methoxychroman (4) ( 1200 pg/ml) . The spiked serum was then measured as unknown sample to evaluate the interference 111 centchroman

Tabl e 3-Specificity of centchroman anti body (AS-2) as determined by solid ph ase EIA

Crossrcacti ng compou nds

Centchroman (2a)

Cis Ccntchroman ( I)

7-Desmcthyl Centchroman (2b)

3,4-/rans-2,2-dimethyl-3-phenyl-

4-(p- hydroxyl)-7-methoxychroman (4 )

% Crossreactivi ty

100

< 1.00

3.42

1.40

Tabl e 4-Recoverics of rat serurn. spiked with centchroman by enzyme immunoassay

Centchroman added to pooled rat serum (pg/ml )

600

1200

2400

% Recovery (Mean ±S D)

86 .7 ±8.5

92.9±7.5

92.7±5.7

Table 5-Intra- and interassay variation of solid phase EIA and RIA

Concentration n %Coefficient of variance (CV ) (pg/ml) EIA RIA

Intra-assay 650 5 8. 1 10.6

1200 5 I 1.8 14.9

2400 5 7.1 12.2

Inter-assay 650 5 9.9 7.4

1200 5 14. 1 I 1.5 2400 5 5.9 12.2

SRIVASTAVA & GROVER: FORMATION OF ANTIBODIES TO CENTCHROMAN 223

Table 6-Crossreactivity of antisera determined by radioimmunoassay using radio labeled centchroman (5d)

Crossreacting compounds % Crossreactivity

Centchroman (2a)

Cis Centchroman (I)

7-Desmethyl Centchroman (2b)

3 ,4-t rans-2 ,2-d i methyl-3-phenyl-

4-(p-hydroxyl)-7-methoxychroman (4)

measurement, in the presence of these metabolites . Presence of these compounds caused only marginal increase ( <6%) in centchroman concentration.

Radioimmunoassay Binding characteristics

The Centchroman- 125I tracer (5d) showed >90% immunoreactivity at I : 100 dilution of antiserum (AS-2). The non-specific bindings were <5.0% when approximately 5000-10000 cpm were used. Titer of the antiserum corresponding to about 30% binding of the radioactive centchroman tracer was l :2000. Since direct serum was used in the RIA, standard curves were generated in rat serum spiked with centchroman between the concentrations 625 pg/ml to 20 ng/ml.

Analytical characteristics The sensitivity of the RIA was 625 pg/ml of serum.

The crossreactivity of RIA, evaluated by the method described for EIA, is presented in Table 6. Spiked recoveries, determined by using rat serum spiked with centchroman at three different concentrations 625, 1250, and 2500 pg/ml , ranged between 96 .8 to I 02.56% (Table 7) . The intra- and inter-assay determined at three concentrations of centchroman, shows CV < 15% (Table 5).

Correlation between solid phase EIA and cent­chroman radioimmunoassay

In 40-control rat serum samples (supplemented with centchroman in concentration range of 300 pg/ml to 20 ng/ml) centchroman was measured by solid phase EIA and radioimmunoassay. Comparison of the values obtained by the two methods showed a strong correlation with r=0.9 (p<O.OO I) .

Discussion I · II k h · · f h h 12 14 15 t ts we nown t at posttton o t e apten · · ·

used for conjugation to earner overwhelming influence on the antisemm. The most exclusively

protein have specificity of studied small

AS2 AS3

100

<1.00

3.15

<1.00

100

3.50

36.0

< 1.00

Table ?-Recoveries of rat serum spiked with centchroman by radioimmunoassay

Centchroman added to pooled rat serum (pg/ml)

625

1250

2500

% Recovery (Mean ±SO)

102.6 ± 7.7

96.8 ± 11.1

97.6 ± 11.9

molecules are steroid hormones. Derivatives of several steroid molecules at virtually all-possible postttons were prepared and antibodies were generated against these derivatives. In some cases, very specific antisera could be generated, which permitted straight quantitation of the hormone in straight semm. There is no general mle where one could predict the specificity of the antibodies to a small molecule. However a few attempts have been made to correlate antibody specificity and stmcture of steroid hapten derivatives. One such hypothesis is based on the observation that conjugates made at farthest position of the molecule, towards which the specificity is required, gives the most specific antibodies. In an attempt to mtmmtze the crossreactivity of centchroman antibodies with DMC, which could be an active metabolite of centchroman, we used centchroman derivatives that had linkage to BSA that was farthest from the ring-A, thus, maximally exposing ring-A of the centchroman molecule for antibody production. It was estimated that the antibodies produced against such immunogens would show maximum specificity towards ring-A, consequently avoiding any cross reactivity towards DMC.

Three HRP tracers were used to monitor four antisera. All three tracers showed different immunoreactivities towards different antisera, and only those formats were pursued for assay development that gave a reasonably high signal and low background. In the liquid phase EIA screening, antiserum AS-I and AS-2 showed mmtmum

224 INDIAN J BIOCHEM B!OPHYS, VOL. 37, AUGUST 2000

crossreactivity towards DMC. We selected AS-2 for solid phase development due to the higher cross reactivity of AS-I towards 3,4-traHs-2,2-dimethyl-3-phenyl-4-(p-hydroxyl)phenyl-7-methoxychroman ( 4). The anti serum AS-2 was further purified by affinity chromatography in order to develop so lid phase EIA and RIA . The purified antibody, showed improved specificity in terms of crossreactivity towards DMC (3.4%) and 3, 4-trans-2 , 2-dimethyl-3-phenyl -4-(p­hydroxy)phenyl-7-methoxychroman ( 1.4%) (Table 3). Both solid phase EIA and RIA, exhibited sati sfactory analyt ical performance in terms of sensitivity, reproduc ibility, and analytical recoveries, and could be used for measurement of centchroman in human serum with minor modi fications.

To monitor antiserum rai sed against Sb, two different enzyme labe ls, one at 3-phenyl ring 3, 4-trans-2, 2-dimethyl-3-(m-carboxymethoxy) phenyl -4-(p- pyrrolidinoethoxy) phenyl-7 -methoxychroman­HRP (5c), and other one, a site heterologous label at ?-position 3, 4-trans-2, 2-dimethyl-3-phenyl-4-(p­pyrro l idinoethox y )pheny 1-7 -carboxymethox ychroman -HRP (2e), were used. The antiserum showed very high crossreacti vity with 7-desmethyl centchroman using both enzyme labels 2e and Sc. Since 2e was made at ? -posi ti on, high crossreactivity of AS-3 towards DMC was somewhat ex pected because of the masking of 7-hydroxyl group . However, hi gh crossreact ivity of thi s anti serum with DMC when used with enzyme labe l Sc was intriguing. The only reason we can attribute for thi s crossreactivity is that 7-desmethyl centchroman can mimic the recognition of m-hydroxy l group in 3, 4-trans-2 , 2-dimethyl-3-(m-hydroxyl) phenyl-4-(p-pyrrolidi noethoxy)phenyl-7-methoxychroman (Sa). Hi gh crossreactivity of AS-3 with DMC may not permit use of direct serum, if it is indeed shown as a metabolite in humans . However, given a chromatographic separat ion method to separate DMC and centc hroman , this antibody can be used fo r the quantitation of DMC in biologica l fluid s. The unexpected high crossreactlVlty with cis centchroman was an interest ing observation, and , required more detailed analysis.

To explain the crossreactivity of AS-3 towards cis centchroman, following immunoreactivity patterrr were taken into consideration (Table I) : (i) When enzyme labe l was made with long spacer arm, such as enzyme label 3c, it showed very poor binding with the antisera AS-3. However, when the enzyme labels were made with short spacer arm, such as 2e and Sc,

they showed very poor binding to antisera AS-I and AS-2 . (ii) In EIA using enzyme labe l 3c and antisera AS-I and AS-2, there was no crossreactivity with cis centchroman ( 1) . The important feature was that the attachment of BSA and HRP to the p- position of 4-phenyl ring of centchroman was through an eight or s ix atom spacer. (iii) In heterologous assays, using enzyme tracers Sc or 2e and antiserum AS-3, the cross reaction with cis centchroman was about 50%. In both these cases, the linkage of BSA in immunogens Sb and 2d was via one-atom spacer at p­

position of 3-phenyl ring of centchroman or three­atom spacer in ring-A.

Based upon X-ray crystallographic 17 analysis the following abso lute conformation of centchroman was proposed: a) the pyran ring assume a quasi chair form; b) one of the methyl groups at C-2 takes an axial conformat ion; and c) the orientati on of the phenyl groups at C-3 and C-4 is trans-diequ atorial and these phenyl groups are in a plane perpendicu lar to the chroman ring. Using a computer program ChemSkech (ACD, Toronto, Canada) the three dimensional structures of trans centchroman , flipped forms of trans centchroman (Fig. 4b), and cis centchroman (Fig. 4c) cou ld be drawn which reveals some interesting correlati ons. The three dimensional structures of trans centchroman agrees in all respects with its absolute structure determined by X-ray crystallographic 17 analysis. The pyran ring in flipped trans form (Fig. 4b) and cis form (F ig. 4c) takes a quasi boat structure: (i) one of the methyl groups at C-2 axial (be low the pl ane) and the phenyl groups at C-3 and C-4 assume a trans diaxial conformation ; and (ii ) the phenyl ring at C-4 in the fli pped trans form and cis form are in a plane perpendicular to ring-A. Us ing the Dreiding mode ls of centchroman, similar observations were made.

In immun ogen 3b and label 3c the linkage to BSA and HRP is via 8-atom spacer and in immunogen 6b the linkage to BSA is via 6-atom spacer, so they re tain the conformation proposed fo r centchroman. However, there could be a si tuat ion, where in immunogen Sb, two population of conjugates can exi st, one with trans-diaxi al ;md the other with trans­diequato rial conformation of the two phenyl groups at C-3 and C-4, depending upon the lys ine residues on BSA which are occupied by the centchroman analog Sa. For example, if the molecul e is attached to lys ine in a relatively unhindered position then it may retain the origina l diequatorial conformation. If, however,

SRIVASTAVA & GROVER: FORMATION OF ANTIBODIES TO CENTCHROMAN 225

.... c (A).

' HO

...... ~o ( 8)

HO

H3C-......._O~

Fig. 4-Structures of trans centchroman in quasi-chai r conformation (A ), flipped quasi-chair conformat ion (B), and cis centchroman (C) [In order to clearly show the changes in conformation due to flipping of ring-8, pyrrolidinoethoxy side chain in ring-C was omited, and st ru ctures are visual ized at an angle parallel to the plane of ring-A]

the lysine residue is in a hindered position then the analog Sa, to be able to fit in the crevice of BSA molecule, may flip over to trans-diaxiai conformation with ring-B taking a boat conformation . These two populations of conjugates can produce (in rabbits) conformational spec ific antibodies, one with trans diequatorial and the other with trans diaxial conformation. The antibodies to the trans diaxial conformer will probably crossreact with cis centchroman (I), which has phenyl group at C-3 or C-4 in axial position . The same situation could be applicable in the enzyme labels 2e and Sc. The flipping from rrans diequatorial to trans diaxial conformation during conjugation to BSA in Sb, or to HRP labels 2c and Sc, is possible because the spacer

between the protein and the spacer between the protein and the hapten are in one and three atoms respectively. If this is true, then preparing a label where there is least possibility of the molecule flipping over could partly prove it. The analog Sa was radio iodinated to give the radio labeled compound Sd, which probably will retain ring-B in quasi chair form, and, phenyl groups at C-3 and C-4 retaining the diequatorial conformation. The position of the radioactive iodine group was fixed at para-position in Sd because of its bulk. This line of thinking proved correct, as contrary to enzyme tracer Sc and 2e, the radio iodinated centchroman analog Sd did show very good binding with antisera AS-l and AS-2. A standard curve with both the antibodies could be constructed between 6.25 pg/tube and 200 pg/tube of centchroman (Table 3) with less than I% crossreaction with cis centchroman . Our line of thinking was further proved when antiserum AS-3 against immunogen Sb evaluated with radio iodinated centchroman Sd showed negligible crossreaction (3.5%) with cis centchroman (Table I) , compared to the 50% crossreactivity with cis centchroman when enzyme labels 2e and Sc were used .

Although there are several reports on development of enantioselective antisera 18 to the two optical antipodes and their respective radioimmunoassay of several drugs, but to best of our knowledge, this is the first report of the production of antibodies specific for particular conformation of a drug molecule.

In conclusion , the study describes the importance of site of conjugation of centchroman molecule to carrier protein on the specificity of antibodies against anticipated metabolites. In add ition , by using a combination of specific antiserum and heterologous enzyme or radio labe led tracer sensttJve immunoassays could be developed and applied for the quantitative measurement of centchroman in serum. Furthermore, data presented in the study provides evidence for the production of conformation specific antibodies to centchroman.

Acknowledgement The authors wish to thank former director of CDRI

Prof. B N Dhawan for encouragement and AKS thanks ICMR, New Delhi for providing the financial support. Some of the work described here formed part of the dissertation submitted by AKS in partial fulfilment of requirements for a Ph.D. degree.

226 INDIAN J BIOCHEM BIOPHYS, VOL. 37, AUGUST 2000

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