Stereochemistry Of Drug Metabolism Warfarin

Trans Trans Trans Trans Cis Cis Cis Cis

RS R

'■The antiserum was generated from a conjugate of an analog of compound Fig. 5[9a], Source-. Wing and Hammock (41).

cyclopropane ring (compare Fig. 5 [9c] vs. [9d] or [9c] vs. [9e] or [9f]). Although the authors attribute this to the closer proximity of the 4' position to the conjugation link (41), the 4' position is well removed from the link. An alternative explanation would be the flexibility of rotation around the 4' O—C bond compared to the rigidity of the cyclopropane ring.

7. Nicotine

Although /-nicotine is the predominant natural isomer, the occurrence of some d-nicotine makes it of interest to develop enantioselective assays for this compound. A review of nicotine and cotinine assays has been given (42), An early report of an RIA for /-nicotine described an immunogen prepared from 6-aminonicotine. This compound was converted to 6-(p-aminobenzamido)nicotine and conjugated to albumin {Fig. 4 [8b]) by diazotization (43), The enantiomeric purity of the 6-aminonicotine was not discussed, and the synthesis of 6-aminonicotine is reported to cause racemization (44). However, the antiserum eventually obtained showed only 6% cross-reaction with d-nicotine (43).

A racemic nicotine analog, ra « s-3 - h y d roxy methy 1 - n i coti ne, was converted to the hemisuccinate, which was conjugated to protein to form an immunogen (Fig. 4 [8c]). The resulting antiserum was used with tritiated /-nicotine as radioligand. With this radioligand the assay was highly selective for /-nicotine, with less than 0.01% cross-reaction with d-nicotine. Similar enantioselectivity is claimed for /-cotinine (42).

Hybridomas producing monoclonal antibodies to S-nicotine were obtained from mice immunized with conjugated racemic 3'hydroxy methyl-nicotine. Affinity constants were around 108 M_1 with 4% cross-reaction to K-nicotine (67).

D. Development and Use of Antisera for Both Enantiomers

1. Introduction

In not all instances in which racemic drug is administered is only one enantiomer of interest. Even when the major portion of the activity is associated with a single enantiomer, knowledge of concentrations of the other enantiomer may be of interest because of potential for toxicity or other side effects. Furthermore, it would be anticipated that the best selectivity would be achieved if both immunogen and labeled ligand were enantiomerically equivalent and optically pure. The enantiomeric immunogen would favor formation of enantioselective antibodies, and the enantiomerically pure labeled ligand would further enhance enantioselectivity by allowing the analyst to observe and use those antibodies with highest affinity for the enantiomer. A few examples of this approach have been published.

2. Warfarin

A method for the determination of both warfarin enantiomers based on enantioselective immunoassay has been reported (45). Warfarin analogs (Fig. 6) containing a 4'-carboxyethyl group (as the methyl ester) were converted to their diastereoisomeric camphor-sulfonates (Fig. 6 [12b]). These were separated chromatographically. Base-catalyzed hydrolysis then removed the camphor-sulfonyl group to yield the pure enantiomeric warfarin analogs. These were individually conjugated to bovine serum albumin by a mixed anhydride procedure to yield an immunogen (Fig. 6 [12c]). For radioligands, halogenated warfarin derivatives were resolved in a similar manner and then reduced with tritium gas to yield radioligands (Fig. 6 [12d]) of high optical purity (25-31 Ci/mmol). To demonstrate that racemization had not occurred under the catalytic conditions of the reduction, the tritiated S enantiomer was mixed with unlabeled R,S-warfarin. The i/-10-camphor-sulfonates were synthesized and separated chromatographically, and it was shown that the tritium in the diastereoisomer containing the R-warfarin enantiomer was less than 1% that in the diastereoisomer containing the S-warfarin enantiomer.

[121

[121

R3 = T *Chiral center

Figure 6 Warfarin, analogs, and immunogen.

R-warfarin exhibited a cross-reaction of only about 0.3% with the S-antiserum and S radioligand. S-warfarin cross-reacted to the extent of 3% with the R-warfarin antiserum and R radioligand. Various warfarin metabolites were shown to have low cross-reactions, with the exception of 4'-hydroxy warfarin. This latter compound is not a human metabolite, although it is found in rats in low concentration after a single dose of warfarin. The method could be used to determine the half-life of the individual enantiomers in rats given racemic drug; the resulting ratios of half-lives were in accord with those previously reported after administration of individual enantiomers. This enantioselective immunoassay was used in humans to demonstrate that vaccination against influenza did not change the relative pharmacokinetics of warfarin enantiomers (46).

The development of the warfarin immunoassays illustrates several points that are of value in development and use of enantioselective assays. In assays of this type, not only must enantioselectivity be considered, but also the cross-reaction with metabolites is still of importance. As in any RIA, high-specific-activity radioligand is required for the best sensitivity. The use of optically pure radioligand is a further advantage in enantioselectivity. The standard samples used for competitive binding assays must also be essentially optically pure. Otherwise, misleadingly high cross-reactions may be observed.

Two rabbits were challenged with each immunogen, and all four rabbits gave highly enantioselective antisera, thus indicating that the use of enantiomerically pure immunogens can be relied on to generally produce antisera of high enantioselectivity.

Unpublished results of immunoadsorption studies indicate that the low cross-reactions between enantiomers are probably not due to the presence of small amounts of antiserum selective for the opposite enan-tiomer. Such antibodies might be generated if an animal responds strongly to a very small amount of the opposite enantiomer present as an impurity. It was observed that even a low cross-reaction may still result in some inaccuracies, particularly in instances in which one enantiomer is present in much higher concentrations than the others. Also, cross-reaction curves are not generally parallel. Thus, in the assay of R- and S-warfarin in rat plasma, it was necessary to set up procedures for correction of the observed cross-reaction. In the rat, the R isomer, which yields the highest cross-reaction with the opposite enantiomer, is also the one with higher plasma concentration and longer half-life. The observation that corrections are necessary, and the earlier problems discussed with antisera to racemic immunogens constitute a strong argument for preparing and using antisera to both enantiomers of a drug.

3. Pentobarbital and Its Analogs

A class of barbiturates possessing the 2'-pentyl side chain ail exhibit similar optical isomerism. These include pentobarbital (Fig, 7 [13a]), thiopental, thiamylal, and secobarbital (Fig. 7 [13b]), Differences in pharmacological activity between enantiomers of these compounds have been shown in humans (47) and animals (48).

R and S forms of pentobarbital have been synthesized (49,50) and alkylated to yield eventually the corresponding JV-irans-crotonic acids. The latter were conjugated to bovine serum albumin, and the resulting conjugates (Fig. 7 [13c]) used to immunize rabbits. Enantiomerically pure tritiumlabeled R- and S-5-propyl-5-(2 '-pentyl)-barbituric adds {Fig. 7 [13d]) were made by catalytic reduction of the enantiomers of secobarbital with tritium gas, for use as radioligands. The resulting antisera were quite selective, with only about 1% cross-reaction of each antiserum with the opposite enantiomer and less than 1% cross-reaction with the hydroxy metabolite of pentobarbital (51). The resulting system could also be used to analyze the other closely related 2'-pentyl barbiturates. For example, the S isomer of secobarbital showed 83% cross-reaction with the antiserum to S-pentobar-bital and only 2% cross-reaction with the R antiserum and could be used to analyze these isomers in human subjects given racemic secobarbital (52). The assay methodology was checked by adding the values for the two enantiomers of pentobarbital and comparing them with those from gas/ liquid chromatography (GLC), which did not resolve the enantiomers (68).

(Pentobarbital)

(Secobarbital)

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