Stereoselectivity in Bioequivalence Studies of Nortriptyline

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Stereoselectivity in Bioequivalence Studies of Nortriptyline To the Editor: A conference report from “Bio-International ‘92”l commented in the following terms on the possible impact of stereochemistry in bioequivalence studies: “...The use of specific analytical methods was briefly discussed. Two aspects must be taken into account (1)systemic availability of the two enantiomers and (2) systemic clearance. It was felt that if Figure 1 the two enantiomers are documented to show no difference in these two pharmacokinetic characteristics, a nonstereoseTable 1-Geometric Mean Data for Concentration Dependent Parameters lective method may be used ...” A survey of some 1291 articles of Nortriptyline, Unseparated 10-Hydroxynortriptyline, (0-10-Hydroxynortriptyline, and (a1 0-Hydroxynortriptyline on bioequivalence issues published over the past quarter century, however, failed to reveal a single stereoselectivestudy AUCa,, (ngih mL-’) Ga(ngimL) in which bioequivalence was assessed by the standard confidence interval a p p r ~ a c h . ~The , ~ lack of objective data was Test Reference Test Reference addressed in a symposium dedicated to issues surrounding Nortriptyline bioequivalency of racemic drugs in which Jamali4 stated 1146.0 1113.2 26.4 Mean (n= 23) 25.8 “...only publication of peer-reviewed data will enable us to Minimum 364.6 465.8 11.9 13.3 decide as to the relevance of the concept of stereochemistry Maximum 2747.3 2684.2 44.6 46.9 in the assessment of bioequivalence.” In a recent study to Unseparated 10-Hydroxynortriptyline evaluate the bioequivalence of two conventional release 1269.0 1252.6 34.9 33.4 Mean (n= 23) formulations of nortriptyline, therefore, the 10-hydroxy me15.4 Minimum 737.2 643.5 12.6 tabolite was monitored by a stereoselective method in order Maximum 2339.3 2502.3 88.8 96.3 to provide data on the impact of geometric isomerism in the ( 4 - 10-Hydroxynortriptyline bioequivalence decision making process. 29.6 Mean (n= 23) 1098.8 1090.1 30.7 Nortriptyline is a n example of a drug which does not itself 13.7 Minimum 620.0 556.2 10.1 exist in isomeric forms but into which isomerism is introduced 90.0 2160.9 2303.4 85.4 Maximum by metabolic oxidation of the carbon atom a t position 10 of (4-10-Hydroxynortriptyline the tricyclic ring system. Figure 1 shows the E-10 and 2-10 3.7 Mean (n= 23) 123.8 125.6 3.9 geometric isomers, each of which exists as R and S optical 1.4 Minimum 10.9 21.5 1.6 isomers (not illustrated). (E)-10-Hydroxynortriptylineis the Maximum 8.3 323.2 340.0 9.7 major form present in the urine after administration of nortriptyline to humans, the 2-isomer contributing only 5-22% of the total amount of 10-hydroxy nortriptyline In the present study, serial blood samples were harvested excreted in urine.5 Moreover, (2)-10-hydroxylationaccounted over 192 h after administration of test (Geneva Pharmaceufor only less than 20% of the hydroxylating activity of human ticals Inc.) and reference (Sandoz Pharmaceuticals Corp.) liver microsomes.6 The formation of (E)-10-hydroxynortripcapsules each containing nortriptyline (75 mg) to 23 healthy tyline is mediated in a highly stereoselective manner by an male volunteers in a randomized two-phase, two-sequence hepatic cytochrome P450 2D6 isozyme (CYP2D6)to form the cross-over design with a 4 week wash out period between (-)-enanti~mer,~ which is the predominant isomer found in phases. Plasma concentrations of (=t)-(E)-lO- and (2)-10the urine of patients mediated with nortriptylineP On the hydroxynortriptyline and the parent drug were measured by other hand, neither (+)-(E)-10-hydr~xynortriptyline~ nor the a validated HPLC method similar t o a published procedure (2)-10-hydr~xynortriptyline~ isomers are formed by CPY2D6, for haloperidol,1° except that an ultraviolet detector was and it appears that formation of (-)-(E)-10-hydroxynortrip- substituted for the coulometric detector used in the haloperidol tyline in intestinal mucosae is also mediated by enzyme(s) assay. The results (Table 1)showed that the area under the other than CYP2D6.7 Despite differences in their pharmaplasma concentration versus time curve to the last measurable cokinetic profiles, however, both geometric isomers of 10concentration (AUCl,,t) and C,, values of (El-10-hydroxynorhydroxynortriptyline appear to possess pharmacological actriptyline after administration of both test and reference tivity,E and evidence that (E)-10-hydroxynortriptylinemay be formulations were comparable with those of the parent drug, an antidepressant with less pronounced side effects than whereas corresponding values for (2)-10-hydroxynortriptyline nortriptyline has been reviewed by Nordin and c o - ~ o r k e r . ~ were approximately 10-fold lower. The mean t,,, values for These data suggest that, in the absence of evidence to the all analytes fell in the range 7-9 h (Table 2) which suggested contrary, a bioequivalence study on formulations of nortripthat the pharmacokinetics of the parent drug may have been tyline should take into account the parent drug and a t least rate limited by absorption, while the kinetics of the metabothe total (*)-(E)-10-hydroxyisomers. It could be argued that lites may have been limited by rate of formation. This concern ideally the enantiomers of (E)-10-hydroxynortriptylineshould was echoed by the fact that the half-life values calculated from have been measured separately, but the substantial costs to terminal slopes of the plasma concentration versus time in the present curves for racemic (E)-10-hydroxynortriptyline the company involved in the production of reference samples study (Table 2) were approximately 4-fold longer than those of the pure enantiomers and the development of enantioreportedg after oral administration of the pure (-)- and (+Iselective assay methodology could not be justified in the enantiomers of (E)-10-hydroxynortriptylinet o healthy huabsence of such a requirement by the regulatory authority. 0 1995, American Chemical Society and American Pharmaceutical Association

0022-3549/953184-1265$09.00/0

Journal of Pharmaceutical Sciences / 1265 Vol. 84, No. 10, October 1995

Table 2-Arithmetic Mean (kSE) for Time Dependent Parameters for Nortriptyline, Unseparated 10-Hydroxynortriptyline, (&iQHydroxynortripyline, and (2)-1 0-Hydroxynortriptyline

Test

Reference

Nortriptyline 7.6 f0.5 35.6 k 2.6

8.7 ? 0.6 36.8 ? 2.8

Unseparated 10-Hydroxynortriptyline

*

7.4 f 0.4 41 .O? 6.3

7.7 0.9 38.7 k 4.2 (0-l O-Hydroxynortriptyline 7.3 k 0.6 42.6 ? 4.6 (4-1 0-Hydroxynortriptyiine 9.0 k 1.2

7.2 ? 0.4 48.3 k 7.4

Unable to calculate

Unable to calculate

References and Notes

9.0 ? 0.9

Table 3 4 0 % Confidence Intervals for Nortriptyline, Unseparated 10-Hydroxynortriptyline,(lFj-10-Hydroxynortripyline,and (4-10-Hydroxynortriptyline 90% cis

ANOVA CV%a

Nortriptyline In AUClast In CIn(b,/AUCiaSi)

95.8-105.6 95.2-105.9 93.5-106.7 Unseparated 10-Hydroxynortriptyline In AUCI,~ 92.2-103.4 In C k x 92.8-112.1 In(CdAUCiast) 96.0-1 13.6

9.4 10.2 12.8 11.1 18.2 16.2

(4-10-Hydroxynortriptyline In AUCIal

In C, In (Cax/AUCasl)

In A L L t In C, In(CdAUC1d a

91.a1032 916 - 1 11.4 95.0-1 13.3

11.2 18.9 16.9

(a1 0-Hydroxynortriptyline 79.4-1 12.3 95.9-1 10.8 92.6-128.7

33.3 13.9 31.7

Calculated from the residual mean square.

mans. Thus the use of 90% confidence intervals based on partial areadl would not be appropriate in the present context. It was not possible to calculate half-life values for (2)-10-hydroxynortriptyline(Table 2) because of undulations in the terminal phase of the plasma concentration versus time curves, possibly arising as a result of biliary recycling. The 90%confidence intervals of AUCl,,t, C ,,,, and C,J AUClastfell within bioequivalence limits of SO-125% for the parent drug, total 10-hydroxynortryptyline, and (+)-(E)-lOhydroxynortriptyline (Table 3), as did the confidence interval of,C for (f)-(2)-10-hydroxynortriptyline.The ANOVA CVs for C,, and C,,/AUClaSt for (f )-(2)-10-hydroxynortriptyline, however, were ’30% (Table 3), which falls within the definition of “highly variable.”’ This phenomenon was a likely cause of the failure of the 90% confidence intervals of AUCl,,t and Cmax/AUC~,,tfor (&)-(2)-10-hydroxynortriptyline to fall within preset bioequivalence limits. Moreover, the high withinsubject variability on these parameters was largely due to the participation in the study of three individuals who had much lower AUClaStvalues (mean n = 3, n g h mL-l, test 29, ref 37) than the other subjects (mean n = 20, ng/h mL-l, test 179, ref 172). Removal of data from the three incongruous subjects resulted in a reduction of the ANOVA CV to <20% and the 90% confidence interval calculated for AUCl,,t and CmaXl AUCl,,t for the remaining twenty subjects fell within the present bioequivalence limits of SO-125%. The removal of data from a bioequivalence study, however, is not acceptable. 1266 1Journal of Pharrnaceutica/ Sciences Vol. 84, No. 10, October 1995

The suggestion was made12 and accepted a t the “BioInternational 94” meeting held in Munich, Germany, in June 1994 (proceedings to be published) that a mechanism be developed for widening the bioequivalence limits for drugs (or metabolites) with ANOVA CVs ’30%. In conclusion, the data suggest that the use of stereoselective methods was unnecessary in the present bioequivalence cross-over study on two formulations of nortriptyline. The study does reaffirm, however, that the analyte(s) to be taken into consideration and the bioequivalence limits to be applied to highly variable analytes must be decided a priori and not a posteriori.

1. Blume, H. H.; Midha, K. K. J . Pharm. Sci. 1993,82,1186-1189. 2. Schuirmann, D. J . Pharmacokinet. Biopharm. 1987, 15, 657680. 3. Westlake, W. J. J . Pharm. Sci. 1972, 61, 1340-1. 4. Jamali, F. J. Clzn. Pharmacol. 1992, 32, 930-4. 5. Mellstrom, B.; Bertilsson, L.; Sawe, J.; Schultz, H.-U.; Sjoqvist, F. Clin. Pharmacol. Ther. 1981, 30, 189-193. 6. Mellstrom, B.; Bertilsson, L.; Birgesson, C.; Goransson, M.; von Bahr, C. Drug. Metab. Dispos. 1983, 11, 115-119. 7. Dahl, M.-L ; Nordin, C.; Bertilsson, L. Ther. Drug Monit. 1991, 13, 189-194. 8. Young, R. Psychopharmacol. Bull. 1991,27, 521-532. 9. Nordin, C.; Bertilsson, L. Clin. Pharmacokinet. 1995, 28, 2640. 10. Midha, K. K.; Cooper, J. K.; Hawes, E. M.; Hubbard, J. W.; Korchinski, E. D.; McKay, G. Ther. Drug Monit. 1988,10,177183. 11. Midha, K. K.; Hubbard, J . W.; Rawson, M.; Gavalas, L. Eur. J . Pharm. Scz. 1994,2,351-363. 12. Midha, K. K.; Hubbard, J . W.; Yeung, P. K.; Ormsby, E.; McKay, G.; Hawes, E. M.; Korchinski, E. D.; Gurnsey, T.; Rawson, M.; Schwede, R. In Bio-International: bioavailability , bioequivalence and pharmacokinetics; international conference of F.I.P. “BioInternational ‘92” held in Bad Homburg, Germany, May 2022, 1992; K. M. H. Blume, Ed.; Medpharm Scientific Publishers: Stuttgart, 1993 pp 53-68.

Acknowledgments The authors thank Mr. Trevor Gurnsey for excellent technical assistance.

KAMAL K. MID HA^, JOHNW. HUBBARD, GORDONMCKAY, MAUREEN RAWSON,AND ROGERSCHWEDE~

College of Pharmacy & Nutrition University of Saskatchewan Saskatoon, Sask Canada S7N 5C9 +Geneva Pharmaceuticals 2555 W. Midway Blvd. P.O. Box 446 Broomfield, CO 80038-0446 Received May 22, 1995. Accepted for publication June 20, 1995. JS950206B