Radioimmunoassay for the N-Oxide Metabolite of Chlorpromazine in Human Plasma and Its Application to a Pharmacokinetic Study in Healthy Humans

Radioimmunoassay for the MOxide Metabolite of Chlorpromazine in Human Plasma and Its Application to a Pharmacokinetic Study in Healthy Humans P. K. F. YEUNG', J. W. HUBBARD*,E. D. KORCHINSKI~, AND K. K. MIDHA*' Received February 5, 1987, from the *Colleges of P!armacy and Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO. Accepted for publication July 9, 1987. Present address: College of Pharmacy, Dalhousie University, Halifax, NS, B3H 3J5, Canada. Abstract 0 Antibodies specific to chlorpromazine Noxide (CPZNO) were produced in rabbits immunized with a hapten-bovine serum albumin conjugate, which was prepared by linking the 7-position of the phenothiazine ring of the metabolite to the protein via a 4-carbon bridge. An extraction radioimmunoassay (RIA) was developed using this antiserum and shown to have adequate sensitivity and specificity for determination of plasma concentrations of CPZNO in the presence of chlorpromazine (CPZ) and its major metabolites. It was used together with the previously developed RlAs for CPZ, chlorpromazine sulfoxide (CPZSO), and 7-hydroxychlorpromazine(7-OHCPZ)to study the pharmacokinetics of CPZ and these metabolites in five healthy volunteers after they received a single 50-mg oral dose of CPZ. It is interesting to note that peak plasma concentrations of CPZNO were considerably higher than CPZ, and the apparent elimination half-lives of this metabolite were shorter than those of CPZ.

It is well recognized that the metabolism of chlorpromazine (CPZ) plays an important role in the overall clinical effectiveness of CPZ in the treatment of schizophrenia and related Chlorpromazine metabolism is extremely complex. It has been estimated that there are 168 potential metabolites of CPZ, and more than 30 of these have already been identified in human and/or animal species.' Some metabolites which are currently thought to be of clinical importance are 7-hydroxychlorpromazine (7-OHCPZ1, chlorpromazine sulfoxide (CPZSO), N-monodesmethylchlorpromazine (NORICPZ),and chlorpromazine N-oxide (CPZNO). Chlorpromazine N-oxide is one of the major CPZ metabolites. Steady-state plasma concentrations of CPZNO have been reported to be -50% of the CPZ concentrations in schizophrenic patient^,^ and CPZNO has been described as either active or inactive depending on the models used. It was reported to be inactive in the inhibition of [3H]spiperone binding to rat brain dopamine receptors in vitro, and inactive in reversing amphetamine- or apomorphine-induced stereotyped behavior in rats in V ~ V O . In ~ other experiments, CPZNO was described as active in altering the electroencephalogram (EEG) pattern, and in elevating brain homovanillic acid (HVA) concentrations in rats in vivo and in an isolated rat brain mode1.5.6 Some evidence4.6 suggests that reduction of CPZNO to CPZ occurs in vivo; so, some of the pharmacological activities observed with CPZNO in vivo could be contributed by CPZ as a result of the conversion. The analysis of CPZNO in body fluids or tissue homogenates is problematic for a number of reasons. N-Oxides are notoriously thermolabile and decompose during gas chromatograph~.'-~ More recent evidence has shown that CPZNO can be reduced to CPZ during the extraction of plasma through the generation of reducing agents which arise when plasma proteins are digested with strong alkalis such as sodium hydroxide, thereby leading to spuriously high apparent levels of CPZ and correspondingly low values for the Noxide.'" Chlorpromazine N-oxide is even more vulnerable to OO22-3549/87/1OOO-0803$0 1.OO/O 1987, American Pharmaceutical Association

0

artifactual reduction during the extraction of alkalinized whole blood wherein a portion of the CPZ so formed undergoes subsequent oxidation to CPZSO.11 Chlorpromazine N oxide appears to reside almost entirely in the plasma fraction of whole blood with little penetration into the red blood cells.12Consequently, CPZNO can be extracted successfully from separated plasma by methods which avoid the use of strongly alkaline conditions.10J2 Thus, while it has long been known that artifacts can arise at all stages in the handling of biological samples or extracts containing CPZ and metabol i t e ~the , ~ discovery ~ of the prevalence of the N-oxide metabolite in clinical plasma samples has greatly increased the analytical challenge. The successful development of sensitive RIA procedures for CPZ,14 CPZS0,l5 and 7-OHCPZI6 in these laboratories will permit us to carry out pharmacokinetic studies after the administration of single doses of CPZ to healthy subjects. This report describes the development of a new RIA for CPZNO in plasma and its application, together with the RIAs reported previously, in the measurement of CPZNO, CPZ, CPZSO, and 7-OHCPZ in the plasma of five healthy men after each received a single 50-mg oral dose of CPZ.

Experimental Section M a t e r i a l d t a n d a r d samples of CPZ and its metabolites were received as gifts from Rhone-Poulenc Pharm Inc., Montreal, Quebec, Canada, and Dr. A. A. Manian of the National Institute of Mental Health, Rockville, MD, respectively. Ring-tritiated CPZ hydrochloride (New England Nuclear Canada, Ltd., Lachine, Quebec, Canada), 3-methoxycarbonylpropionyl chloride (Figure l; 2; Aldrich Chemical Company, Milwaukee, WI), 30% hydrogen peroxide solution (Fisher Scientific Canada, Edmonton, Alberta, Canada), rnchloroperoxybenzoic acid (Aldrich Chemical Company, Milwaukee, WI), and bovine serum albumin (BSA; Sigma Chemical Company, St. Louis, MO) were purchased commercially. Solvents used were of high-performance liquid chromatography (HPLC) grade (BDH Chemical Canada Ltd., Saskatoon, Saskatchewan, Canada). Thin-layer chromatography (TLC) aluminum sheets (20 x 20 cm) precoated with silica gel G60 (0.2-mm thickness; BDH Chemical Canada Ltd., Saskatoon, Saskatchewan, Canada) and preparative TLC glass plates (20 x 20 cm) precoated with silica gel G-200 (2.0mm thickness; Brinkman Instruments, Inc., Westbury, NY) were purchased commercially. In most cases, the TLC sheets were developed once with the appropriate solvent system (single development). For preparative purposes, the TLC plates were developed, dried under nitrogen, and then developed again with the same solvent system. This repeated development process was continued until desired separation of the required product from impurities was obtained. Compounds on TLC sheets (or plates) were visualized under ultraviolet light (UV)or a h r spraying the sheets (or plates) with Forrest's reagent (a mixture of concentrated sulfuric acid, absolute ethanol, and ferric chloride, 100:300:1).All R, values reported in this work are based on the values obtained with analytical TLC sheets. For liquid scintillation counting, the scintillation fluid (Ready Solve-MP, Beckman Instruments, Inc., Fullerton, CA) was obtained commercially, and the radioactivity was determined by a liquid Journal of Pharmaceutical Sciences / 803 Vol. 76, No. 10, October 1987

scintillation counter equipped with automatic quench compensation (LKB Rackbeta model 1215, Fisher Scientific Canada, Edmonton, Alberta, Canada). A HPLC-UV system was used for purification and isolation of [3HICPZN0. The HPLC-UV system consisted of a liquid chromatographic pump (model 45, Waters Associates, Milford, MA), a valve-loop injector fitted with a 1-mL loop (Rheodyne model 7120, Technical Marketing Assoc., Ottawa, Ontario, Canada), a 250 x 4.6 mm i.d. 5-pm cyanobonded column (Spherisorb CN, Altex, Beckman Instruments, Toronto, Ontario, Canada), and a variable wavelength UV detector set a t 254 nm (model 480, Waters Associates). The system was operated a t ambient temperature and a t a flow rate of 1.5 ml/min, with a mobile phase composed of 10% sodium acetate buffer (0.05 M, pH 6) in methanol. Several stages were involved in the development of the RIA for CPZNO. Briefly, an immunogen was prepared and used to immunize rabbits in order to obtain antibodies. Radioactive CPZNO was prepared for use as a tracer. Once the RIA for CPZNO was developed, it was applied to the study of the pharmacokinetics of CPZNO in healthy men given a single 50-mg oral dose of CPZ. The effectiveness of the RIA in measuring CPZNO in plasma directly was compared with its performance in measuring CPZNO levels in extracts of plasma. Each of these stages is described in detail under appropriate headings below. synthesis of 7-(3Synthesis of Immunogen (Figure 1)-The methoxycarbonylpropiony1)CPZ (Figure 1, 3) and the subsequent hydrolysis to yield 7-(3-carboxypropionyI)CPZ (Figure 1, 4) was carried out as described previ0us1y.l~Carbon-13 nuclear magnetic resonance ("C NMR) spectra obtained for 3 in deuterated chloroform using tetramethylsilane as reference showed a downfield shift of the aromatic carbon a t the 7-position of the phenothiazine ring (C, of NMR spectra were kindly CPZ, 123.0 ppm; C7 of 3,132.5 ppm). obtained and interpreted by Dr. K. Bailey of the Health Protection Branch, Health and Welfare Canada, Ottawa, Canada; a Bruker Spectrospin instrument operating a t 200 MHz was used.) This downfield shift suggested that the Friedel-Crafts acylation had occurred a t the 7-position of the phenothiazine ring system. The chemical shift assignments of CPZ were based on a literature report.lR To a solution of 4 (Figure 1; 1.0 g, 2.4 mmol) in 50 mL of methanol and 4 mL of 0.6 M sodium hydroxide solution was added 0.3 mL of a 30% hydrogen peroxide solution (Fisher Scientific Canada, Edmonton, Alberta, Canada). This mixture was heated a t 50 "C in a water bath for 1.5 h. The pale yellow solution was cooled to room temperature, and the methanol was removed under reduced pressure. To the remaining aqueous portion was added 25 mL of water, and the pH was adjusted to 5.4 a t which point the mixture became cloudy. The mixture was allowed to stand a t 4 "C overnight to permit an oily residue to separate. The supernatant fraction was decanted and discarded, and the residual oil was dried under reduced pressure in

4

C

13

Flgure 1-Outline of the synthesis of a hapten for chlorpromazine Noxide and its bovine serum albumin (BSA) conjugate. 804 1 Journal of Pharmaceutical Sciences Vol. 76, No. 10, October 1987

the presence of potassium hydroxide pellets. A sample of this residual oil was analyzed by TLC using a solvent system of methano1:chloroforrn:aqueous ammonium hydroxide (1:l:O.Ol). The TLC showed that there were two major spots and two minor spots: one of the major spots (R, = 0.26) corresponded to unreacted 4 (Figure l ) , the other major spot ( R , = 0.12) corresponded to the N-oxide product 5 (Figure 1). The identities of the two minor spots ( R , = 0.21 and 0.07, respectively) were not determined because there was insufficient material. The crude oil was purified by preparative TLC using a solvent system of acetone:methanol:glacialacetic acid (1:2:0.02) until the product contained a single spot ( R , = 0.18). The N-oxide product ( 5 , Figure 1) was recovered from TLC plates by scraping the appropriate band and stirring the scraped silica gel powder with a mixture of chloroform and methanol (1:l) for 3 h a t room temperature. The suspension was then filtered and the solvent was removed under reduced pressure. The residual oil was solidified by triturating with petroleum ether (bp 30-40 "C) to yield a yellowish solid (0.2 g, 19%. uncorrected mp > 200°C with decomposition starting to occur a t 150 "C). Attempts to crystallize this material were unsuccessful. This material was used in the coupling reaction without further purification. Mass spectra were obtained with a VG7070HE double-focusing mass spectrometer which was equipped with electron-impact ionization, ammonia-chemical ionization, and fast-atom-bombardment ionization. Neither electron-impact (EI, 70 eV) nor ammonia chemical ionization (CI) mass spectra (direct probe, 200 "C) showed molecular ions (M)*' at m/z 434/436, but gave ions a t m/z 373/375 (EI mass spectrum) or m/z 3741376 (CI mass spectrum), which corresponded to one of the Cope elimination productsy of the N-oxide product 5 (Figure 1; EI [(MI" -C,H,NO]; CI [(M + 1)' -C2H,NO]). The quasi molecular ions (M + 1)' a t m/z 4351437 were observed in the spectrum obtained by fast-atom-bombardment ionization. In order to increase the sample volatility so that further mass spectral interpretations could be made, the methyl ester of 5 (Figure 1)was prepared by heating 5 in methanol containing 5%of concentrated hydrochloric acid a t 60 "C for 3 h with subsequent purification by analytical TLC using a solvent system of methano1:chloroform:aqueous ammonimum hydroxide (1:l:O.Ol; R , of 5 = 0.12; R, of the methyl ester = 0.50). The EI mass spectrum (70 eV) of the purified methyl ester of 5 was consistent with the assigned structure: (M)+' a t m/z 448/450 (absent); mlz 432/434 [(M)" -01; m/z 387/389 l(M)+'-C,H,NOI; m/z 346/348 [(M)'' -C5H,,NO]. The UV spectrum of the methanolic solution of the acid ( 5 , Figure 1)showed two absorption maxima (A maxl = 272 nm, A maxa = 287 nm) which were identical to the absorption maxima of 4 (Figure 1). The hapten ( 5 , Figure 1) was covalently linked to BSA by the water soluble carbodiimide method,'7 in which -13 mol of 5 were coupled to each mol of BSA as determined by the UV difference spectral method.19 Synthesis of ['Hlbenzene Ring-Labeled Chlorpromazine N-Oxide-A solution of ring tritium-labeled CPZ hydrochloride (New England Nuclear Canada, Ltd., Lachine, Quebec, Canada) in ethanol (300 pL, 17 C i h m o l , 1 mCi/mL) was dried at 35 "C under a stream of nitrogen. The residue was dissolved in 1 mL of hydrochloric acid (0.1 M). The solution was alkalinized by adding 0.2 mL of a 1.2 M sodium hydroxide solution, and then extracted with diethyl ether (3 x 1mL). The combined ether extract was evaporated to dryness at 35°C under a stream of nitrogen to yield tritiated CPZ as the free base. The CPZ free base was dissolved in 1 mL of methylene dichloride, and the solution was cooled to -70 "C in a dry ice and acetone bath. To the solution was added 0.1 mL of a solution of m-chloroperoxybenzoic acid (Aldrich Chemical Company, Milwaukee, WI) in methylene dichloride (2.0 pg,0.012 pmol). The mixture was allowed to stand a t -70°C for 0.5 h, and then was washed with a 0.5 M sodium carbonate solution (2 x 1mL), followed by water (2 x 1mL). Finally, the solvent was evaporated at 35 "C under a stream of nitrogen. The residue was redissolved in 0.2 mL of methanol, and aliquots of the methanolic solution (0.1 mL) were injected into the above-mentioned HPLC-UV system. Ring tritium-labeled CPZNO was collected in the fraction between 3.6 and 4.5 min. To determine the specific activity of the tracer, aliquots of the collected fraction were injected into the HPLC system. The amounts of tritiated CPZNO were determined by comparing the peak area of the tracer with those obtained from injections of known amounts of unlabeled CPZNO. The tritiated CPZNO peak was collected, and

-

the radioactivity was determined by a liquid scintillation counter. The specific activity was calculated to be -17 CiImmol. The total amount of radioactivity recovered as tritiated CPZNO was -14%. The tracer was stable in the mobile phase for up to six months when stored a t -20 "C. Working tracer solution was prepared on the day of assay by diluting an aliquot of the stock solution with a 3% BSA solution, such that 0.25 mL of the working tracer solution contained -10 000 cpm. The radiochemical purity of the tracer was assessed by TLC using a solvent system (ch1oroform:methanol:aqueousammonium hydroxide; 3:1:0.02) in which the R, value of tritiated CPZNO was 0.45. Radioactivity corresponding to CPZNO was compared with the total radioactivity on the TLC sheet. The radiochemical purity of the tracer used in the RIA was maintained a t >go%. Pilot experiments using unlabeled CPZ as starting material were carried out as described for the synthesis of tritiated CPZNO. The EI mass spectrum of the HPLC isolated product was identical to that of standard CPZNO: (M)+' a t m/z 334I336 (absent); d z 318I320 [(M)+' -01; mlz 304/306 [(M)+' -CH20]; mlz 273/275 [(M)" -C2H,NOl; m/z 232/234 [(M)"-CsH,2NOl. Immunization-Four New Zealand white rabbits, -4 months old, were each immunized intradermally with 1 mg of the hapten-BSA conjugate emulsified with 0.5 mL of Freund's complete adjuvant (Difco Laboratories, Detroit, MI) and 0.5 mL of isotonic saline. The rabbits were re-immunized at two-week intervals with the same amount of immunogen emulsified with incomplete adjuvant (Difco Laboratories, Detroit, MI). After four intradermal injections, binding activity to [3H]CPZN0 was detected in the sera of all the rabbits. Before the rabbits were sacrificed, they were each given two booster doses of 1 mg of immunogen in 0.2 mL of isotonic saline via the ear vein in two-week intervals. The harvested antisera were lyophilized, and the lyophilized antiserum of one of the rabbits was used in the CPZNO assay. Radioimmunoassay Procedure-The lyophilized antiserum (10 mg) was reconstituted in 8 mL of 0.2 M phosphate buffer (pH 7) containing 0.02% sodium azide as a preservative. This stock solution was stable for more than one year when stored a t 4 "C. On the day of the analysis, the stock antiserum solution was diluted 10 times with the same buffer solution to provide the working antiserum solution (final concentration 0.0125% w/vh This concentration was the working titre of antiserum a t which -306 of the tritiated CPZNO added was bound to the antiserum in the absence of unlabeled CPZNO. To each 12 x 75-mm polystyrene tube (Fisher Scientific Canada, Edmonton, Alberta, Canada) containing 0.1 mL of plasma sample or standard was added 0.1 mL of Red Cross bag plasma, 0.2 mL of phosphate buffer, 0.25 mL of working tracer solution (-10 000 cpm), and 0.2 mL of working antiserum solution. The mixture was incubated a t 4 "C for 1 h. To this solution was added 1 mL of cold dextrancoated charcoal suspension prepared in our laboratory according to the procedure of Powell et a1.20 The suspension was mixed (vortex from Fisher Scientific Canada, Edmonton, Alberta, Canada) for 5 8 , and then incubated a t 4°C for another 10 min. The mixture was centrifuged (1720 x g, 4 "C, 10 min) and the supernatant fraction was decanted into 10 mL of the scintillation fluid which was mixed and counted. A standard curve was constructed on each day of analysis by plotting the logit values of BIB, on the y-axis and the log values of plasma concentrations of CPZNO on the x-axis, where B and B, are the antibody-bound radioactivity in the presence and absence of unlabeled CPZNO, respectively. Logit BIB, is defined as

(1) The RIA was normally carried out in triplicate unless otherwise specified. Plasma concentrations of CPZ, CPZSO, and 7-OHCPZ were determined by RIAs previously described.l+ls Extraction of Chlorpromazine and Metabolites from PlasmaTo a 15-mL test tube with a polytef-lined screw cap (Fisher Scientific Canada, Edmonton, Alberta, Canada) was added 3.0 mL of plasma sample or standard and 0.2 mL of phosphate buffer (0.2 M, pH 7.2). The sample was gently mixed (vortex) and then extracted with 8.0 mL of hexane for 30 min. It was centrifuged at 400 x g for 5 min a t room temperature, and 7.0 mL of the organic layer containing mainly CPZ was transferred to a 10-mL screw-capped tube and evaporated to dryness a t 55 "C under a stream of nitrogen. To the

remaining aqueous layer was added 7.0 mL of diethyl ether, and, after mixing for 30 min, the contents were centrifuged (400 x g) for 5 min a t room temperature. The organic layer (7.0 mL), containing mainly 7-OHCPZ, was transferred to a 10-mL screw-capped tube and evaporated to dryness a t 55 "C under a stream of nitrogen. The remaining plasma layer was then extracted with 5.0 mL of methylene dichloride in a rotating mixer (Multi-Purpose Rotator, model 151, Scientific Industries Inc., Bohemia, NY) for 30 min, and then centrifuged (1720 x g) at 0 "C for 15 min. The organic layer (5.0 mL) containing CPZNO and CPZSO was transferred to a 10-mL screwcapped tube and evaporated to dryness a t 45 "C under a stream of nitrogen. The residues were each reconstituted in 1.0 mL of a 7.5% BSA solution in phosphate buffer (0.2 M, pH 7), and 0.2 mL of the reconstituted samples was analyzed by the RIAs as described above except that Red Cross bag plasma was not needed in the assay procedure. The RIAs for the extracted samples were carried out in duplicate during routine analysis. Plasma S a m p l e e A f t e r a n overnight fast, five male healthy volunteers (aged 20-30 years) were each given a single 50-mg oral dose of CPZ (Largactil syrup; gift from Rhone-Poulenc Pharm Inc., Montreal, Quebec, Canada), together with 250 mL of water. No food was allowed until 4 h after dosing. Blood samples from the antecubital vein were collected in green stopper, heparinized evacuated glass tubes (Vacutainers, Becton Dickinson Company, Mississauga, Ontario, Canada), with care being taken to avoid contact of blood with the rubber stoppers. Samples were taken a t 0 (just before dosing), 0.25,0.5,1.0, 1.5,2.0,2.5,3.0,4.0,6.0,8.0,12.0,24.0,32.0, and 48.0 h after drug administration. The plasma was separated immediately and stored a t -20 "C in plastic scintillation vials (Fisher Scientific Canada, Edmonton, Alberta, Canada) until analysis. Pharmacokinetic Calculations-Elimination half-lives or apparent elimination half-lives were determined by the quotient In 2/k, where the elimination rate constant (k)was calculated from the last available slope of the log plasma concentration-time curve of each analyte. The area under the plasma concentration-time curve (AUC,") was calculated by the linear trapezoidal rule up to the last measured plasma concentration (CIaeJand extrapolated to infinity by adding the quotient C,,&.

Results Sensitivity and Specificity of the Chlorpromazine NOxide Radioimmunoassay-The sensitivity of the RIA, with or without extraction, was <0.5 ng/mL. The standard curve of the RIA without extraction (referred to as 'direct RIA' in subsequent discussion) was linear (r2 = 0.9990) between the concentrations 0.25 and 20.0 ng/mL (Table I). The RIA after extraction of CPZNO from plasma (referred to as extraction RIA in subsequent discussions) also had the same limit of quantitation of 0.25 ng/mL, although the upper linear limit was reduced to 10 ng/mL (r2 = 0.9975, Table I). Table CStandard Curves for Direct Radiolmmunoassayand Extraction Radiolrnrnunoassay of Chlorpromazine NOxide In Plasma ~~

~~

Extraction RIAb

Direct RIA'

Concentration, ng/mL 0.25 0.5 1.o 2.5 5.0 10.0 20.0

Logit s/5c 2.788 2 0.014 2.154 ? 0.032 1.368 t 0.022 0.356 2 0.005 -0.393 2 0.003 - 1.056 t 0.095 -1.621 2 0.016

Logit S / b c 2.289 2 1.431 2 0.477 2 -0.306 2 -0.995 ? -1.807 2 -d

0.032 0.033 0.020 0.018

0.040 0.054

'n = 3; r = 0.990; slope = -2.379; mean %CV = 2.25. b n = 4; r = 0.9975; slope = -2.497; mean %CV = 3.46. 'Logit B 5 = In [B&l WE,,]where 8 i s radiotracer bound in the presence of cold drug and S,IS radiotracer bound in the absence of cold drug. dLarge %CV at this

concentration.

Journal of Pharmaceutical Sciences / 805 Vol. 76, No. 10, October 1987

The cross reactivities of the CPZNO antiserum to CPZ and metabolites were determined according to the criteria of Abraham.21 The antiserum did not cross react significantly with any of the CPZ-related species tested (Table 11). The possible interference of the RIA by CPZ and other major metabolites was assessed by the following experiments. Samples of Red Cross outdated bag plasma spiked with 1 ng/mL of CPZNO alone were assayed in parallel with aliquots of the same bag plasma spiked with 1 ng/mL of CPZNO plus 5 ng/mL of each of CPZ, CPZSO, and 7-OHCPZ. In the presence of five times excess of these potentially interfering compounds, the assayed concentrations of CPZNO were significantly inflated (p < 0.05, t test) when they were determined by the direct RIA. However when the spiked samples were analysed by the extraction RIA procedure, the assay values were not affected by the presence of large excess of these CPZ-related compounds (Table 111). Thus it was decided to use the extraction RIA procedure for the pharmacokinetic study. Effect of Bag Plasma versus Fresh Plasma-In order to determine the effect of plasma endogenous materials on the extraction RIA, the amounts of antibody-bound radioactivity in the absence of unlabeled CPZ or metabolites (B, values) were determined in Red Cross outdated bag plasma (five bags) and fresh plasma obtained from 10 volunteers. There were no statistically significant differences (p > 0.05, t test) between the B, values of the two types of plasma (bag plasma, B , = 23.1 2 1.8%; individual fresh plasma, B, = 26.4 2 3.6%). The coefficient of variation (CV) of the B, values between the individual fresh plasma was 6.7%, which was similar to the intra-assay variations (-6%). These data indicated that endogenous materials, either in bag plasma or fresh plasma from healthy volunteers, did not interfere with the extraction RIA. The interassay variations of the extraction RIA were <15%. Table ICCross-Reaction Profiles of the Chlorpromazlne NOxlde Antiserum '

Compound

Cross-Reaction, %

Chlorpromazine Koxide Chlorpromazine 7-Hydroxychlorpromazine Chlorpromazine sulfoxide

100 <1 <1 <1 <1 <1 <1 <1

KMonodesmethylchlorpromazine KDidesmethylchlorpromazine

Chlorpromazine N,Sdioxide 7-Hydroxychlorpromazine- Oglucuronide

'Values are expressed as percent cross reaction which was calculated by the method of Abraham (ref 16). Table Ill-Determination of Plasma Chlorpromazlne NOxlde Concentrations In the Absence and Presence of Chlorpromazine and Other Major Metabolites

Concentration

Concentration

9"c"p"z"?:A:!

Added as cpzhro' + 7-OHCPZ].1 ng/mL ng/mL; 5 ng/mL

Dikffetn;;pf Test)

each Direct RIAa (n = 3) Extraction RIAb (n = 6)

1.03 ? 0.12' 0.97

2

0.09

1.33 2 0.05

p < 0.05

0.04

p > 0.05

0.98

2

'CPZhro was measured directly in plasma without prior extraction. ' C P Z N was measured after extraction from plasma, as described in

the Experimenfal Secrion. 'Mean concentration.

?

SD of the assayed plasma

806 / Journal of Pharmaceutical Sciences Vol. 76, No. 10, October 1987

Plasma Concentrations of Chlorpromazine, 7-Hydroxychlorpromazine, Chlorpromazine Sulfoxide, and Chlorpromazine N - O x i d d h l o r p r o m a z i n e was readily absorbed following oral administration. Measurable concentrations of CPZ were found in most volunteers a t the first sampling time (0.25 h). The plasma concentrations of CPZ began to rise and reached a maximum between 1 and 4 h (mean T,, 2.1 h ) after drug administration. The maximum plasma CPZ concentrations (C,,,) were, on the average, 11.3 ng/mL, although large interindividual variations were observed (CV 2 100%; Table IV). Plasma concentrations of CPZSO and CPZNO were also measurable a t the first sampling time in most of the volunteers, whereas free 7-OHCPZ was not detected in these early plasma samples. This hydroxylated metabolite became detectable in three of the five volunteers 0.5 h after the oral dose, and was measurable in all the volunteers 1.0 h after drug administration. The mean T,, values for free 7-OHCPZ, CPZSO, and CPZNO were 2.7,2.0, and 1.9 h, respectively (Table 111). Highest plasma concentrations were observed for CPZNO in that the mean C,,, of CPZNO (24.4 ng/mL) was more than two times that of CPZ. The mean C,,, of CPZSO (11.1 ng/mL) and CPZ were , offree 7-OHCPZ (3.7 ng/mL) similar, whereas the mean C was considerably lower than that of CPZ (Table 111). There were also large interindividual variations in the plasma concentration maxima of the metabolites (CV of free 7OHCPZ, 37.8%; CV of CPZSO, 18.9%; CV of CPZNO, 54.1%), although they were considerably smaller than those of CPZ (CV 2 100%; Table IV). Plasma concentrations of CPZ and its primary metabolites appeared to follow multiphasic decline patterns after the 50mg oral dose. In most volunteers, CPZ (415) and free 7OHCPZ ( 5 6 ) were still measurable in the 48-h plasma samples, but in only one volunteer was CPZNO detectable a t this sampling time. In three other subjects, CPZNO was measurable up to 32 h after dosing, while in one individual, the N-oxide was not detectable in plasma samples after 12 h. There was similar intersubject variation in the times of the last detectable concentrations of CPZSO (48 h, 2/5; 32 h, 3/5). The mean elimination half-life value for CPZ (10.9 h) was comparable to the mean apparent elimination half-life value calculated for CPZSO (11.2 h). By contrast, the mean apparent elimination half-life of free 7-OHCPZ (24.5 h) was more than twice as long as that of CPZ, whereas the mean apparent elimination half-life value obtained for CPZNO was 6.7 h (Table IV). On the average, the areas under the plasma concentration-time curves, calculated from 0 h to infinity (AUCZ), of free 7-OHCPZ, CPZSO, and CPZNO were -70,87, and 116%, respectively, of that of CPZ. However, large interindividual variatiQnswere again observed for the AUC," values (Table IV). A semi-log plot of the plasma concentrations versus time of one of the volunteers is shown in Figure 2. Aliquots of the plasma were hydrolyzed by means of a crude glucu1ase:sulfatase preparation (pglucuronidase type H-2, crude solution from Helix pomatia containing p-glucuronidase and sulfatase activities; Sigma Chemical Company, St. Louis, MO) before analysis by the extraction RIA for 7-OHCPZ. Subtraction of the corresponding level of free 7OHCPZ from the value so obtained gave a n estimate of the level of conjugated 7-OHCPZ in each plasma sample. Table IV shows that the mean T,,, for conjugated 7-OHCPZ occurred earlier than that for free 7-OHCPZ, and that the conjugated 7-OHCPZ had the highest values for C,,, and AUC; of any of the analytes measured. Nevertheless, the mean apparent elimination half-life of the 7-OHCPZ conjugate(s) was -1/3 that of free 7-OHCPZ, which is consistent with the rapid excretion of the phase I1 metabolite(s) in the urine and possibly in the bile.

Table IV-Phannacoklnetlc Parameters of Chlorpromazlne and Metabollteo In Flve Healthy Volunteers after Receiving a Single 50-mg Oral Dose of Chlorpromazlne

CPZ L,h E C-, ng/mLb b,+, h

AUC;, ngh/mL

2.1 2 1.3' 11.3 2 11.4 10.9 2 6.1 102.0 _t 118.2d

7-OHCPZ (Free) 2.7 2 3.7 2 24.5 2 72.2 2

2.0 1.4 15.2 20.3

CPZSO 2.0 2 11.1 2 11.2 2 88.5 2

0.5 2.1 9.2 25.6

CPZhEO 1.9 2 24.4 2 6.7 2 118.8 2

'Time to reach maximum plasma concentrations. *Maximum plasma concentration. cValues are mean corresponding geometric mean: 65.25 ngh/mL (24.27% CV).

Discussion Development of Antibodies-The development and evaluation of the RIAs for CPZ, CPZSO, and 7-OHCPZ have been described in detail in previous publicationsl"16 and therefore they will not be discussed here. In order to produce antibodies for a small drug molecule, it is essential that the molecule be covalently linked to a macromolecule. A most commonly used carrier macromolecule is BSA, a protein with a molecular weight of -70 000. It is believed that the approach chosen to couple the drug molecule to the carrier antigen is crucial in determining the specificity of the antibodies. According to the principle suggested by Parker22 and the experience gained by the authors in developing RIAs for the phenothiazine antipsychotics, antisera raised against drug molecules are most specific for the portion of the molecule most distal to the point of attachment to protein. Thus when CPZ, CPZSO, or 7-OHCPZ was covalently linked to BSA via the dimethylaminopropyl side chain, antisera produced against these conjugates were most specific to the phenothiazine ring portion of the molecule, but were indiscriminate towards the tertiary amine and side-chain modified anologues, such as the primary and secondary amine metabolites of these species.1&I6On the other hand, the most distinguishable functional group of CPZNO is the tertiary amine N-oxide moiety which is located at the dimethylaminopropyl side chain of the CPZ molecule. Thus, in order to produce antibodies most specific to the N-oxide function, a suitable site for attachment to BSA is the phenothiazine ring system. Since a reactive functional group is not available in this region for direct coupling to the protein, a carboxylic acid functional group was introduced 100,

0

20

40

T h e (h)

Figure 2- Plasma concentrationsof chlorpromazine and metabolites in a healthy volunteer (#3) after a single 50-mg oral dose of chlorpromazine. Key: (A)chlorpromazine; (A)chlorpromazine N-oxide; ( 0 )conjugated 7-hydroxychlorpromazine;(0) free 7-hydroxychlorpromazine;(H) chlorpromazine sulfoxide.

2

0.5 13.2 1.4 44.5

7-OHCPZ (Conjugated) 1.9 2 0.4 39.1 t 20.2 8.5 2 5.4 234.5 2 132.6

SD of five healthy volunteers. dThe

into the phenothiazine ring system by means of a FriedelCrafts acylation reaction as described previ0us1y.I~In the previous workI7 it was speculated, but not proven, that Friedel-Crafts acylation of CPZ occurred at the 7-position of the phenothiazine ring system. Examination of the hapten by 13C NMR in the present study confirmed that acylation of CPZ had occurred a t the 7-position. The haptenic molecule containing the carboxylic acid function (5, Figure 1 ) was covalently linked to BSA via an amide bond by a watersoluble carbodiimide method.17 The purity and chemical identity of the hapten (5, Figure 1) are also very important in the production of antibodies specific to CPZNO. If the hapten is contaminated with impurities, the antibodies produced may recognize the impurities as well as CPZNO. For these reasons the chemical integrity and purity of the hapten were evaluated by TLC, NMR, UV, and MS prior to coupling to BSA. Specificity and Sensitivity of the RadioimmunoassayThe antiserum obtained with this approach appeared to be specific according to the criteria suggested by Abraham.17 None of the compounds tested showed significant cross reactions with the antiserum (Table 11).However, since the RIA was to be used to determine CPZNO concentrations in clinical plasma samples which always contain CPZ and a plethora of other metabolites in addition to CPZNO, it was considered necessary to evaluate the specificities of the CPZNO RIA in the presence of CPZ and other metabolites. As shown in Table 11, in the presence of a mixture of CPZ, CPZSO, and 7-OHCPZ (each in five times excess), the apparent plasma CPZNO concentrations as determined by the direct RIA were significantly inflated (p C 0.05, t test). Thus, the extent of cross reaction determined by the criteria of Abrahamz1 alone is not adequate to establish assay specificity. The knowledge of the nature and amounts of crossreacting metabolites or other species can help in designing experiments either to overcome or reduce their interferences. The problem of interference from drug and metabolites in the present assay was overcome by selectively extracting CPZ and its metabolites from plasma, and then analyzing the extracted samples by each of the RIAs.In the extraction RIA procedure, analysis of CPZNO was not affected by the presence of CPZ and the other metabolites (Table 11). The RIA described here is at present the most sensitive analytical method for detection of CPZNO with a quantitation limit of 0.25 ng/mL. The assay made it possible, for the first time, to monitor plasma CPZNO levels for up to 48 h after the administration of a single oral dose of 50 mg of CPZ. ,, and AUC: values for Table IV shows that the mean C CPZNO were higher than the corresponding values for the parent drug, although the mean apparent elimination halflife for CPZNO was shorter than that of CPZ. Craig and coworkers3 reported that plasma levels of CPZNO were 3050% of those of the parent drug in a group of patients given CPZ a t daily dosage levels at least 10-fold higher than the 50mg single dose administered in the present study. It is difficult to make direct comparisons between these studies Journal of Pharmaceutical Sciences / 807 Vol. 76,No. 10, October 1987

because of the vast differences in dosages, but it is not unreasonable that steady-state Cmin levels of CPZNO in plasma might be lower than those of CPZ in view of the shorter apparent elimination half-life of the metabolite. Reliability of the Radioimmunoassay-Also to be considered is the question of assay reliability. It is clear that in the quantitation of CPZ and metabolites in plasma, no analytical method is free from problems. For example, in the GLC-MS method employed by Craig and co-workers,3 the use of sodium hydroxide during the extraction procedure may have led to conversion of some CPZNO into CPZ,l0 resulting in an elevation of CPZ concentrations at the expense of CPZNO. On the other hand, it is possible that there may have been interference in the extraction RIA procedure through cross reaction(s) of the antiserum with unknown metabolites in the plasma. In a preliminary validation study, the extraction RIA was compared with an HPLC method in the measurement of CPZNO levels in four healthy subjects after the administration of a single oral dose of 100 mg of CPZ.23 Plasma levels of CPZNO determined by RIA were either comparable to, or lower than, those determined by HPLC. This suggests that interference from unknown metabolites was unlikely to have been a factor in the measurement of CPZNO by extraction RIA after the administration of a single oral dose of CPZ. Nevertheless it must be borne in mind that the pattern of metabolites in the plasma of patients medicated chronically with CPZ may differ from the single-dose profile in healthy subjects. Consequently, it will be necessary to compare the performances of the extraction RIA and a chromatographic method in the measurement of steady-state levels of CPZNO in patients in order to complete the validation process. Pharmacokinetics of Chlorpromazine and its Metabolites-The application of modern analytical methods to the measurement of phenothiazine antipsychotic drugs and key metabolites in plasma has led to improvements in both assay reliability and sensitivity. These developments have made it possible to generate plasma concentration-time curves for parent drug and metabolites after the administration of low, single, oral doses to healthy subjects, which in turn has led to some interesting problems in the interpretation of pharmacokinetic data. For example, in order to obtain definitive pharmacokinetic parameters it would be ideal if each metabolite could be administered separately to the volunteers, preferably parenterally as well as orally. Since this ideal is impractical, it is necessary to deal with the pharmacokinetic treatment on the basis of data which are available. Hence, in the present study, we have used the term “apparent elimination half-life” in comparing the elimination kinetics of the metabolites. Each “apparent elimination half-life” value was calculated from the elimination rate constant of the last elimination phase measured. We have avoided use of the word “terminal” since it is the sensitivity of the analytical method, rather than any pharmacokinetic consideration, which limits the ability to measure the late phaseIs) of e l i m i n a t i ~ nHence, . ~ ~ on the basis of data available on the parent drug and three primary metabolites measured in the present study, CPZNO had the highest mean C,,, value (24.4 ng/mL), twice that of the parent drug, and the shortest mean apparent elimination half-life (6.7 h), approximately half that of CPZ (Table IV). The mean C,,, and apparent elimination half-life values for CPZSO were comparable to those of CPZ, while free 7-OHCPZ had the lowest C,, (3.7 ng/mL) and the longest mean apparent elimination half-life (24.5 h ) of any of the analytes measured. It must be emphasized, however, that the antiserum for 7-OHCPZ measures the sum of the concentrations of 7-OHCPZ plus 7-OH-Ndesmethyl CPZ,16 so the plasma level pharmacokinetic parameters reported here are hybrid values representing total 808 /Journal of Pharmaceutical Sciences Vol. 76, No. 10, October 1987

plasma primary and secondary metabolites. Furthermore, there is wide intersubject variation, such that these relationships between mean pharmacokinetic parameters do not apply per se to any individual subject. For example, Figure 2 shows the individual profile of subject #3 in whom the C, of CPZSO is much greater than that of the parent drug, while of CPZ is comparable to that of free 7-OHCPZ. the C, In conclusion, the present study has demonstrated that the extraction RIA of CPZNO is applicable to single-dose pharmacokinetic studies. After oral administration of CPZ, its N oxide metabolite is produced in significant concentrations.

References and Notes 1. Usdin, E. CRC Crit. Reu. Clin. Lab. Sci. 1971, 2, 347-391. 2. Cooper, T. B. Clin. Pharmaokinet. 1978,3, 14-38. 3. Crai , J . C.; Gruenke, L. D.; Hitzeman, B. A.; Holaday, J.; Loh,

hf

M. In Phenothiazines and Structurally Related Drugs: Basic and Clinical Studies; Usdin, E.; Eckert, H.; Forrest, I. S., Eds.; Elsevier North Holland: Holland, 1980; pp 129-132. 4. Lewis, M. H.; Widerlov, E.; Knight, D. L.; Kilt, C. D.; Mailman, R. B. J. Phnrmacol. Ex Ther 1983,225,539-545. 5. Alfredsson, G.; Wiesef’ F.-A..; Skett, P. Psychopharmacology

1977,53, 13-18. 6. Krieglstein, J.; Rieger, H.; Schutz, H. Eur. J . Pharmucol. 1979,

-fifi-, -363-3717 -- - . -.

7. Cope, A. C.; Trumbull, E. R. Organic Reactions; Wiley: New York, 1960; vol. 11, p 317. 8. Craig, J. C.; Mary, N. Y.; Roy, K. K. Anal. Chem. 1964, 36, 1142-1 143. 9 . Essien, E. E.; Cowan, D. A.; Beckett, A. H. J. Pharm. Phnrmacol. 1975,27, 3 3 4 3 4 2 . 10. Hubbard, J. W.; Cooper, J. K.; Hawes, E. M.; Jenden, D. J . ; May, ~~~~

~~

~

P. R. A.; Martin, M.; McKay, G.; Van Putten, T.; Midha, K. K. Therap. Dru Monitor 1985, 7, 222-228. 11. McKay, G.; r J K ;Hawes, E. M.; Hubbard, J. W.; Martin, M.: Midha. K. Ffheherau. Drug Monitor 1985. 7. 472-477. 12. Hawes,E.’M.; Hubbard, J. W.7 Martin, M.; McKay, G.; Yeung, P. K. F.; Midha, K. K. Therap. Drug Monitor 1986,8, 37-41. 13. Turano, P.; Turner, W. J.; Donab-, D. In Phenothiazines and Structurally Related Drugs; Forrest, I. S.;Carr, C. J.; Usdin, E.; Eds.; Raven: New York, 1974; 315-322. 14. Midha, K. K.; Loo,J. C. K.; Huglard, J. W.; Rowe, M. L.; McGilveray, I. J. Clin. Chem. 1979,25, 166-168. 15. Yeung, P. K. F.; Hubbard, J. W.; Cooper, J. K.; Midha, K. K. J. Pharmacol. Ex Ther. 1983,226, 833-838. 16. Yeung, P. K. McKay, G.; Ramshaw, I. A.; Hubbard, J . W.; Midha, K. K. J. Pharmacol. Exfi Ther. 1985,233,816-822. 17. Hubbard, J. W.; Midha, K. K.; cGilveray, I. J.; Cooper, J. K. J. Pharm. Sci. 1978, 67, 1563-1571. 18. Jovanovoic. M. V.; Biehl, E. R. J. Heterocycl. Chem. 1984, 21,

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Iff

1589-1592. 19. Erlanger, B. F.; Boret, F.; Beiser, S. M.; Liberman, S. J. Biol. Chem. 1957,228, 713-727. 20. Powell, B.; Garola, R. E.; Chamness, G. C.; McGuire, W. L. Cancer Res. 1979,39, 1678-1682. 21. Abraham, G. E. J. Clin.Endocrinol. Metah. 1969.29, 866-870. 22. Parker, C. W. Radioimmunoassay of Biological Active Comounds; Prentice-Hall: New Jersey, 1976; pp 4-23. 23. b i d h a , K. K.; Hubbard, J. W.; Cooper, J . K.; Gurnsey, T.;

Hawes, E. M.; McKay, G.; Chakraborty, B. S.;Yeung, P. K. F. Therap. Dru Monitor., in press. 24. Hubbard, J.fV.; Canes, D.; Midha, K. K. Arch. Gen. Psychiatry 1987,44, 99-100.

Acknowledgments The Medical Research Council of Canada, Program Grant PG-34, and studentshi to P K F. Yeung are atefully acknowledged. The authors thank Er. K.‘Bailey of the Heaf6h Protection Branch, Health and Welfare Canada, for obtainin and inte retin the I3C NMR, Dr. A. A. Manian of the National fnstitute o%lent$ Health for the enerous supply of authentic samples of CPZ metabolites, and Drs. M. Hawes and G. McKay of the College of Pharmacy, University of Saskatchewan, for their helpful discussions. The Canadian Red Cross is acknowledged for the generous gift of outdated plasma used in this study. Part of the material in this paper has been presented a t the 45th International Congress of Pharmaceutical Sciences Meeting, Montreal, Canada, September, 1985.

8.