Radioimmunoassay for the 7-Hydroxy Metabolite of Trifluoperazine and its Application to a Kinetic Study in Human Volunteers

Radioimmunoassay for the 7-Hydroxy Metabolite of Trifluoperazine and Its Application to a Kinetic Study in Human Volunteers MANICKAMARAVAGIRI, EDWARDM. HAWES, AND

KAMAL K.

MIDHA'

Received May 6, 1985, from the College of Pharmacy, University of Saskatchewan, Saskatoon, Saskatchewan, S7N OW0 Canada. for publication August 14, 1985. Abstract 0A hapten derivative of the 7-hydroxymetabolite of trifluoperazine, 7-hydroxy-l0-[[3-[4-(2-carboxyethyl)-l-piperazinyl]propyl]]9-tri-

fluromethyl-1OKphenothiazine, was synthesized and coupled to bovine serum albumin. Immunization of New Zealand white rabbits with this drug-protein conjugate resulted in the production of antisera, one of which was subsequently utilized in the development of an RIA procedure. The described RIA for the first time enables the quantitation of the 7-hydroxy metabolite of trifluoperazine in human plasma after oral administration of single and therapeutic doses of trifluoperazine, in which 20 pg of the nonconjugated 7-hydroxy metabolite in 200 pL of plasma can be measured with a CV of <3% in B/Bo readings. Similar results were obtained by this assay procedure with or without the selective extraction of the 7-hydroxy metabolite and in the presence or absence of a large excess of trifluoperazine and other suspected major metabolites, such as the sulfoxide and v'-oxide metabolites. This RIA procedure, together with a previously developed RIA for trifluoperazine, was used to directly determine plasma concentrations of trifluoperazine and its 7-hydroxy metabolite after administration of a single 5-mg oral dose of trifluoperazine to six healthy male volunteers. The mean f SD for the peak concentration (C,,,,,)the , time to ,, C the area under the curve from 0 to 24 h and, the apparent terminal elimination half-life for the 7-hydroxy metabolite were found to be 0.86 2 0.2 ng/mL, 6.2 2 1.6 h, 11.1 f. 4.9 ng . himL, and 10.6 r 5.7 h, respectively. The antiserum used to measure the 7-hydroxy metabolite of trifluoperazine crossreacted significantly with the 7-hydroxy metabolites of fluphenazine and prochlorperazine and, thus, can be used to develop RIA procedures for these and, perhaps, other 7-hydroxy metabolites of piperazine type phenothiazine drugs.

The phenothiazine antipsychotic agents are extensively metabolized to numerous metabolites by various routes, which include aromatic hydroxylation at the 7-position of the phenothiazine ring, ring S-oxidation, N-dealkylation, and Noxidation. The importance of each individual metabolite, with respect to therapeutic and/or toxic effects is not clearly delineated. The 7-hydroxy metabolites are reputed to contribute to antipsychotic activity. In fact, in the case of chlorpromazine, when equal oral doses of chlorpromazine and its 7-hydroxy metabolite were compared in five chronic schizophrenics, the 7-hydroxy metabolite was found to be at least as effective as chlorpromazine.' Also, some workers have demonstrated that the clinical response of schizophrenic patients receiving chlorpromazine correlates better with plasma level ratios involving active drug-inactive drug (such as chlorpromazine plus the 7-hydroxy metabolite-sulfoxide metabolite) rather than with the steady state levels of chlorpromazine.z6 With the piperazine-type, phenothiazine, antipsychotic agent trifluoperazine (l),the 7-hydroxy metabolite (2) has been identified as a metabolite in humans7 and animals.8 The importance in clinical therapy of the presumed major metabolites of trifluoperazine, including this 7-hydroxy compound, is unknown. The major reason for this is the slow development of suitable, sensitive, and specific analytical methods to measure the concentrations of metabolites in the 1196 / Journal of Pharmaceutical Sciences Vol. 74, No. 11, November 1985

Accepted

plasma of patients under treatment with trifluoperazine. This slow development can be largely attributed to the reputed instability and adsorptive loss of phenothiazines and their metabolites in all stages of sample handling, and the low plasma levels encountered due to the low doses of trifluoperazine given to patients. However, recently a n RIA was r e p ~ r t e d which ,~ enabled the determination of 60 pg of the sulfoxide metabolite of trifluoperazine in 200 p L of plasma with a CV of <3%. This RIA procedure was used in a single dose study involving healthy volunteers to determine for the first time the plasma concentrations of the sulfoxide metabolite after administration of trifluoperazine. The ultrasensitive biological method of RIA has advantages over chemical methods of analysis, such as GC-MS, in that extraction of the biological sample may not be necessary and because such methods are more amenable to routine clinical monitoring. This paper reports a rapid, sensitive, and specific RIA to determine for the first time plasma concentrations of the 7-hydroxy metabolite of trifluoperazine. By using this method and a previously reported RIA for trifluoperazine,lo the concentrations of trifluoperazine and the 7-hydroxy metabolite were directly determined in plasma samples up to 24 h after the administration of a single 5-mg oral dose of trifluoperazine to six healthy volunteers.

I

CH2CH2CH2-

1

n

W - C H ,

2

Experimental Section Materials and Methods-The 7-hydroxy, sulfoxide, N4'-oxide, N demethyl, N-demethyl-7-hydroxy, and N-demethyl sulfoxide metabolites were synthesized in this laboratory by modified literature procedures.11-13 The other phenothiazines and their metabolites were donated (Smith, Kline and French Laboratories, Philadelphia, and RhBne Poulenc Pharmaceuticals, Montreal, Canada). The N methyl tritiated 7-pyranyloxy derivative of trifluoperazine was custom synthesized by Amersham Corporation, Oakville, Canada. Both the phenothiazine ring tritiated and N-10 side chain N-methyl tritiated derivatives of trifluoperazine were purchased from commercially available sources (Nuclear Research Centre, Negev, Israel and New England Nuclear, Lachine, Canada, respectively). All solvents were reagent and/or HPLC grade and were used without further purification. Bovine serum albumin was obtained from Sigma Chemical Company, St. Louis, MO. The dextran-coated charcoal and the various buffers were prepared in this laboratory whenever required. Liquid scintillation counting, in which a commercial cocktail (Ready-Solve H P h , Beckman Instruments, Fullerton, Canada) was used, was performed in a counter equipped with automatic quench compensation (LKB RackBeta, model 415, Fisher Scientific Company, Toronto, Canada). Plastic sheets, precoated with 0022-3549/85/1100-1 196$01.OO/O 0 1985, American Pharmaceutical Association

silica gel (250 pm thickness), with or without a fluorescence indicator for TLC, and silica gel (60-200 mesh) for column chromatography were procured from commercially available sources (Terochem Laboratories, Limited, Edmonton, Canada and J. T. Baker Chemical Company, Phillipsburg, NJ, respectively). All reagents for organic synthesis were purchased from Aldrich Chemical Co., Milwaukee, WI. Organic solvents were evaporated using a Buchi Rota-Vapor, model RE 120, Brinkmann Instruments, Rexdale, Canada. TLC spots were observed under shortwave UV light. Melting points of synthesized compounds were determined with a Gallenkamp melting point apparatus and are uncorrected. 'H NMR spectra were determined in MezSO-d6 on a Varian T-60 instrument with tetramethylsilane as the internal reference. Lowresolution electron-impact mass spectrometry (EI-MS) of probe samples were recorded on a VG Micromass MM16F instrument, a t 70 eV, equipped with a VG 2025 data system. All the EI-MS and 'H NMR spectra of synthesized compounds were consistent with assigned structures. Microanalyses for samples dried over phosphorus pentoxide a t 60°C under reduced pressure were performed at the Department of Chemistry, University of Saskatchewan. Quantitative UV spectrophotometry was performed using a Pye Unicam SP 1700 instrument. Radiolabeled tracers were purified by a HPLC system consisting of a solvent delivery system (M45; Waters Associates, Milford, MA), a valve loop injector fitted with a 2-mL loop (model 7120; Rheodyne, Berkley, CAI, a radial compression separation system (Z module; Waters Associates), a CI8 radial pack column (10 pM), a mobile phase composed of 10% sodium acetate buffer (0.05 M, pH 6.5) in methanol, and a fixed wavelength (254 nm) Perkin-Elmer model LC15B UV detector.

~ H ~ C H ~ C H ~ C I

3

Hogjr;~cF, I

ylamine; 100:5:5). Further purification involving use of a silica gel column and a dich1oromethane:methanol:diethylamine(80:15:5) solvent mixture gave 4a as a pale brown semi-solid; m.p. of the dihydrochloride, 224-226°C; 'H NMR 6 1.9 (t, 2, propyl central CH,), 2.5 (m, 14, piperazine methylene groups, CHz-piperazine, CHzCHz-ester), 3.3 (s, 3, ester CH3), 3.9 (t, 2, CH2-phenothiazine), and 7.1 ppm (m, 6, ArH); MS miz 495 (M+'). Anal.-Calc. for CZ4Hz8F3N3O3S.2HC1: C, 50.7; H, 5.32; N, 7.39. Found: C, 49.9; H, 5.55; N, 7.18. The remaining portion (0.4 g) of the crude ester 4 was hydrolyzed by refluxing in methanol (30 mL) with 3 M NaOH (2 mL) for 1.5 h. To remove the pyran protecting group, the mixture was adjusted to pH 2 with 5 M HCI solution. The mixture was then adjusted to pH 7.0 with saturated NaZCO3solution. The product was extracted into dichloromethane (5 x 20 mL), and the dried (MgSO,) extract was evaporated under reduced pressure to give the desired hapten 5 (0.32 g, 96% yield) as pale brown scales, m.p. 184-186"C, which showed one spot on TLC (R, 0.3; benzene:methanol:diethylamine; 1001O:lO); 'H NMR 6 1.9 (t, 2, propyl central-CH2), 2.5 (m, 14, piperazine methylene groups, CHz-piperazine, CH2CHP-acid),3.9 (t, 2, CH,-phenothiazine), and 7.1 ppm (m, 6, ArH); MS at miz 481 (M+'). Preparation of the Immunogen ( 6 h T h e hapten 5 was coupled to bovine serum albumin by the mixed-anhydride method.14 However, due to the insolubility of 5 in the dioxane used in this method, the following procedure was necessitated. To a solution of hapten (0.25 g, 0.51 mmol) in dry dimethylformamide (2 mL), isobutyl chloroformate (70 wL, 0.51 mmol) was added, and the resultant mixture was stirred for 60 min. This mixture was then added to a solution of bovine serum albumin (0.36 g) in a water:dimethylformamide mixture (15:5). The pH was maintained between 8.5 and 9.5 by the addition of 2 M NaOH solution. The mixture was stirred overnight at 4°C and then dialyzed, first against bicarbonate buffer (0.1 M; pH 8) a t 4°C for 4 h, changing the buffer every 1 h, then against acetate buffer (0.1 M; pH 5.2) a t 4°C for 4 h, changing the buffer every 1 h, then against running distilled water a t 25°C for 6 h, and finally against distilled water for 20 h at 4"C, changing the water every 4 to 6 h. The clear aqueous solution of the immunogen 6 was then lyophilized and obtained as a fibrous crystalline white solid (0.35 9). A blank was prepared by subjecting the bovine serum albumin to the same coupling conditions, but without the hapten. The number of hapten residues coupled per mole of bovine serum albumin conjugate was determined by the UV method14 to be 23.

1

HcNH,

Scheme I

\1

HCI

Nal

Synthesis of the Hapten, 7-Hydroxy-l0-[[3-[4-(2-~arboxyethyl)l-piperazinyl]propyl]]-2-trifluoromethyl-lO~-phenothiazine (5kTo a solution of l0-(3-chloropropyl)-7-tetrahydropyranyloxy-2-trifluoromethyl-10H-phenothiazine(3) (0.5 g, 1.1 mmol)13 in methyl ethyl ketone (20 mL) was added l-(2-methoxycarbonylethyl)piperazine (0.2 g, 1.2 mmol) and NaI (50 mg). The mixture was heated under reflux overnight. The organic solvent was evaporated, and the residue was taken up in dichloromethane. The organic phase was dried over MgSO, and the solvent was evaporated under reduced pressure to yield 4 (0.55 g, 84% yield) as a crude yellow oil. TLC (benzene:methanol:diethylamine, 100:5:5) of this oil showed an intense spot (R, 0.6) 4 and a very weak spot (R, 0.8) 3. The identity of 4 was indirectly established by conversion of this oily 7-tetrahydropyranyloxy compound into its solid 7-hydroxy derivative, 4a. A portion of the oil, 4, (100 mg) was dissolved in 2 M HCI solution (10 mL). Compound 3 was subsequently removed by ether (3 x 20 mL) extraction. The aqueous layer was neutralized to pH 7.58.0 by adding saturated Na2C03 solution and then extracted with dichloromethane (4x 15 mL). The dried (anhydrous MgSOJ extract on evaporation under reduced pressure gave a pale brown semi-solid which showed a single spot on TLC ( R f0.4; benzene:methanol:dieth-

CH~CHZCH~CI

3 Scheme I1

Synthesis of the 7-Hydroxy Derivative of [3H]trifluoperazine, 7Hydro~y-l0-[3-(4-[~HJmethyl1-piperazinyl)-propyl]-2-trifluoromethyl-10H-phenothiazine(9)-The tritiated N-methyl derivative of the 7-hydroxy metabolite of trifluoperazine, supplied with a pyran protected hydroxyl group (10-[3-(4-[3H,]methyI-l-piperazinyl)propyll-7-tetrahydropyranyloxy - 2 - trifluoromethyl-1 OH-phenothiazine) (%),was custom synthesized according to Scheme 11. The N-demethyl starting material (l0-[3-~l-piperazinyI~propylJ-7-tetrahydropyranyloxy-2-trifluoromethyl-1OH-phenothiazine) (7), a previously reported Journal of Pharmaceutical Sciences / 1197 Vol. 74, No. 11, November 1985

compound,12 was prepared in our laboratory by a new route involving N-alkylation of piperazine with 3. Whenever tracer, 9, was required, the commercially supplied tritiated compound, 8, was readily deprotected by the following procedure. To the commercially supplied solution of 8 (200 pL, 1 mCi/mL) was added 8 M HCl solution (30 pL). The resultant mixture was stirred well, basified by the addition of saturated Na2C03 solution, and purified by injection into an HPLC system. The specific activityg of the purified tracer, 9 was found to be 80 Ciimmol. The unlabeled 7-hydroxy compound was added to this tritiated material to give a specific activity of 50 Ci/mmol. This latter tracer was the material subsequently utilized in the development of RIA procedures for the 7-hydroxy metabolite of trifluoperazine. The purity of the tracer was checked: whenever required for RIA, by TLC (Rf0.6;benzene:methanol:diethylamine,100:5:5).The purity was always >95%. The working solution of the tracer was made by diluting the freshly collected tracer from the HPLC system with 0.01 M HCI, such that the diluted solution contained 3000-3500 cpm/pL. Immunization-Eight New Zealand white rabbits (Animal Resources Centre, University of Saskatchewan, Saskatoon, Canada) each received a primary immunization of 1 mg of immunogen emulsified with 0.5 mL of Freund's complete adjuvant (Gibco, Grand Island, NY) and 0.5 mL of isotonic saline. Each dose was administered as four intradermal injections on both sides of the dorsal column. Thereafter, a t 2-week intervals, the rabbits were intradermally immunized with 1 mg of immunogen, except that 0.5 mL of Freund's incomplete adjuvant was added. After five intradermal injections, the sera of all eight rabbits showed binding activity to the [3H]7-hydroxytrifluoperazine tracer. The rabbits were then given five intravenous booster doses of 1mg of the immunogen in 0.25 mL of isotonic saline, administered via the ear vein at 2-week intervals. The six rabbits, which produced antisera of adequate titer (1in 3000 to 1in 6000), were sacrificed and each antiserum was lyophilized and stored a t -20°C. The lyophilized antiserum of one of the rabbits was used for the RIA of the 7-hydroxy metabolite of trifluoperazine. Direct Radioimmunoassay Procedure-A stock solution of the 7hydroxy derivative of trifluoperazine was made by dissolving the 7hydroxy compound in 0.01 M HCl. Subsequently, standard solutions containing 0.1, 0.25, 0.5, 1, 2.5, 5, and 10 ng of the 7-hydroxy metabolite/mL were prepared in pooled plasma (Canadian Red Cross, Saskatoon). The working antiserum solution was prepared by dissolving the lyophilized antiserum (1 mgi25 mL) in phosphate buffer (0.2 M; pH 7.2), and 250 pL of this solution was added to each assay tube. To each polystyrene tube (12 x 75 mm) containing a 200 pL plasma sample (spiked or from a volunteer), was added 250 pL of phosphate buffer (0.2 M, pH 7.2) and 5 pL of the solution containing the 7-hydroxy derivative of [3Hltrifluoperazine (15,000-17,500 cpm). The contents of this tube were mixed (Delux Mixer Scientific Products, IL) for 5 s and the antiserum solution (250 pL) was added. All the additions were carried out in an ice bath. The tubes were mixed (Delux Mixer) for 5 s, incubated in a water bath (Dubnoff Metabolic Shaking Water Bath, Precision Scientific, IL) at 37°C for 1 h, cooled in an ice bath for 10 min, and chilled dextran-coated charcoal suspension (1mL) was added. Each tube was again mixed for 5 s and subsequently incubated for 30 min at 4°C. The samples were then centrifuged (Beckman model TJ6 Centrifuge, 1720xg for 10 min at 4"C), and the supernatant solution from each tube was decanted into a scintillation vial (Fisher Scientific Company) containing 10 mL of scintillation cocktail, mixed well, and counted for 5 min. Each plasma sample, spiked standard or unknown, was assayed in triplicate. A pair of tubes, without the addition of antiserum, served as a control for nonspecific binding. The calibration curve was constructed by plotting logit values of B/Bo on the y-axis and log values of the 7-hydroxy metabolite plasma concentrations on the x-axis, where B and Bo are the antibody-bound radioactivity in the presence and absence of the 7-hydroxy metabolite of trifluoperazine, respectively. Logit B/Bo is defined as:

logit B/Bo = log e

B/Bo 1 - B/Bo

Extraction Radioimmunoassay Procedure--To each 15-mL teflon-coated screw-capped glass test tube was added a 1-mL plasma sample (spiked or from a volunteer) and saturated Na2C03 solution 1198 /Journal of Pharmaceutical Sciences Vol. 74, No. 1 7 , November 1985

(100 pL). Each tube was sequentially extracted with 1% isopropyl alcohol in pentane (5 mL) and ether (2 x 5 mL). Each bulked ethereal extract was evaporated to dryness. The residue was dissolved with 0.01 M HCl (200 pL) and reconstituted to 1 mL by the addition of blank-pooled plasma (0.8 mL, Canadian Red Cross). This reconstituted extract was then analyzed as in the above-described direct RIA procedure. The efficiency of the above extraction procedure for the 7-hydroxy metabolite of trifluoperazine was checked by repeating the extraction procedure with spiked samples containing known amounts of the tritiated metabolite. Of the total radioactivity added, -90% was found to be extracted from samples containing 0.1-5.0 ng/mL of the 7-hydroxy metabolite of trifluoperazine. Plasma Samples-Six healthy male volunteers, after an overnight fast, were each given a 5-mg dose orally of trifluoperazine (Stelazine, Smith Kline and French Laboratories) with 50 mL of water (the protocol was approved by the local Ethics Committee on Human Experimentation). Blood samples were withdrawn by venipuncture at 0 (predose) and 0.5, 1, 1.5, 2, 3, 4.5, 6, 8, 12, and 24 h postdose. Care was taken to avoid contact between the blood and the rubber stoppers of the evacuated heparinized tubes (Vacutainer, Becton and Dickenson, Mississauga, Canada) used for blood collection, as spurious results have been found in the RIA determination of trifluoperazine when blood has been allowed to touch the rubber stopper of these evacuated tubes.16 The plasma was immediately separated and stored a t -20°C until analysis by the procedures described above for the 7-hydroxy metabolite of trifluoperazine and the previously reported RIA method for trifluoperazine.lO The areas under the plasma concentration-time curves from 0 to 24 h (AUCo24) were calculated by the linear trapezoidal method. Plasma halflives were estimated from the raw data using the equation: tllz = O.693/Ke1,where kel is the calculated rate constant of elimination.

ResuIts Assay Specificity and Sensitivity-The extent of tracer binding at zero concentration of the 7-hydroxy metabolite of trifluoperazine was determined at 30, 60, 90, and 120 min and at temperatures of 4°C and 37°C for the first incubation step in the assay procedure. The optimum conditions for the assay thus found was an incubation time of 60 min at 37°C) where the Bo was -40% of total tracer added. The optimum time for the second incubation step was similarly found to be 30 min at 4°C when incubation times of 10,20,30,40,50,and 60 min were investigated. The assay has the sensitivity to measure 20 pg of the 7hydroxy metabolite of trifluoperazine when 200-pL plasma samples are used. Under the assay conditions described above, the standard curve was linear from 0.1 to 10 ng of the 7-hydroxy metabolite/mL of plasma. A typical calibration curve in this range was defined by the equation: logit y = -2.3 logl$ + 0.87 (r2 = 0.9995). A pilot study in two volunteers indicated that plasma concentrations of the 7hydroxy metabolite were low (<5 ng/mL) following administration of a 5-mg oral dose of trifluoperazine to healthy volunteers; therefore, a working calibration curve of 0.1 to 5 ng/mL was used for the plasma concentration determinations of the samples described above. These unknown concentrations were determined by running a calibration curve with each set of unknown samples. Concentrations of the 7hydroxy metabolite below 0.1 ng/mL are estimates. The specificity of the antiserum was initially assessed by the criteria of Abraham.lGThe antiserum did not cross-react markedly with trifluoperazine or the available, supposed, major metabolites of trifluoperazine, including the sulfoxide, N-demethyl, and N4 -oxide metabolites. However, as expected, there was significant cross-reactivity with the N-demethyl-7-hydroxy metabolite of trifluoperazine, as well as with the 7-hydroxy metabolites of other piperazine-type phenothiazines (Table I). In order to check cross-reactivities in a manner more closely resembling the clinical situation, plasma samples

Table I-Cross-Reactions" of Antiserum Against the 7-Hydroxy Metabolite of Trifluoperazine Cross-Reaction, Compounds Tested %

7-Hydroxy metabolite of trifluoperazine Trifluoperazine Sulfoxide metabolite of trifluoperazine N4'-Oxidemetabolite of trifluoperazine N-Demethyl metabolite of trifluoperazine N-Demethyl sulfoxide metabolite of trifluoperazine N-Demethyl-7-hydroxymetabolite of trifluoperazine 7-Hydroxy metabolite of triflupromazine 7-Hydroxy metabolite of fluphenazine 7-Hydroxy metabolite of prochlorperazine 7-Hydroxy metabolite of chlorpromazine 7,E-Dihydroxy metabolite of perphenazine

100

Table Ill-lntra- and Interassay Variance of the Radioimmunoassay for the 7-Hydroxy Metabolite of Trlfluoperazlne B/Bo Values when Spiked with the 7-Hydroxy Metabolite, ng/mL of Plasma

<1 <1 <1 <1 <1

Interassay variancea Mean

27 16 76 52

intra-assay variance Mean f SD

f SD Yo

cv,

cv, %

8 <1

aCross-reactionswere assessed by the criteria of Abraham (ref. 16). containing the 7-hydroxy metabolite of trifluoperazine over the standard curve range were additionally spiked with trifluoperazine and its other suspected major metabolites a t five times the concentration of the 7-hydroxy metabolite. The 7-hydroxy metabolite concentrations were subsequently determined by the direct RIA procedure. These experiments were repeated at least four times, and the values obtained were compared with those for the 7-hydroxy metabolite standard curve samples prepared a t the same time in the usual manner. There were no significant differences (p > 0.05) in the values obtained for the 7-hydroxy metabolite among samples of the same concentration of the 7-hydroxy metabolite, with or without trifluoperazine, with or without the sulfoxide metabolite, and with or without the N4-oxide metabolite when compared using the unpaired t test. Furthermore, similar results were obtained when trifluoperazine and the sulfoxide and N4 -oxide metabolites were all added together a t five times the 7-hydroxy metabolite concentrations to plasma samples containing the 7-hydroxy metabolite over the concentration range of the standard curve (Table 11). Assay Precision: Intra- and Interassay VariationsBoth intra- and interassay variations were determined with spiked plasma standards prepared according to the described procedure. Interassay variations were calculated from the B/Bo data obtained for the assay on six separate days of analysis, whereas intra-assay variations were determined from B/Bo data obtained on a single day. The CV was <3%for both intra- and interassay determinations over the range of 0.1 to 5 ng/mL (Table 111). Plasma Concentrations of Trifluoperazine and Its 7Hydroxy Metabolite--Plasma samples obtained after administration of single 5-mg oral doses to six healthy volunteers were assayed by direct RIA procedures, which can

5

2.5

1

0.5

0.25

0.1

32.8

49.7 0.9 2.0

70.5 1.8 2.5

84.2 1.3 1.6

91.3 0.8 1.0

95.4 0.5 1.0

49.9 0.6 1.2

71.7 0.8 1.1

84.4 0.4 1.6

91.9 0.9 1.0

96.5 1.2

0.7 2.0 32.4

0.4 1.2

1.2

'

a Interassay variance was calculated from assay readin s of plasma standards obtained on six different days of analysis. Intra-assay variance was calculated from five assay readings of plasma standards obtained on a single day of analysis.

accurately determine trifluoperazine and its 7-hydroxy metabolite a t concentrations as low as 0.25 and 0.1 ng/mL, respectively. The sensitivities of the assays were such that plasma concentrations of both species could be accurately determined in all samples obtained a t the last time point (24 h) postdose. However, trifluoperazine plasma concentrations could not be accurately determined in samples obtained at 0.5 h for three volunteers, while using the more sensitive assay for the 7-hydroxy metabolite, this was the case at the 0.5 and 1 h time points for five and one volunteer(s), respectively. The plasma concentration-time profiles obtained for the six volunteers showed considerable intersubject variations in the concentrations of both trifluoperazine and its metabolite (the mean curves are shown in Fig. 1). The mean C, t,, and AUCo-24obtained for trifluoperazine in the six volunteers are in agreement with the previously reported values obtained by RIA following administration of 5-mg single oral doses of trifluoperazine to healthy volunteers.9J7 The AUCo-24 ranged from 17.5 to 35.6 ng h/mL for trifluoperazine and from 4.8 to 15.6 ng WmL for the 7-hydroxy metabolite. The values of AUCo-24 for the metabolite were, on the average, 54.1% of those of the parent drug (range, 13.5-83.8%). The time required to reach the peak concentrations (t,,,), which ranged from 2 to 6 and from 4.5 to 8 h for trifluoperazine and its 7-hydroxy derivative, respectively, was longer for the metabolite in all six volunteers. The peak concentration (Cm& which was greater for trifluoperazine than its 7-hydroxy metabolite in all six volunteers, ranged from 0.52 to 1.10 ng/mL for the hydroxy compound and from 1.05 to 2.75 ng/mL for trifluoperazine (Table IV). In all six volunteers, the plasma concentration of the 7-hydroxy metabolite after the C,, was reached de-

-

Table ll-Determination by Direct Radioimmunoassayof the 7-Hydroxy Metabolite in Plasma Samples Spiked with a Fivefold Excess of Trifluoperazine, the Sulfoxide Metabolite, or the N4 -Oxide Metabolite or all Three of These" Plasma Concentration of the 7-Hydroxy Metabolite of Trifluoperazine, ng/mL * SD Compound(s)Added n 5 2.5 1 0.5 0.25 0.1 Trifluoperazine Sulfoxide metabolite of trifluoperazine N'"-Oxidemetabolite of trifluoperazine Trifluoperazine plus its sulfoxide and p'-oxide metabolites

4

4.9 f 0.4 5.3 f 0.4

2.4 2.5

4 4

5.0 f 0.4 5.2 2 0.4

2.4 2.5

4

f

k

0.2 0.1

* 0.2 2

0.2

* 0.07

0.48 f 0.06 0.50 f 0.04

0.24 0.23

0.02

0.04

0.09 0.1 1

f

0.90 f 0.06

f

0.03

1.00 t 0.10 1 .OO 0.08

0.45 f 0.06 0.44 2 0.06

0.25 k 0.04 0.22 f 0.04

0.11 0.10

f

0.02 0.01

0.90

f 0.16

*

f

f

"At each standard curve concentration, the mean values found for samples containing added trifluoperazine and/or metabolite(s) did not differ significantly (p > 0.05) from the standards containing only the 7-hydroxy metabolite determined in the same experiment by the unpaired t-test. bConcentration of the compound is five times the concentration of the analyte.

Journal of Pharmaceutical Sciences / 1199 Vol. 74, No. 11, November 1985

5'0

z

1

If I

11 I

5

10

20

15

25

30

TIME. h

Flgure 1-Mean plasma trifluoperazine (0)and the 7-hydroxy metabolite (A)concentrations in six healthy volunteers after single 5 mg oral doses of trifluoperazine dihydrochloride. Each point represents mean f SD.

clined monoexponentially, and the tli2 from C,,, to 24 h ranged from 5.1 to 18.5 h. The plasma concentrations of the 7-hydroxy metabolite of trifluoperazine for all plasma samples from the six volunteers were also determined by the extraction RIA procedure. When the values obtained from the direct RIA procedure 01) were plotted against these values ( x ) , a straight line was obtained which could be defined by the equation, y = ~ ( 1 . 2 6 ) + (-0.1) (r2= 0.864). The 95%confidence bounds (k0.28) of the slope (1.26) of this regression line were calculated, and the slope was not significantly different from 1.0 (p < 0.05). These results indicate that the direct and extraction RIA procedures compare favorably.

Discussion The synthetic route to a hapten, which could be utilized to develop antibodies to the 7-hydroxy metabolite of trifluoperazine, required protection of the 7-phenolic group. The use of the tetrahydropyranyl protecting group enabled N-alkylTable IV-Pharmacokinetic

ation and hydrolysis reactions to be carried out, with facile removal of the protecting group with acid in the final step (Scheme I). This route allowed synthesis of the desired hapten, 5 , where the N-methyl group of the 7-hydroxy metabolite of trifluoperazine was replaced by a n N-(2-carboxyethyl) group. Recrystallization of this aliphatic acid derivative proved difficult and, therefore, the identity of the dihydrochloride salt of the methyl ester, derivative 4a, of the hapten was established by 'H NMR, EI-MS, and elemental analysis. The organic synthesis of 7-hydroxy derivatives of phenothiazine drugs require multiple steps from starting materials which are themselves not readily accessible. Therefore, in view of published r e p o r t ~ ~concerning ~J~ the microsomal hydroxylation of chlorpromazine and the availability to us of two types of [3Hltrifluoperazine, a n attractive source of the desired tracer required for the RIA of the 7-hydroxy metabolite of trifluoperazine appeared to be a similar incubation with aged sheep liver microsomes of these available tritiated derivatives. Unfortunately, the ring labeled trifluoperazine gave a 7-hydroxy derivative with high, nonspecific binding (-50%) when incubated with plasma, whereas the [N-C3H& labeled trifluoperazine derivative gave the 7-hydroxy derivative in poor yields (-5%). However, since this latter tracer desirably gave low nonspecific binding when incubated with plasma, a tracer with the tritium label in the N-CH3 group was prepared synthetically (Scheme 11).The labeled tetrahydropyranyl-protected compound, 8, was stored a t -20°C until required, and the desired tracer, 9, was obtained by deprotection with acid and subsequent purification by HPLC. The tracer had identical chromatographic properties (TLC and HPLC) to a synthetically prepared unlabeled sample.12 The development of this reported RIA procedure was based on the successful approach taken to separately develop RIA procedures for trifluoperazine20.21 and its sulfoxide metabolite? in which the drug molecule was linked to bovine serum albumin via a two-carbon bridge attached through the N-10 side chain of either the trifluoperazine structure or the sulfoxide metabolite structure. Thus, a n RIA was developed where the antisera was raised to an immunogen 6 with a 7hydroxyphenothiazine nucleus containing a n N-10 side chain linked to bovine serum albumin. The antisera raised to such a n immunogen should be specific to the ring portion of the drug molecule, and in fact, the antiserum reported here did not cross-react significantly with trifluoperazine or its sulfoxide metabolite. Also, the antiserum cross-reacted less significantly with the tested 7-hydroxy metabolites of aliphatic type phenothiazines, namely chlorpromazine and triflupromazine. On the other hand, there was significant crossreactivity with the available 7-hydroxy metabolites of other

Parameters' of Trifluoperazine and its 7-Hydroxy Metabolite

AUCO-24

ng.h/mLC

years 1 2 3 4 5 6

Mean t S.D

77.5 74.5 79.0 79.4 87.3 73.2 78.5 5.0

20 27 20 23 20 23 22.2 2.8

Met.b

Drug

Met.

Drug

Met.

Drug

6 8 6 8 4.5 4.5 6.2 1.6

3 6 3 6 2 3 3.8 1.7

1.1 0.86 1.06 0.87 0.52 0.76 0.86 0.2

1.58 1.05 1.88 1.79 2.75 1.59 1.80 0.56

13.5 15.5 12.0 15.6 4.8 5.3 11.1 4.9

17.5

18.5 19.5 24.0 35.6 22.5 22.9 6.7

AUCO-24

Drug, YO

Met.

Drug

77.1 83.8 61.7 65.0 13.5 23.6 54.1 28.9

7.0 16.8 10.0 18.5 6.1 5.1 10.6 5.7

7.2 15.0 10.3 10.5 16.1 20.8 13.3 4.9

a Pharmacokinetic parameters of trifluoperazine and its 7-hydroxy metabolite in the plasma of six healthy volunteers after a 5 mg oral dose of trifluoperazine. The antisera used to measure trifluoperazine cross-reacts with the 7-hydroxy and Kdemethyl metabolites of trifluoperazine to the extent of 24 and 26%, respectively. *met. = metabolite. cThe AUCSz4 was calculated by the linear trapezoidal method. dThe plasma half-life was calculated by the equation f7,+ = 0.693/ke,,where kelwas estimated by using the plasma concentrations between ,,C, and 24 h.

1200 / Journal of Pharmaceutical Sciences Vol. 74, No. 1 7 , November 1985

piperazine type phenothiazines, that is, fluphenazine and prochlorperazine (Table I). Therefore, the antiserum can be used for the development of quantitative RIA analyses of these 7-hydroxy metabolites in plasma. There is a previous report of the identification of the 7hydroxy metabolite of trifluoperazine in the urine of patients under treatment with trifl~operazine.~ In that report, the metabolite was extracted after glucuronidase and sulfatase treatment of the urine and, therefore, the metabolite is likely found to be predominantly excreted in the conjugated form. However, in plasma level monitoring, it would be preferable to measure the free levels of this presumably active metabolite. This first report of an RIA procedure can measure 20 pg of the unconjugated 7-hydroxy metabolite in a 200 pL plasma sample, thus obviating the need for large blood samples. Similar results were obtained for the assay irrespective of whether the procedure was carried out directly on plasma or on plasma extracts. Thus, a plot of BIB0 values obtained by the direct RIA procedure for plasma standards, covering the range from 0.1 to 5.0 ng of the 7-hydroxy metabolitelml, versus those obtained by the extraction RIA procedure for the same plasma standards gave a slope of 0.985 and a correlation coefficient of 0.999. Since there were no significant changes in binding when plasma standards of the 7-hydroxy metabolite over the standard curve concentration range were spiked with trifluoperazine at concentrations five times those of the metabolite, it is unlikely that trifluoperazine will interfere with the determination of this metabolite in samples from healthy volunteers or patients treated with trifluoperazine. Likewise, the available supposed major metabolites of trifluoperazine in humans, that is, the sulfoxide and N4 -oxide compounds, failed to demonstrate significant cross-reactivity either when added at five times the concentration of the 7-hydroxy metabolite to spiked plasma standards of 7-hydroxy compound or by the criteria of Abrahamle (Tables I, 11). As expected, the N demethyl metabolite did not cross-react according to this criterion,16 whereas the N-demethyl-7-hydroxy compound did cross-react significantly with the antiserum. Whether or not the levels of the N-demethyl-7-hydroxy metabolite in plasma samples are high enough to interfere in the determination of concentrations of the 7-hydroxy metabolite awaits the study of the levels of this secondary metabolite after single-dose and chronic administration of trifluoperazine to humans. Further proof for the specificity of the RIA procedure for the 7-hydroxy metabolite was obtained by comparison of the data for extraction RIA with that for direct RIA, determined for the same plasma samples from the volunteers dosed with a single dose of trifluoperazine. This favorable comparison between the direct and extraction RIA procedures indicates that physiological material and metabolites of trifluoperazine, not extractable in ether or 1%isopropyl alcohol in pentane, do not appreciably interfere in the determination of the 7-hydroxy metabolite. Furthermore, this experiment indirectly indicates that the polar conjugates of the 7-hydroxy compound do not cross-react with the antiserum, since conjugates are present in the plasma of volunteers who have ingested small single doses of trifluoperazine in concentrations which are, on average, approximately twice those of the nonconjugated metabolite.22 Pharmacokinetic parameters for the 7-hydroxy metabolite of trifluoperazine have not been previously reported. The appreciably longer mean t,,, obtained for the metabolite as compared with the parent drug (6.0 1.8 compared with 3.8 t 1.7 h), and the inability of the ultrasensitive assay for the 7-hydroxy metabolite to accurately measure plasma concentrations at the 0.5 h sampling point in five of the six volunteers, are indicative of a lack of presystemic metabolism of trifluoperazine by aromatic 7-hydroxylation. This

*

contrasts with the comparatively rapid appearance of the sulfoxide metabolite, which is probably due to extensive metabolism during presystemic absorption of trifluopera~ i n e Also . ~ in contrast to the sulfoxide metabolite, where plasma concentrations subsequent to the 0.5-h time point were always greater than trifluoperazine concentrations,s the 7-hydroxy plasma concentrations were always less than the parent compound measured in the same sample. In fact, for the 7-hydroxy metabolite, the mean C,, and AUCo-24for the six volunteers were approximately half of the corresponding mean values for trifluoperazine. However, there was considerable intersubject variation in these comparisons as indicated by the percent AUCo-24 7-hydroxytrifluoperazinel AUCo-24trifluoperazine ratio range of 13.5-83.8 for the six volunteers. The mean apparent elimination half-life of trifluoperazine was longer than the 7-hydroxy metabolite, which is more polar and presumably more extensively excreted as conjugates. However, for two of the volunteers, the apparent elimination half-life of the metabolite appeared to be longer than the parent drug. This was also the case in a previous studyz3on comparison of the apparent elimination half-lives of chlorpromazine and its 7-hydroxy metabolite in two healthy volunteers after administration of a low, single, oral dose of chlorpromazine and measurement of plasma concentrations by RIA. However, pharmacokinetic data obtained from the RIA measurement of plasma concentrations should be viewed with suspicion. Indeed, the antisera used to measure plasma concentrations of trifluoperazine cross-reacts with the 7-hydroxy and N-demethyl metabolites of trifluoperazine to the extent of 24 and 26%,respectively.1° The RIA method for the 7-hydroxy metabolite needs to be compared with a sensitive and specific chemical method for the determination of this metabolite in plasma in order to validate the pharmacokinetic data reported here. However, due to the low plasma levels of the 7-hydroxy metabolite encountered in the present study, it is obvious that such assay development will not be easy. In fact, validation of the RIA by a chemical method using plasma samples from a single-dose study may be inappropriate, such that samples from patients a t steady state may be necessitated. In the meantime, due to the specificity demonstrated in this report, the RIA for the 7hydroxy metabolite of trifluoperazine can be used to measure plasma concentrations in patients responding and not responding to chronic treatment with trifluoperazine. The resulting data, along with plasma levels determined in the same patient samples by RIA for trifluoperazine,lO and its sulfoxideg and N4-oxidez2metabolites, will assist in determining if any relationship(s) exists between these plasma concentrations andlor resultant plasma concentration ratios and therapeutic response and/or side effects.

References and Notes 1. Kleinman, J. E.; Bigelow, L. B.; Rogol, A,; Weinberger, D. R.; Nasrallah, H. A.; Wyatt, R.J.; Gillin, C. J. in “Phenothiazines and Structurally Related Drugs: Basic and Clinical Studies”; Usdin, E.; Eckert, H.; Forrest, I. S., Eds.; ElseviedNorth Holland: New York, 1980; pp 275-278. 2. Bunney, B. S.; Aghajanian, G. K. Life Sci. 1974, 15, 309-319. 3. Phillipson, 0. T.; McKeown, J. M.; Baker, J.;Healey, A. F. Br. J. Psvchiat. 1977. 131., 172-184. 4. MicKay, A. V.’P.;Healey, A. F.; Baker, J. Br. J . Clin. Pharmacol. 1974, 1, 425-430. 5. Sakalis, G.; Chan, T. L.; Sathananthan, G.; Schooler, N.; Goldsberg, S.; Gershon, S. Commun. Psychopharmacol. 1977,1, 157~

~~~

1 GG

6 . S;i;’urai, Y.; Takahashi, R.; Nakahara, T.; Ikenaga, H. Arch. Gen. Psvchiat. 1980.37. 1057-1062. 7. Fagarasan, M.; Fagarasan, E. Rev. Chim. 1983,34, 1133-1134; Chem. Abstr. 1984,100, 114438n. 8. Schmalzing, G. Drug Metab. Dispos. 1977, 5, 104-115. Journal of Pharmaceutical Sciences / 1201 Vol. 74, No. 1 7 , November 1985

9. Aravagiri, M.; Hawes, E. M.; Midha, K. K. J . Pharm. Sci. 1984, 73, 1383-1387. 10. Midha, K. K.; Hubbard, J. W.; Cooper, J. K.; Hawes, E. M.; Fournier, S.; Yeung, P. Br. J . Clin. Phurmacol. 1981, 12, 189193.

11. Bre er U: Schmalzing, G. Drug Metab. Dispos. 1977,5,97-103. 12. Nodk,,E. A.;Sharma, H. L.; Taunk, P. C.; Shukla, A. P.; Sadhnani, M. D.: Thambi. S. B.: Mital. R. L. J . Heterocvcl. Chem. 1981.18.1529-1532.’ 13. Shetty, H. U., Ph.D. Thesis; University of Saskatchewan, Saskatoon, Canada, 1983, pp 60 and 99. 14. Erlanger, B. F.; Boerk, F.; Beise, S. M.; Lieberman, S. J . Biol. Chem. 1957,228,713-727. 15. Midha. K. K.: CooDer. J. K.: Lauierre.’ Y. D.: Hubbard. J. W. Can. Med. Assoc. J: 1981, 124, 263. 16. Abraham, G. E. J . Clin. Endocrinol. Metub. 1969,29, 866-870. 17. Midha, K. K.; Korchinski, E. D.; Roscoe, R. M. H.; Hawes, E. M.; Cooper, J. K.; McKay, G. J . Pharm. Sci. 1984, 73,261-263. 18. Brooks, L. G.; Holmes, M.; Forrest, I. S.; Bacon, V. A.; Duffield, A. M.; Solomon, M. D. Agressologie 1971,12,333-342.

1202 / Journal of Pharmaceutical Sciences Vol. 74, No. 11, November 1985

19. Brooks, L. G.; Forrest, I. S. Exp. Med. Sur ery 1971,29, 61-71. 20. Hawes, E. M.; Shetty, H. U.; Cooper, J. K.;%auw, G.; McKay, G.; Midha. K. K. J . Pharm. Sci. 1984. 73.247-250. 21. Midha; K. K.; Hawes, E. M.; Rauw, G.;McKay, G.; Cooper, J. K.; Shetty, H. U. J . Pharmacol. Meth. 1983, 9. 283-293. 22. Aravagiri, M., un ublished results. 23. Yeung, P. K. F.; hcKay, G.; Ramshaw, I. A.; Hubbard, J. W.; Midha, K. K. J . Pharmacol. Exp. Ther., 1983,233, 816-822. I

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I

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-

Acknowledgments The Medical Research Council of Canada 0 erating Grants (MT7838 and PG-34) are gratefully acknowledgecf We are thankful to Dr. G. McKa and K. Hall for runnin the mass spectra and the Canadian ReiCross for the enerous gigs of plasma. We would also like to thank Mr. R. E. Tee$ Department of Chemistry, University of Saskatchewan for performing the microanalyses.