Metabolism and Distribution of 1-['4C]Alprazolam in Rats WlLLiAM R. BANKS'*, HIROKAZUYAMAKITA'O, AND GEORGE A. DIGENIS*' Received ril 19, 1991, frwn the 'Division of Medicinal Chemistry and Pharmaceutics, Coll e of Pharmacy, Univers of Ken*, Lexington, KY 4053682. Accepted for publication September 18, 1991. Present addresses: *%teringMedical Center, epartment of Nuclear MedicinelPositron Emission Tomography, Kettenng, OH 45459, and 5Pharmaceutic Research Laboratory, Tanabe Seiyaku Company, Ltd., Osaka, Japan.

r!

''C-labeled aiprazolam (1-['*C]APZ) as a model for l-(''CMPZ, we evaluated the tissue distribution of total radioactivityand assessed the contribution of the polar metabolites of APZ (ahydroxmethyl- and 4-hydroxy-APZ)to radioactivity levels in the plasma and brains of rats over the course of 1 h. The Modistribution data showed that the uptake of radioactivityby rat brain was 0.31%of the injected dose per gram of tissue weight at 10 min postinjection. Pretreating rats reduced the levels in brain to 0.21% of the injected dose at 10 min but had little effect on the distribution of radioactivity in plasma and other tissues studied. Analysis of the metabolites in plasma and brain homogenates by an extraction-thin-layerchromatography-liquidscintillation method revealed that >94% of the radioactivity in the rat brain was due to 1-[i4C]APZover the course of 1 h. APZ, therefore, is stable to metabolic transformations in the rat brain, and the polar metabolites are readily conjugated and excreted so that their cerebral uptake is minimal. Abrtnct 0 Using

Many receptor-based radiopharmaceutical agents with positron-emitting isotopes have been designed for in vivo positron emission tomography (PET) imaging studies of the benzodiazepine-Saminobutyric acid (GABA) chloride ionophore complex. Included in these studies were the agonists ["C]flunitrazepam,l ["Clsuriclone,2 and the widely exploited antagonist ["ClR015-1788 (['lClflumazenil).~ Additionally, radiolabeled analogues of iodoflunitrazepams and R0160154 (iomazenill7 have been successfully prepared for singlephoton-emission computerized tomography studies of this same receptor class. Recently, we disclosed the radiosynthesis of a "C-labeled triazolobenzodiazepine, alprazolam (APZ),in which the label was incorporated within the heteroaromatic triazole nuc1eus.B-10 All ligands mentioned are subject to oxidative metabolism; however, APZ is unique, because the metabolites formed retain the radioisotope (Scheme I). It is well-known that N-

I

Cn'\rN*rr

I

I m '

m'

I

m '

4 Q H A L m

m '

m '

Experlmental Section

ALRuOHALpRAzouM

Dose and Route of Adminiatrati~n-l-['~C]APZ was synthesized as previously deecribed'a and had a specific activity of 29.74 mCi/

Schema &Partial metabolism of APZ (structure shown in boxed area). 0022-3549/92l~797$02.50/0 Q 1992, American Pharmaceutical Assodcltion

and 0-alkylated xenobiotica undergo peripheral deactivation in the form of a hepatic microsomal enzyme-mediated oxidative dealkylationll or hydrolytic cleavage. With respect to 1,4-benzodiazepines, the immediate product is the N-hydroxymethyl metabolite; this metabolic process results in the release of the label into the pool of one-carbon compounds.12 A second susceptible site of oxidation for classical benzodiazepines and triazolobenzodiazepines is ring hydroxylation at the diazepine methylene carbon to produce a pharmacologically active metabolite, which, for classical benzodiazepines, may or may not retain the radiolabel. Similarily, the antagonist, ROE-1788, when labeled with carbon-11, undergoes rapid and extensive biotransformation, distributing the radioactivity among several ligand-derived species.4 The analogous iodinated ligand R016-01547 may have a similar metabolic fate, albeit the isotope remains attached to the parent structure. Attempta to create kinetic models of the behavior of such ligands in the brain are impeded by the distribution of cerebral radioactivity among several ligandderived compounds.4 Furthermore, compromises in PET image quality may arise because of the possibility of increased nonspecific binding of these species or the possible presence of radioactivity in a form that is chemically unrelated to the injected tracer ke., oxidatively produced C 0 2 W . We speculated that an alternative strategy would reduce the impact of labeled metabolites.S.9 The obvious choice was a ligand that has high aflinity for the receptor and yet can be labeled in a metabolically stable position. Furthermore, metabolites derived from the parent tracer should be restricted from crossing the blood-brain barrier. The metabolic profile of APZ,14,16 which indicates that the compound has an intermediate (10-11-h) systemic elimination rate,16 attracted ua to label this clinically significant sedative hypnotic agent with carbon-11 for PET studies of the benzodiazepine receptor. In vitro studies have shown that several of the hydroxylated metabolites [i.e., a-hydroxymethyl-APZ (a-OH-APZ) and Phydroxy-APZ (4-0H-APZ)I possess aflinity for the receptor.16 It has also been shown, with a sensitive gas chromatographic asaay,16 that APZ is the sole chemical species in the rat brain 1 h after intraperitoneal dosing (2.5 mgkg). This study was undertaken to substantiate this claim16 and to assess the biodistribution of 1-[l4C1APZ as a model for l-["CIAPZ in rats. The time courae of the levels of the two metabolites and APZ in plasma and brain and the impact of a pretreating dose on the distribution of radioactivity were also investigated at 10, 30, and 60 min postiqjection. The duration of the study was chosen to mimic the duration of a typical PET imaging protocol with carbon-11 (half-life, 20.4 mid .

Journal of Pharmaceutical SciencesI 797 Vol. 81, No. 8, August 1992

mmol (95.94 flilrng). The material was dissolved in propylene glycol:H,O (50:50, v/v) such that each animal received -11 f l i via the femoral vein (average dose, 11.08 pCi or 0.115 mg) in 0.5 mL of propylene glycol:H,O. Rats in the pretreatment group were given APZ (Xanax; Upjohn Company, Kalamamo, MI) by intravenous (iv) injection at a dose of 0.89 mgkg (0.22 mg in 0.13 mL of propylene glycol:H,O) 30 min prior to receiving the radioactive material. AnimaleTwenty-four male Sprague-Dawley rats (Harlan Industries, Indianapolis, IN), weighing 265-315 g, were used. All animals had free access to food and water until the time of the experiment and were lightly anesthetized with ether prior to receiving urethane intraperitoneally (1200 mgkg). After anesthetization, the femoral vein was surgically exposed for injection. The rats subjected to the pretreating dose had both femoral veins exposed. After injection, the body temperature was maintained with a light source and a reflective surface until serial sacrifice at 10, 30, and 60 min postinjection. Collection and Counting of Tissue S a m p l e e A t the appropriate time, samples of blood were taken from the abdominal aorta, and the animals were sacrificed via cervical dislocation. The brain, heart, lungs, liver, kidneys, spleen, testes, eyes, stomach, and intestines were removed in their entirety, blotted, placed into tared vials, sealed, weighed, and stored frozen until needed. After thawing, samples of each tissue were accurately weighed (nominally 150-200 mg), placed into scintillation vials, and dissolved in a quaternary ammonium tissue-solubilizing agent (Soluene; 1.0 mL). The rate of dissolution was enhanced by heating the sealed vials at 50 "C in a shaking water bath. Once the sample had dissolved, glacial acetic acid (0.2 mL) was added to quench any chemiluminescence. Scintillation cocktail (Scintiverse II; 15 mL; Fischer Scientific Company, Fairlawn, NJ) was added, the vials were shaken until samples were homogeneous, and the radioactivity was quantitated with a Packard Tricarb 2200 CA liquid scintillation counter. The radioactivity in the sampled tissues was expressed as percent injected dose per gram of tissue (% id/g; Table I). Analysis of Metabolites in Plasma and Brain-Whole blood was centrifuged, and the plasma was decanted and stored frozen until the time of assay. Sections of the whole brain were carefully weighed, homogenized [Teflon (polytetrafluoroethylene)on gleeal on ice with normal saline to make a 30% (w/v) homogenate, and again stored frozen until required. Development of Thin-Layer Chromatographic (TLC) Procedure and Treatment of S a m p l e d u l t u r e tubes were spiked with 10 pg each of Apz and its metabolites, a-OH-APZ and 4-OH-APZ. Increasing amounts of l-[14ClApz (44.6, 108.2, 180.4, 353.3, 521.4, 930.4, 1806.8,4030.9, and 8199.1 disintegrations per minute) were added to all but the blank tube. The carrier solvent was evaporated under nitrogen with gentle heating. To each tube was added 0.1 mL of either plasma or 30% brain homogenate; this was followed by vigorous mixing for 1min. To1uene:ether (1.0 mL; 75:25, v/v) was added, and each tube was agitated for an additional 1min. The two-phase system was centrifuged for 10 min; this was followed by the transfer of 0.75 mL of the organic layer to separate tubes. The samples were concentrated to dryness under nitrogen with gentle heating. To each Table I-Tiaam

tube was added CHzC1, (0.05 mL) with gentle agitation, and the solution was spotted onto a 20 x 20-cm', analytical-grade silica gel TLC plate (Whatman 6KF, 250 p with F254 indicator) that had been sectioned into l-cm lanes. This procedure was done three times to ensure maximal recovery of the analytes. The plate was eluted with CHC1,:EtOH:MeOH:triethylamine(100:7.5:4 1).In this system. optimal separation of all analytes was achieved [retardation factors (R for APZ, a-OH-APZ, and 4-OH-APZ were, respectively, 0.42, 0.3$ and 0.271. Once eluted, the compounds were visualized under UV light, and 1.0-cm' zones around the particular &value and the origin were scraped into scintillation vials. A solution of MeOH:H,O (0.1 mL; 50:50, v/v) and a suspension of the gel in scintillation-grade silicon dioxide (Cab-o-Sil; Fluka Chemical Company, Hauppauge, NY) and Scintiverse II(15.0 mL) were added. The samples were left at room temperature for 2 days prior to counting to allow the luminescence of the silica gel fluorescence indicator to decay. The efficiency of extraction of l-["C]APZ from both sample media was calculated by comparing the ratio of the recovered radioactivity with identical standards of known radioactivity that were not treated with plasma or brain homogenate; the extraction efficiencies were 84.2 t 6.7% for plasma and 91.2 f 3.8% for brain (n = 9). With this extraction procedure, linear resulta were obtained for both media in the range 2.0-392.3 ng/mL (r = 0.997 for plasma; r = 0.999 for brain). The actual samples obtained from experimental rats were treated identically. All samples were analyzed in duplicate, and each plate was treated with a blank sample and a blank spiked with a known amount of l-["C]APZ as a check of the extraction efficiency.Because the efficiency of extraction of metabolites was unknown, because of the lack of "C-labeled metabolites, the residual pellets were always analyzed for residual total carbon-14 radioactivity. This analysis was achieved by aspirating the residual to1uene:ether and disrupting the pellet by adding fresh solvent (0.1 mL); agitating, centrifuging, and aspirating the organic supernatant; and finally solubilizing the brain residue with Soluene. Addition of Scintiverse 11 and acetic acid (0.2 mL), followed by liquid scintillation analysis, revealed only background levels of radioactivity. The results from the plasma and brain extractions were used to normalize the total radioactivity levels determined from the tissue distribution study to provide estimates of the contribution of each analyte to these values (Tables II and 111).

Results and Discussion After iv administration of 1-[l4C1APZ, the distribution of radioactivity into the tissues was rapid (Table I). "he apparent maximal uptake in the brain and plasma occurred within 10 min. An extensive uptake of radioactivity was noted in the liver and kidneys. In this connection, APZ undergoes considerable hepatic oxidative deactivation, producing polar metabolites that, in addition to the parent compound, are rapidly excreted. The increased intestinal uptake of radioactivity with time may indicate biliary excretion and possible entero-

Distribution of Carbon-14 Radloactlvlty after hr Admlnl8tratl011of l-['%]APZ' Radioactivity ("h id/g) at indicated Times (min) after Dosing

Tissue

Brain Heart Lungs Liver Kidneys Spleen Intestinesb Stomachb Eyes Testes Plasma Brain:Plasmac ~~

Control

Pretreatment

10

30

60

10

30

60

0.307 f 0.035 0.817 f 0.030 0.642 f 0.021 2.036 f 0.203 1.43 f 0.062 0.555 f 0.026 0.768 f 0.087 0.172 t 0.075 0.167 f 0.033 0.180 f 0.030 0.252 t 0.008 1.22

0.097 f 0.017 0.403 2 0.053 0.342 t 0.064 1.792 t 0.114 0.964 f 0.137 0.307 f 0.031 0.830f 0.342 0.479 f 0.237 0.100 f 0.017 0.126 t 0.017 0.157 f 0.022 0.617

0.039 f 0.009 0.190 f 0.042 0.175 f 0.030 0.932 t 0.071 0.541 f 0.088 0.149 f 0.015 1.33 5 0.87 0.331 2 0.188 0.050 f 0.004 0.076 f 0.009 0.086 f 0.014 0.453

0.205 f 0.013 0.665 f 0.035 0.507 f 0.016 2.32 f 0.168 1.25 f 0.041 0.526 f 0.079 0.656 f 0.027 0.315 f 0.059 0.134 f 0.021 0.154 f 0.012 0.215 2 0.015 0.953

0.088 2 0.010 0.406 f 0.020 0.311 f 0.010 1.563 2 0.109 0.998 f 0.047 0.350 f 0.052 1.41 f 1.38 0.432 f 0.206 0.079 2 0.0078 0.126 f 0.010 0.164 f 0.014 0.536

0.053 f 0.006

~

~~

~

~

0.246 f 0.019 0.204 f 0.024 1.037 t 0.121

0.777 t 0.086 0.185 f 0.011 1.51 f 1.14 0.273 t 0.007 0.051 f 0.008 0.096 f 0.013 0.098 f 0.006

0.543

~~~

'All time points represent averages of four rats, except for the pretreatmentgroup at 10 min, which representsaverages of three rats; values are expressed as mean f standard deviation. Contents included. Values are ratios of carbon-14 radioactivity in brain to that in plasma. 798 I Journal of Pharmaceutical Sciences Vol. 81, No. 8, August 1992

T a b Il-Rdloactlvlty Irolated irom Bnln Homog.nrt# mnd Plumam

Tissue and COmpoUnd

Brain 4-OH-APZ

*OH-APZ APZ Plasma

COH-APZ

AH-APZ APZ

Percent Isolated Radioactlvityb at Indicated Timer, (min) after Dosing Control Pretreatment 10 30 60 10 30 60 2.31 0.35 2.10 3.82 0.18 1.37 (0.17) (1.25) (2.30) (0.25) (0.99) (3.46) 0.77 1.69 1.20 0.51 1.19 0.62 (0.36) (0.25) (1.82) (0.94) (0.66) (2.67) 99.31 97.03 95.39 98.80 95.25 94.11 (3.92) (2.00) 3.91) (1.21) (1.33) (2.71) 21.54 32.48 31.74 15.08 20.94 (2.41) (3.80) (7.1) (0.81) (4.82) 3.69 5.37 4.85 2.96 4.83 (0.47) (1.35) (1.20) (1.09) (1.09) 75.20 60.45 59.38 79.06 74.91 (2.33) (4.08) (5.63) (1.74) (4.81)

37.15 (3.60) 2.05 (1.48) 60.70 (4.81)

.All values represent the average (standard deviation) of four rats, except forthe pretreatment group at 10 min, for which three were used. Values were calculated from the sum of all radioactivitiesisolated from the TLC plate. TIM. IIcclrbon-14 FiadloclctMty In Bmln and P l a u a~ b r Comctlon for Motobdlteo'

Radioacthrity (Yo id/g)b at Indicated Times (min) after

Dosing

Tissueand Compound

control

10

Brain W-ARZ oOH-ARZ

APZ Plasma W-APZ

oOH-APZ APZ

30

60

loc

Pretreatment 30 60

0.0007 (0.0001) 0.0013 (0.0001) 0.203 0.305 0.094 (0.035) (0.004) (0.020) (0.010) O.ooo6 0.0013 (0.Mx)l) (0.0001) 0.0016 0.0012 (0.0001) (0.OOol)

O.ooo9

(0.Oool) 0.0005 (0.0001) 0.038

0.0018 0.002 (0.0001) (O.Oo0) 0.0007 O.OOO9 (0.0001) (0.0001) 0.084 0.050 (0.003) (0.001)

0.051 0.027 0.032 0.034 (0.0026) (0.002) (0.011) (0.001) 0.0075 0.0076 0.0046 0.010 0.006 (O.Ooo3) (O.OoO5) (O.oO03) (0.006) (0.000) 0.189 0.095 0.051 0.170 0.122 (0.005) (0.004) (0.003) (0.005) (0.005) 0.054 (0.002)

0.036

(0.001) 0.002 (0.000) 0.059 (0.002)

Calculated by multiplying the percent isolated values by the % id/g. Values in parentheses are standard deviations (n = 4). n = 3. hepatic recirculation of the drug and/or its metabolites. This increased uptake has been noted in mice atter iv and oral doses of l-['"CIAPZ.1~Zecca and Ferrarios reported a similar phenomenon when [12sIlflunitrazepam was given iv to rats. They reported a n intestinal uptake of 3.5% id/g at 15 min, which increased to 7.2% id/g at 120 min. In general, the pretreating dose had little effect on uptake or elimination of radioactivity from most organs, except the brain. At 10 min postdosing, 0.307 and 0.205% idlg (total radioactivity) was present in the brain in the control and pretreatment groups, respectively (significant at 95% level, p c 0.054, two-sample t test). The plasma levels, however, remained essentially the same at the 10-min point (p < 0.190). An extraction and TLC system was used to estimate levels of l-["C]APZ and the two major metabolites in brain and

plasma (Tables I1 and 111). Because PET measures only total radioactivity, we wanted to synthesize a ligand that was metabolically stable in the sense that cerebral radioactivity would primarily represent the ligand and no metabolites. Additionally, the label should remain intact such that no radioactivity would be due to a chemical species other than the pharmacologically active one. The goal of this study was to show, during the course of 1h by a typical imaging protocol for carbon-11, that the major compound in the rat brain is APZ. In vitro studies have shown that the metabolites a-OH-APZ and COH-APZ have &ties for the benzodiazepine receptor, albeit slightly lower than that of APZ (&ities were 3.4, 4.4, and 24 nM for A M , a-OH-APZ, and COH-APZ, respectively, from a ['Hlflunitrazepam-binding inhibition assayla). a-OH-APZ is pharmacologically active in mice.16 However, in humans, a-OH-APZ achieves only insignificant levels in serum aRer oral dosing.20 The distribution of APZderived radioactivity in plasma and brain, expressed as a percentage of isolated radioactivity, is shown in Table II. The efficiency of extraction of APZ was >84% from plasma and 91% from brain homogenates. Correction of total radioactivity levels for metabolites resulted in substantial contributions of drugderived materials to levels of radioactivity in plasma (Table III). The metabolite levels in plasma appeared to reach steady state, with a-OH-APZ never reaching >9.0% of the concentration of APZ (3.9 and 5.9% for control and pretreatment, respectively, at 10 min; rising to as high as 9.0 and 3.4% of the APZ concentration at 60 min for control and pretreatment, respectively). COH-APZ reached higher concentrations (28.5 and 18.8%for control and pretreatment, respectively, at 10 min; rising to as high as 52.9 and 61.02% of simultaneous APZ levels at 60 min for control and pretreatment, respectively). Pretreating may have had a n effect on the appearance of metabolites and their ratios. For example, at 10 min, the level of COH-APZ was -40% lees for the pretreated rats (relative to control). The total radioactivity levels in the brain corrected for metabolite contributions (Tables 11 and 111) indicated that APZ accounted for >94% of the radioactivity in the brain during the c o r n of 1 h after iv administration. Although metabolites may have been present in insignificant levels, their appearance seemed to increase slightly with time. Because l-[14ClAPZwas the fastest eluting species, residual counts may have appeared at lower R, values because of trailing of l-['%]APZ (allowing for variability in these measurements). In mice, the apparent maximal brain uptake of carbon-14 radioactivity occurred as early as 1 min after iv dosing of l-[14CclApz (2.33 mgkgl.21 Therefore, the time points uaed in this study with rats might have corresponded to the postdistribution phase of APZ in the brain; the data for concentration in brain were fit (with R-STRIP) to a monoexponential model. Apparent rates of elimination (A) from the brain and plasma were calculated (Table IV). Faster apparent elimination of 1-[14C]AF%from brain was observed for the control group, with a mean K of 0.054min-' compared with 0.035 min-' for the pretreated rats. Lower radioactivity levels in the brain of pretreated rats were expeded, in part because of partial blockage of specific and nonspecific binding sites by the nonradioactive APZ in the brain. The uptake system for benzodiazepines may be a nonspecific system based on passive dihion.22 Hantraye et al.29 showed that increasingthe dose of nonradioactive RO181788 cowected with ["C]R015-1788 decreased the maximal uptake in the human brain in a doedependent fashion. However, no comment was made on the decrease in the rate of washout from the brain as a function of increasing carrier concentration, as was evident from the data.= Miller et al.22 Journal of Pharmaceutical Sciences I 799 Vol. 81, No. 8, August 1992

Tabk IV-Pammeters fw Ellminetkn of APZ from Plesma and Brain'

Parameter Maximum amount,

Parameter Value for Elimination from Brain

Parameter Value for Elimination from

Plasma Control Pretreatment Control Pretreatment 0.305'

0.203'

0.189'

0.170'

0.518

0.281

0.251

0.210

0.054

0.035 19.79

% id/g

Theoretical maximum amount, % id/gd k, min-'

rln, mine

12.92

0.029 23.52

0.020 34.97

Values were estimated by fitting to a monoexponentialdecline. 'p > significant at 95% level. 'Not significant. Estimated from y-intercept value. * Half-life. a

0.049;

and Shinotoh et a1.6 claim that saturating doses of loraze am Hljand doses of clonazepam during the in vivo evaluation of [! and [11C]R015-1788do not alter the nonspecific uptake of the radioligand into the rat brain relative to controls (i.e., no apparent alterations in the cerebral blood flow (CBF) or blood-brain barrier permeability to the radioligand). The decreased uptake and elimination from the brain also could be due to reduction in CBF resulting from the pretreating dose. Similar compounds, when administered iv, lower CBF. For example, iv administration of lorazepam (4 mgkg) to monkeys decreased CBF to 72%of control measurements." Nugent et al.26 showed that diazepam (0.3 mgkg) decreased CBF to 50% of the control value a t 30 min after iv administration. Similarly, midazolam, an imidazolobenzodiazepine, at iv doses of 0.2 mgkg reduced CBF from 104 to 55 mL. min-1 g-1.26 Clearly, a decrease of CBF could d e c t the time to reach the maximal amount of APZ entering the brain by slowing the rate of entry into the brain. Similarly, the value of k for APZ would be altered, and the result is prolongation of the washout phase. Saturable metabolism is another possible factor in the differences in the maximal levels in brain and plasma and k values. If the microsomal enzyme system responsible for the hepatic oxidative hydroxylations at positions 1 and 4 of APZ is saturated by the pretreating dose, then clearly the systemic clearance of the 14C-labeleddrug would be impeded. Indeed, this inhibition was observed in the profiles of the levels of drug in plasma versus time (Table IV); the k values were lower for the pretreated rats relative to controls when fitted to the monoexponential model. The lower k values resulted in a gradual increase in the retention of radioactivity with respect to the pretreated animals over time (Table I). The levels of radioactivity in plasma at 30 and 60 min were slightly higher in the pretreatment group compared with the control group. When the % id/g values for plasma were normalized with the percent isolated radioactivity values from Table 11, the contributions to radioactivity in plasma from 1-['4ClAPZ were greater for the pretreated rats relative to control (22.1 and 13.6% greater for 30 and 60 min, respectively; Table 111).This result indicated again that the enzymatic system responsible for the deactivation and/or conjugation of APZ was slowed af'ter pretreatment with nonradioactive APZ and allowed more of the parent tracer to appear. This finding is important because receptor specificity studies uae saturability as a criterion. Consequently, animal or human studies to determine the effect of iv APZ on CBF should be undertaken. The cerebral kinetics of 1-["CIAPZ may have to be corrected to obtain true estimates of dosedependent saturation. Preliminary data from Tewson et a1.26 indicate that APZ, when given iv (1.0 mg), had little effect on

.

800 I Journal of Pharmaceutical Sciences Vol. 81, No. 8, August 1992

altering CBF in normal human subjects. The lack of metabolites in the brain is important, because both metabolites are known to bind to the benzodiazepine receptor in vitro and to elicit activity in mouse models. For APZ, the doses required to protect 50%of the tested mice from leptazol- and nicotine-induced seizures were 0.13 and 0.056 mgkg (intraperitoneal), whereas for a-OH-APZ, values of 0.22 and 0.08 mgkg, respectively, for the same models were found.16 The data from this study indicate that the pharmacological activity after iv administration of APZ may be due solely to intact APZ and not to circulating metabolites. Insignificant metabolite levels in the rat brain suggest their rapid conjugation and excretion as polar phase 11 metabolites. The lower levels of a-OH-APZ,relative to 4-OHAPZ, indicated that a-OH-APZ was initially formed in lesser amounts. However, Eberta et al.14 showed that the major metabolite in human urine after a 2-mg oral dose is a-OHAPZ (17%of administered dose). Smith and Kroboth27 showed that 4-OH-APZ is present in human serum to a higher degree relative to a-OH-APZ after a 2-mg oral dose of APZ. The results of this study, as well as the earlier studies cited, support the classification of triazolobenzodiazepines as drugs with short or intermediate half-lives, probably because of their rapid oxidative metabolism and conjugation at the methyl group. Because deactivation and elimination were efficient enough to prevent polar metabolites from entering the brain, APZ meets the metabolic requirements of an ideal ligand. In the absence of minimal species variabilities, complications arising from labeled active metabolites may be avoided. Future cerebral pharmacokinetic analysis of l-["CIAPZ, therefore, may be simplified, because the arterial input to the brain is easily corrected to represent intact I-["CIAPZ. Additionally, compromises in image quality and contrast will be avoided, because APZ is the sole radioactive species in the brain during the study.

References and Notes 1. Comar, D.; Maziere, M.; Cepeda, C.; M o t , J. M.; Menini, C.; Naquet, R. Eur. J . Pharmacol. 1981, 75, 21-26. 2. Frost,J. J.; Wagner, H. N.; Dannals, R. F.;Ravert, H. T.;Wilson,

A. A.; Links, J. M.; Rosenbaum, A. E.; Trifiletti, R. R.; Snyder, S.S.Eur. J . Pharmacol. 1986, 122, 381-383. 3. Samson, Y.;Hantraye, P.;Baron, J. C.; Sousealine, F.; Comar, D.; Maziere, M. Eur. J . Pharmacol. 1985. 110. 247-251. 4. Halldin,.C.; Stone-Elander, S.; Thorell, J.;.Persson, A.; Sedvall, G. Appl. Radiut. Zsot. 1988,39, 993-997. 5. Shinotoh, H.; I 0 , M.; Yamada, T.; Inoue, 0.;Suzuki, K.; Itoh, T.; Fukuda, H.; Jamasaki, T.; TaBno, Y.; Hirayama, K. Psycho-

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Acknowledgments We atefully acknowled e the financial sup ort and the generous gift o ~ e t a b o h t e sand autfentic APZ by the 8pjohn Company.

Journal of Pharmaceutical Sciences I 801 Vol. 81, No. 8, August 1992