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`0090-9556/97/2505-0573–583$02.00/0
`DRUG METABOLISM AND DISPOSITION
`Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
`
`Vol. 25, No. 5
`Printed in U.S.A.
`
`DISPOSITION AND METABOLISM OF OLANZAPINE IN MICE, DOGS, AND
`RHESUS MONKEYS
`
`EDWARD MATTIUZ, RONALD FRANKLIN, TODD GILLESPIE, ANTHONY MURPHY, JOHN BERNSTEIN, ANDRE CHIU,
`TERRY HOTTEN, AND KELEM KASSAHUN
`
`Department of Drug Metabolism (E.M, R.F, T.G, A.M, J.B, A.C, K.K), Eli Lilly and Company,
`Lilly Corporate Center, and Lilly Research Centre (T.H)
`
`(Received September 12, 1996; accepted January 30, 1997)
`
`ABSTRACT:
`
`Olanzapine (OLZ) is a novel antipsychotic agent with a high affinity
`for serotonin (5-HT2), dopamine (D1/D2/D4), muscarinic (m1–m5),
`adrenergic (␣1), and histamine (H1) receptors. The pharmacokinet-
`ics, excretion, and metabolism of OLZ were studied in CD-1 mice,
`beagle dogs, and rhesus monkeys after a single oral and/or intra-
`venous dose of [14C]OLZ. After oral administration, OLZ was well
`absorbed in dogs (absolute bioavailability of 73%) and to the extent
`of at least 55% in monkeys and 32% in mice. The terminal elimi-
`nation half-life of OLZ was relatively short in mice and monkeys
`(⬃3 hr) and long in dogs (⬃9 hr). In mice and dogs, radioactivity
`was predominantly eliminated in feces; but, in monkeys, the major
`route of elimination of radioactivity was urine. Dogs and monkeys
`excreted in urine, respectively, 38% and 55% of the dose over a
`168-hr period, whereas the fraction of the dose excreted in urine of
`mice over the collection period (120 hr) was 32%. OLZ was subject
`to substantial first-pass metabolism; at the tmax, OLZ accounted
`for 19%, 18%, and 8% of the radioactivity, in mice, dogs, and
`monkeys, respectively. The ratio of AUC OLZ to AUC radioactivity
`was, respectively, 10%, 14%, and 4% in mice, dogs, and monkeys.
`The principal urinary metabolites in mice were 7-hydroxy OLZ
`
`glucuronide, 2-hydroxymethyl OLZ, and 2-carboxy OLZ accounting
`for ⬃10%, 4%, and 2% of the dose. Metabolites that were present
`in urine in lesser amounts were 7-hydroxy OLZ, N-desmethyl OLZ,
`and N-desmethyl-2-hydroxymethyl OLZ. In dogs, the major metab-
`olite accounting for ⬃8% of the dose was 7-hydroxy-N-oxide OLZ.
`Other metabolites identified were 2-hydroxymethyl OLZ, 2-carboxy
`OLZ, N-oxide OLZ, 7-hydroxy OLZ, and its glucuronide and N-
`desmethyl OLZ. The major metabolite in monkey urine was N-des-
`methyl-2-carboxy OLZ, and accounted for ⬃17% of the dose. In
`addition, N-oxide-2-hydroxymethyl OLZ, N-oxide-2-carboxy OLZ,
`N-desmethyl-2-hydroxymethyl, 2-carboxy OLZ, and 2-hydroxy-
`methyl OLZ were identified in monkey urine. Thus, in mice and
`dogs, OLZ was metabolized through aromatic hydroxylation, allylic
`oxidation, N-dealkylation, and N-oxidation reactions. In monkeys,
`OLZ was biotransformed mainly through double oxidation reac-
`tions involving the allylic carbon and methyl piperazine nitrogen.
`Whereas the oxidative metabolic profile of OLZ in animals was
`similar to that of humans, animals were notable for not forming
`appreciable amounts of the principal human metabolite (i.e. 10-N-
`glucuronide OLZ).
`
`was N-glucuronidation. OLZ also underwent oxidative metabolism
`through N-oxidation, N-demethylation, and 2-alkyl hydroxylation.
`This study describes the comparative absorption, pharmacokinetics,
`and metabolism of OLZ in mice, dogs, and monkeys. The studies were
`conducted after the administration of [14C]OLZ.
`
`OLZ1 (fig. 1) is a new antipsychotic drug with a thienobenzodiaz-
`epinyl structure. OLZ displays a broad pharmacological profile with
`potent activity at dopamine (D1/D2/D4), serotonin (5-HT2A/2C), mus-
`carinic (especially m1), histamine (H1) and adrenergic (␣1) receptors
`(1, 2). The receptor binding profile of OLZ is very similar to cloza-
`pine, although OLZ is a more potent inhibitor of these receptors.
`In clinical studies with patients suffering from schizophrenia or
`schizophreniform disorder, OLZ was effective in the treatment of both
`positive and negative symptoms of schizophrenia, with a low inci-
`dence of extrapyramidal side-effects (3–5). Antipsychotic efficacy of
`OLZ was demonstrated in the dose range of 5–20 mg/day.
`The disposition and metabolism of OLZ after a single oral dose to
`healthy volunteers has recently been reported (6). OLZ was well
`absorbed and extensively metabolized. The primary metabolic route
`
`1 Abbreviations used are: OLZ, olanzapine; LSC, liquid scintillation counting;
`Cmax, maximum plasma concentration; tmax, time to maximum concentration;
`AUC, area under the plasma concentration-time curve; LC-MS/MS, liquid chro-
`matography-tandem mass spectrometry; CID, collision-induced dissociation; IV,
`intravenous.
`
`Send reprint requests to: Dr. Kelem Kassahun, Department of Drug Metab-
`olism, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center,
`Mail Drop 0825, Indianapolis, IN 46285.
`
`Materials and Methods
`Reference Compounds and Other Materials. The following compounds
`were synthesized at Lilly Research Laboratories: OLZ (2-methyl-4-(4-methyl-
`1-piperazinyl)-10H-thieno[2,3-B][1,5]benzodiazepine), [4,10a-14C2]OLZ
`([14C]OLZ; radiochemical purity, 98.7%; specific activity, 26.2 ␮Ci/mg),
`4⬘-N-desmethyl OLZ (N-desmethyl OLZ, 2-methyl-4-(1-piperazinyl)-10H-
`thieno[2,3-B][1,5]benzodiazepine), 4⬘-N-oxide OLZ (N-oxide OLZ, 4-(2-
`methyl-10H-thieno[2,3-B][1,5]benzodiazepin-4-yl)-1-methylpiperazine-1-
`oxide), 2-hydroxymethyl OLZ (4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-
`B][1,5]benzodiazepine-2-methanol), 2-carboxymethyl OLZ (methyl 4-(4-
`methyl-1-piperazinyl)-10H-thieno[2,3-B][1,5]benzodiazepine-2-carboxylate),
`7-ethoxy OLZ (7-ethoxy-2-methyl-4-(4-methyl-1-piperazinyl)-10H-thi-
`eno具2,3-B典具1,5典benzodiazepine), and 4⬘-N-desmethyl-2-hydroxymethyl OLZ
`(4-(1-piperazinyl)-10H-thieno[2,3-B][1,5]benzodiazepine-2-methanol). NEE-
`154 Glusulase was purchased from the DuPont Company (Wilmington, DE).
`␤-Saccharolactone was purchased from Sigma Chemical Co. (St. Louis, MO).
`Scintisol was supplied by Isolab, Inc. (Akron, OH). HPLC-grade ammonium
`acetate, acetonitrile, triethylamine, and reagent-grade boron tribromide were
`purchased from Fisher Scientific (Fair Lawn, NJ). 7-Hydroxy OLZ was pre-
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`Analysis of Radioactivity. Total radioactivity in plasma and urine samples
`was determined using LSC after the addition of scintillation cocktail (Aquas-
`sure; New England Nuclear, Boston, MA). Feces was suspended in 5%
`aqueous sodium lauryl sulfate solution, and aliquots of the dried homogenate
`were combusted in a sample oxidizer. The fecal sample prepared in this
`manner was counted after addition of Aquassure. Mice carcasses were digested
`using alcoholic potassium hydroxide. The homogenate obtained in this manner
`was neutralized with acetic acid, and the radioactivity was determined by LSC.
`Quench correction was conducted by automatic external standardization.
`HPLC Assay of Plasma OLZ. Concentrations of OLZ in mouse, dog, and
`monkey plasma were determined by HPLC (8). In this assay, OLZ and the
`internal standard (2-ethyl homolog of OLZ) are isolated from plasma using a
`mixed-mode, solid-phase extraction, separated with a reversed-phase method,
`and detected electrochemically. The upper and lower limits of quantitation of
`the assay were 100 and 1 ng/ml, respectively.
`Pharmacokinetics. Noncompartmental analysis was used to determine the
`pharmacokinetics of OLZ and radioactivity. Cmax and tmax were assesed by
`visual inspection. The terminal elimination half-life was calculated using the
`relationship 0.693/k, where k is the elimination rate constant. The AUC-–t was
`calculated up to the last time point (t) by the trapezoidal rule.
`In Vitro Protein Binding. [14C]OLZ was dissolved in n-propyl alcohol at
`0.2, 0.02, and 0.002 mg/ml, and an aliquot (15 ␮l) of each concentration was
`added into 2985 ␮l volume control plasma. Plasma samples were then placed
`in a water bath (⬃37°C) for 1 hr. After ultracentrifugation (360,000 g at 37°C
`for 4 hr), the amount of OLZ in the supernatant was determined by LSC. The
`fraction of OLZ bound to protein was calculated from the radioactivity con-
`centrations in the spiked sample and the supernatant.
`Metabolite Isolation. Aliquots of urine samples from each of the orally
`dosed dogs were combined (⬃80 ml total urine) and made basic by the
`addition of 0.1 M ammonium hydroxide (8 ml). Ethyl acetate (300 ml) was
`added, and the phases were mixed by shaking vigorously. The ethyl acetate
`layer was separated and evaporated to dryness in a water bath (40°C) under a
`stream of nitrogen. The aqueous fraction was lyophilized to dryness, dissolved
`in water, and analyzed by HPLC. Pooled mouse urine was also extracted as
`described. For NMR analysis, the 7-hydroxyl-N-oxide metabolite was isolated
`from urine using column chromatography. Approximately 400 g of Amberlite
`XAD-2 resin was packed in a 2.5 ⫻ 30 cm glass column. The remaining urine
`samples from each dog were combined (⬃1.5 liters) and passed through the
`column after preconditioning the column with methanol and purified water.
`The column was washed with water and the radioactivity eluted with methanol.
`Methanolic extracts were concentrated in vacuo at 25°C using a rotary evap-
`orator, and the resulting residue was reconstituted in 70 ml methanol/water
`(1:1) for HPLC analysis. Aliquots (50 ml; 0–24 hr) of urine sample from each
`monkey were lyophilized to ⬃2 ml. The residue was reconstituted in 5 ml
`water:methanol (4:1, v/v) and separated by HPLC.
`Estimation of the Amount of Metabolites. The amount of each metabolite
`in urine was estimated by LSC after isolation by HPLC. An aliquot of the ethyl
`acetate or aqueous extract (50–200 ␮l) was injected into HPLC, and each
`metabolite was collected as it eluted the column. Scintisol (15 ml) was added,
`and the amount of radioactivity was determined by LSC. The total radioactivity
`in the sample was determined by injecting an equal aliquot into the HPLC
`injector and collecting the entire sample before it reached the column.
`Hydrolysis of Conjugates. Glucuronide conjugates (⬃2 ␮g) isolated from
`urine were hydrolyzed to the corresponding aglycone by incubating with
`Glusulase (containing 2,070 units of ␤-glucuronidase and 150 units sulfatase)
`at 37°C for up to 20 hr. Incubations were also conducted in the absence of
`Glusulase and in the presence of ␤-saccharolactone (0.0325 M).
`HPLC Separation of Metabolites. The HPLC system consisted of a
`Beckman pump, NEC controller, Waters Wisp autosampler, Applied Biosys-
`tem UV detector, and a Berthold radiodetector with 150 ␮l yittrium solid cell.
`Aliquots (ⱕ200 ␮l) of concentrated urine or extract were analyzed on a
`Hypersil C18 column (5 ␮m particle size, 0.46 ⫻ 25 cm) using a gradient
`containing A (0.1 M ammonium acetate) and B (1% triethylamine in acetoni-
`trile). The initial solvent composition was 90% A and 10% B. After 2 min, the
`pump was programmed to increase solvent B by 2.5%/min until a proportion
`of 40% A and 60% B was achieved. The mobile phase was maintained for 8
`min at that composition. The flow rate was 1 ml/min. Metabolites were isolated
`
`14C atoms is indicated
`
`FIG. 1.Chemical structure of OLZ; the position of
`by bullet points.
`pared by deethylation of 7-ethoxy OLZ, and 2-carboxyl OLZ was prepared by
`hydrolysis of 2-carboxymethyl OLZ. Approximately 2 mg of each starting
`material was placed in separate siliconized tubes and dissolved in methylene
`chloride (2 ml). The solution was flushed with nitrogen and treated with boron
`tribromide solution (2 ml of 25% solution in methylene chloride). The reaction
`was allowed to proceed at room temperature for 2 hr. Approximately 90% of
`7-ethoxy OLZ was converted to 7-hydroxy OLZ, whereas ⬃50% of 2-car-
`boxymethyl was converted to the corresponding acid as determined by HPLC
`and electrospray LC/MS. N-Desmethyl-2-carboxy OLZ was prepared by oxi-
`dizing the corresponding hydroxy compound using chromium trioxide (7).
`Animal Experiments. All animal experiments were conducted according to
`protocols approved by the Eli Lilly Animal Care and Use Committee. The
`dosing solution used for all animal studies was prepared by dissolving the
`required amounts of OLZ and [14C]OLZ in 1 M HCl and titrating the solution
`to approximately pH 6 by the addition of 0.1 M NaOH. The appropriate
`volume was then obtained by the subsequent addition of water.
`Mouse. Male CD-1 mice were obtained from Charles River Laboratories
`(Wilmington, MA) and acclimatized for 3 days before use. Food and water
`were supplied ad libitum at all times throughout the experiment. For radiola-
`beled excretion study, the mice were divided into three groups, with each
`group containing five mice. Each animal was administered a single oral
`(gavage) dose of OLZ (15 mg/kg containing 420 ␮Ci/kg of [14C]OLZ). Urine
`and fecal samples were collected at 24-hr intervals for up to 120 hr. For
`pharmacokinetic study, four mice were used for each time point and dosed as
`described. Blood was collected and pooled from four mice at 0.5, 1, 2, 4, 7, 12,
`24, 48, and 72 hr after the dose. Plasma was obtained by centrifugation and
`stored at ⫺70°C until analysis. Metabolite identification was conducted in
`urine obtained from mice given a 20 mg/kg dose. Urine samples collected for
`24-hr postdose from eight mice were combined and stored at ⫺70°C until
`analyzed. Additional mice (10) were administered a single oral dose (15 mg/kg) of
`OLZ, and plasma was collected at ⬃1 hr for metabolite identification.
`Dog. Four female beagle dogs (age: 2–4 years; weight: 8.7–13.1 kg) were
`obtained from stock animals maintained at Lilly Research Laboratories and
`placed in individual stainless-steel metabolism cages. Animals were fasted
`overnight before and 2 hr after drug administration. Animals were given a
`single oral (gavage) dose of OLZ (5 mg/kg containing 4 ␮Ci/kg of [14C]OLZ).
`Urine and fecal samples were collected every 24 hr for 168 hr. Blood was
`drawn at 0, 0.5, 1, 3, 6, 12, 24, 48, 96, and 168 hr after dosing. Aliquots were
`withdrawn for determination of radioactivity, and the remainder was centri-
`fuged to obtain plasma. Plasma was also obtained from three female dogs
`given a single intravenous dose of OLZ (5 mg/kg containing 5 ␮Ci/kg
`[14C]OLZ) at 0, 0.08, 0.25, 0.5, 1, 3, 6, 12, 24, 36, 48, 72, 96, 120, 144, and
`168 hr postdose. For identification of plasma metabolites, another group of
`three dogs were dosed orally with OLZ (5 mg/kg), and plasma was collected
`at 3 and 12 hr after the dose.
`Monkey. Young adult rhesus monkeys (2 males and 2 females) weighing
`between 3 and 7 kg were used in the study. Each animal was given a single oral
`(nasogastric) dose of OLZ (5 mg/kg containing 8.9 ␮Ci/kg [14C]OLZ). Blood
`was collected at 0, 0.5, 1, 4, 8, 12, 24, 48, 96, 120, and 168 hr postdose,
`whereas urine and fecal samples were collected every 24 hr up to 168 hr.
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`METABOLISM OF OLANZAPINE IN MICE, DOGS, AND MONKEYS
`
`575
`
`TABLE 1
`Urinary and fecal elimination of radioactivity in mice, dogs, and monkeys after the administration of a single oral dose of [14C]OLZ
`% Dose Excreted
`Dogc
`
`Monkeyd
`
`Time
`
`Mousea,b
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`hr
`0–24
`24–48
`48–120
`120–168
`
`Total
`
`Urine
`
`Feces
`
`Urine
`
`Feces
`
`Urine
`
`Feces
`
`25.0 ⫾ 1.8
`4.0 ⫾ 1.1
`2.9 ⫾ 2.2
`NCe
`
`31.9 ⫾ 2.8
`
`46.7 ⫾ 2.9
`11.6 ⫾ 3.0
`6.0 ⫾ 2.3
`NC
`
`64.3 ⫾ 3.4
`
`20.5 ⫾ 3.7
`11.7 ⫾ 5.1
`5.6 ⫾ 1.0
`0.60 ⫾ 0.13
`
`2.7 ⫾ 2.9
`22.9 ⫾ 13.0
`19.0 ⫾ 9.0
`1.1 ⫾ 0.27
`
`47.6 ⫾ 3.6
`4.8 ⫾ 0.70
`2.0 ⫾ 0.70
`0.23 ⫾ 0.08
`
`6.2 ⫾ 8.7
`14.0 ⫾ 3.4
`8.0 ⫾ 5.8
`0.31 ⫾ 0.10
`
`38.4 ⫾ 2.6
`
`45.6 ⫾ 5.4
`
`54.6 ⫾ 3.7
`
`28.5 ⫾ 5.2
`
`97.4 ⫾ 1.1f
`
`Grand total
`Data represent mean ⫾ SD.
`a N ⫽ 3.
`b % dose recovered from the carcass was 0.6%.
`c N ⫽ 4.
`d N ⫽ 4.
`e NC, not collected.
`f Includes radioactivity from cage washings and carcass.
`g Includes cage washings.
`
`84.3 ⫾ 5.0g
`
`83.1 ⫾ 3.9
`
`by collecting the radioactive eluent as it eluted off the column. Several
`injections were made to obtain a sufficient amount of each metabolite for mass
`spectral identification.
`LC-MS/MS. Isolated metabolites were analyzed by LC/MS and LC-
`MS/MS on a Finnigan MAT TSQ700. Metabolites were introduced into the
`electrospray LC interface using a Waters Model 600 pump. Metabolites were
`separated on an Inertsil C18 column (5 ␮m particle size, 0.46 ⫻ 25 cm) using
`the same gradient as described with 0.05 M ammonium acetate and acetoni-
`trile. Injection volumes ranged from 10 to 200 ␮l. The flow rate was 1 ml/min,
`and the effluent was split such that equal volumes were delivered into the ion
`source and a Raytest Ramona model 5LS radiodetector. MS spectra were
`obtained by scanning from m/z 200 to 600 every second. For CID experiments,
`the collision gas (argon) pressure was maintained at 2.0 m torr, and the
`collision offset voltage was ⫺20 eV. MS and MS/MS spectra were averaged
`for 1 min.
`NMR Spectroscopy. Proton and carbon-13 NMR spectra were recorded in
`d6-DMSO or CDCI3 on a Bruker AMX spectrometer operating at 500 MHz.
`Chemical shifts are reported in ppm relative to tetramethylsilane.
`
`Results
`Excretion of Radioactivity. Mice administered a single oral dose
`(15 mg/kg) of OLZ eliminated 64.3 ⫾ 3.4% (mean ⫾ SD) and 31.9 ⫾
`2.8% of the radioactivity, respectively, in feces and urine over a
`120-hr period (table 1). The majority of the dose (⬎87%) was ex-
`creted during the first 48 hr of dosing. Less than 1% of the adminis-
`tered dose was recovered in the carcasses.
`In dogs, ⬃84% of the radioactivity was recovered after 168 hr, with
`slightly more radioactivity eliminated in the feces (45.6 ⫾ 5.4%) than
`in the urine (38.4 ⫾ 2.6%). Greater than 50% of the dose was
`recovered within 48 of dosing (table 1).
`In monkeys, renal excretion was the primary mode of radiocarbon
`elimination accounting for 54.6 ⫾ 3.7% of the dose. Another 28.5 ⫾
`5.2% of the dose was eliminated via the feces over the same period.
`Greater than 50% of the dose was eliminated in the urine and feces 24
`hr after the dose (table 1). There was no difference between males and
`females with respect to the amount of radioactivity in either the urine
`or feces.
`Pharmacokinetics. Mice. Pharmacokinetic parameters of OLZ and
`radioactivity in mice are shown in table 2. OLZ was quantitated in
`plasma using an HPLC assay with a lower limit of quantitation of 1
`
`ng/ml. The Cmax of OLZ was 421 ng/ml and occurred at 0.5 hr after
`the dose. The corresponding value for radioactivity was 2,260 ng-
`eq/ml and was reached at a much later time (4 hr). At 0.5 hr, OLZ
`accounted for ⬃19% of plasma radioactivity. This is indicative of the
`extensive metabolism of OLZ in the mouse. Similarly, OLZ ac-
`counted for 10% of the total 14C AUC. The plasma terminal half-life
`of OLZ was 3.2 hr. Radioactivity in plasma declined slowly with a
`half-life of 10.6 hr. The plasma radioactivity vs. time curve (fig. 2)
`showed elevated concentrations at both 0.5 and 4 hr, suggesting
`enterohepatic recycling.
`Dogs. The mean Cmax of OLZ was 172 ⫾ 69 ng/ml and occurred
`between 1 and 3 hr in 3 of the 4 animals tested. The fourth animal had
`a tmax of 6 hr. The elimination of OLZ from plasma seemed to be
`biphasic (fig. 3), with the terminal phase displaying a half-life of
`9.2 ⫾ 1.4 hr.
`The mean tmax for radioactivity in plasma was 1 ⫾ 0.0 hr, and the
`Cmax was 949 ⫾ 296 ng-eq/ml. Plasma radioactivity declined with a
`mean half-life of 27.6 ⫾ 12.0 hr. The ratio of AUC OLZ to AUC
`radioactivity was 0.14.
`After a single IV dose of OLZ to three dogs, the mean Cmax and
`AUC for OLZ were, respectively, 871 ⫾ 241 ng/ml and 2,633 ⫾
`1,041 ng * hr/ml. The corresponding values for plasma radioactivity
`were 1,145 ⫾ 195 ng-eq/ml and 18,813 ⫾ 2,598 ng-eq * hr/ml. Thus,
`after an IV administration, at the tmax OLZ accounted for 76% of the
`radioactivity, compared with a value of 18% after an oral dose.
`Because the amount of radioactivity excreted in urine after the oral
`and IV doses was almost the same (38.4% and 39.7% of the dose), the
`decreased bioavailability after oral administration is likely due to
`first-pass metabolism. The ratio of AUC OLZ to 14C AUC was the
`same as that obtained after oral dosing. The absolute oral bioavail-
`ability of OLZ was calculated to be 73%.
`Monkeys. The mean Cmax of OLZ and radioactivity were 60 ⫾ 18
`and 757 ⫾ 169 ng eq/ml, and were reached on average within 1.5 hr
`postdose. Therefore, at the Cmax OLZ accounted for ⬃8% of the
`plasma radioactivity. On the basis of AUC, the fraction of plasma
`radioactivity represented by OLZ was ⬃4%.
`The mean elimination half-life of OLZ was 3.4 ⫾ 1.2 hr. The
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`TABLE 2
`Mean pharmacokinetic parameters of OLZ and radioactivity in mice, dogs and monkeys after the administration of a single oral dose of [14C]OLZ
`Mousea
`Parameter
`Dog
`Monkey
`Dose (mg/kg)
`5
`5
`Cmax (ng or ng-eq/ml)
`OLZ
`14C
`OLZ as % of 14C
`tmax (hr)
`OLZ
`14C
`t1/2 (hr)
`OLZ
`14C
`AUC (ng or ng-eq 䡠 hr/ml)
`1,923 ⫾ 325
`OLZ
`13,405 ⫾ 2,123
`14C
`14%
`10%
`OLZ as % of total 14C
`a Mouse data obtained from pooled plasma, for dogs and monkeys values represent mean ⫾ SD (N ⫽ 4).
`b Points used in the determination of the respective half-lives.
`
`15
`
`421
`2,260
`
`19%
`
`0.5
`4
`
`3.2 (7–12 hr)b
`10.6 (7–48 hr)b
`
`1,522
`15,201
`
`172 ⫾ 69
`949 ⫾ 296
`18%
`
`3.3 ⫾ 2.1
`1 ⫾ 0.0
`
`9.2 ⫾ 1.4 (3–48 hr)b
`27.6 ⫾ 12.0 (24–96 hr)b
`
`60 ⫾ 18
`757 ⫾ 169
`8%
`
`1 ⫾ 0.0
`1.5 ⫾ 1.7
`
`3.4 ⫾ 1.2 (1–12 hr)
`5.3 ⫾ 0.7 (4–12 hr)
`
`536.6 ⫾ 208.3
`14,429 ⫾ 1,572
`4%
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`FIG. 2.Plasma concentration vs. time profiles for OLZ and total
`radioactivity in mice given an oral dose of 15 mg/kg of [14C]OLZ.
`Radioactivity is expressed as mean ⫾ SD (N ⫽ 4), whereas OLZ concen-
`trations were obtained from pooled plasma.
`
`elimination of radioactivity from plasma was biphasic (fig. 4), with
`the initial and terminal phases having half-lives of, respectively, 5.3 ⫾
`0.7 and 98.7 ⫾ 26.5 hr.
`In Vitro Plasma Protein Binding. The plasma protein binding of
`OLZ was similar in the three species studied, with mean binding being
`77%, 75%, and 83% in mice, dogs, and monkeys, respectively. The
`binding was concentration-independent (10–1,000 ng/ml). The extent
`of protein binding was lower in these species than that reported for
`humans at 93% (6).
`Metabolism. Mice. Upon partitioning pooled urine (0–24 hr) be-
`tween ethyl acetate and water, 19% of the radioactivity was extracted
`into the ethyl acetate, whereas 76% remained in the aqueous fraction.
`An aliquot of the aqueous fraction was separated by HPLC with
`radiochemical detection and yielded the chromatogram in fig. 5. The
`corresponding HPLC chromatogram from the ethyl acetate extract is
`shown in fig. 6. The individual peaks were collected and analyzed by
`direct infusion electrospray MS and MS/MS. The following metabo-
`lites were identified in urine of mice by comparing their LC and
`LC-MS/MS properties to those obtained from synthetic standards.
`The metabolite that eluted as peak 1 in fig. 5 was identified as
`
`FIG. 3.Plasma concentration vs. time profiles for OLZ and total
`radioactivity in dogs given an oral dose of 5 mg/kg of [14C]OLZ.
`Data are expressed as mean ⫾ SD (N ⫽ 4).
`
`2-carboxy OLZ on the basis of the similarity of its HPLC retention
`time and product ion spectrum to those obtained from a sample of
`synthetic 2-carboxy OLZ. The positive ion electrospray mass spec-
`trum of the major urinary metabolite (peak 2, fig. 5) exhibited an
`MH⫹ ion at m/z 505, which suggested that the metabolite was the
`glucuronide of a hydroxylated OLZ derivative (Mr OLZ ⫽ 312). The
`product ion spectrum of m/z 505 was dominated by the fragment at
`m/z 329, which is likely due to loss of dehydroglucuronic acid from
`the conjugate. ␤-Glucuronidase hydrolysis of the conjugate resulted in
`7-hydroxy OLZ, confirming the major metabolite in urine as 7-hy-
`droxy OLZ glucuronide. Peaks 3 and 4 were characterized as N-des-
`methyl-2-hydroxymethyl OLZ and 2-hydroxymethyl OLZ, respec-
`tively, by comparison with authentic standards. Six metabolites (fig.
`6) were isolated from the ethyl acetate extract for MS identification.
`2-Hydroxymethyl OLZ, which was also present in the aqueous frac-
`tion, was identified as the component eluting as peak 1 in fig. 6. The
`metabolite that eluted as peak 2 had the same HPLC retention volume
`and MS/MS fragmentation as authentic 7-hydroxy OLZ. The metab-
`olite shown as peak 3 (fig. 6) was identified as N-desmethyl OLZ.
`Unchanged OLZ was also excreted in urine (peak 4, fig. 6). The
`identities of the other radiolabeled components in fig. 6 were not
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`Six metabolites of OLZ were identified in the urine of mice in
`addition to the parent compound. Based on the percentage of the
`radioactivity that was extracted into the ethyl acetate (19%) and the
`percentage remaining in the aqueous fraction (76%), the amount of
`urinary radioactivity accounted for by each metabolite was estimated
`as shown in table 3.
`Dogs. Pooled urine sample from the first 48 hr after dosing was
`used for metabolite identification. The partitioning of radioactivity
`between ethyl acetate and water was similar to that obtained for
`mouse urine with 15% extracted into ethyl acetate and 79% of the
`radioactivity remaining in the aqueous fraction. The HPLC separation
`of the radioactive components in the ethyl acetate and aqueous frac-
`tions is shown, respectively, in figs. 7 and 8. In the ethyl acetate
`extract, 2-hydroxymethyl OLZ, N-oxide OLZ, 7-hydroxy OLZ, N-
`desmethyl OLZ (peaks 1–4; fig. 7) were identified in addition to the
`parent compound.
`The major component in the aqueous fraction (peak 3, fig. 8) had a
`retention time that was different from the available standards. The
`electrospray MS of this metabolite gave an apparent protonated mo-
`lecular ion of m/z 345. MS/MS experiments indicated that the metab-
`olite was an N-oxygenated species with a hydroxyl group on the
`benzodiazepine moiety. Approximately 30 mg of the metabolite was
`isolated from urine using XAD-2 chromatography and further purified
`by HPLC fractionation. The 1H- and 13C-NMR data obtained for the
`metabolite are shown in table 4. The 1H-NMR of the metabolite
`showed a downfield shift of the 4⬘-CH3 to ␦3.08 (␦2.21 for OLZ) and
`was identical to the value obtained for N-oxide OLZ. Similarly, the
`13C-NMR exhibited a downfield shift of the 4⬘-CH3 resonance to ␦
`58.09 (␦ 45.73 for OLZ). Two-dimensional nuclear Overhauser en-
`hancement was used to confirm the exact position of the hydroxyl
`group on the benzene ring of OLZ. The absence of a C-7 proton (␦
`6.83 for OLZ) and the fact that the C-9 proton showed ortho coupling
`only (J ⫽ 8 Hz) indicated the hydroxyl group was at the C-7 position.
`Thus, on the basis of combined MS and NMR data, the major urinary
`metabolite in dogs was identified as 7-hydroxy-N-oxide OLZ.
`
`FIG. 4.Plasma concentration vs. time profiles for OLZ and total
`radioactivity in monkeys given an oral dose of 5 mg/kg of [14C]OLZ.
`Data are expressed as mean ⫾ SD (N ⫽ 4).
`
`confirmed, although MS/MS fragmentation indicated that they were
`OLZ metabolites. The peak eluting between peaks 2 and 3 had an
`apparent MH⫹ ion of m/z 327, 2 Da less than that of 2-hydroxymethyl
`OLZ. This metabolite could possibly be the precursor of 2-carboxy
`OLZ, the 2-formyl derivative of OLZ.
`In plasma, in addition to the parent compound, 2-hydroxymethyl
`OLZ, N-desmethyl OLZ and the glucuronide of a hydroxy OLZ
`metabolite were detected. Also, mass spectral data was obtained that
`indicated the presence of two isomeric glutathione conjugates of OLZ.
`The conjugates exhibited an MH⫹ ion at m/z 618, which upon CID
`fragmentation, gave MH-129⫹—a characteristic loss of glutathione
`conjugates (9), in addition to other fragments consistent with the
`glutathione conjugate of OLZ. The MS/MS data also suggested that
`the glutathione moiety was attached to one of the carbons of the
`benzene ring of OLZ.
`
`FIG. 5.HPLC radiochromatogram obtained from the aqueous fraction of mouse urine.
`1, 2-carboxy OLZ; 2, 7-hydroxy OLZ glucuronide; 3, N-desmethyl-2-hydroxymethyl OLZ; 4, 2-hydroxymethyl OLZ.
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`FIG. 6.HPLC radiochromatogram obtained from the ethyl acetate extract of mouse urine.
`1, 2-hydroxymethyl OLZ; 2, 7-hydroxyl OLZ; 3, N-desmethyl OLZ; 4, OLZ.
`
`The aqueous fraction also contained a metabolite (peak 1, fig. 8)
`that was identified as 2-carboxy OLZ. In addition to 7-hydroxy OLZ
`glucuronide (peak 2, fig. 8), a glucuronide of OLZ was identified
`(peak 4, fig. 8) and characterized as the tertiary N-glucuronide, OLZ
`10-N-glucuronide.
`LC/MS analysis of the XAD-2 extract indicated the presence of
`apparent protonated molecular ions at m/z 432 and 448. The product
`ion spectra of these metabolites indicated that the metabolites might
`be the cysteine adducts of OLZ and N-oxide OLZ. CID analysis of the
`ion at m/z 432 resulted in fragment ions at m/z 345 and 311 that could
`be produced, respectively, from loss of 87 Da as a neutral [CH2AC—
`(NH2)COOH] from the cysteinyl moeity of the conjugate and com-
`plete cleavage of the cysteine residue. The ion at m/z 311 further
`fragmented to an ion at m/z 254. This transition is characteristic of
`OLZ and metabolites (6) and results from loss of 57 Da as
`CH2ACH—NH—CH3 from the methyl piperazine ring of the mole-
`cules. MS/MS analysis (precursor m/z 448) of the putative cysteine
`conjugate of N-oxide OLZ yielded a fragmentation pattern that was
`different from that obtained for the corresponding conjugate of OLZ.
`The fragment at m/z 261 perhaps resulted from the combined loss of
`100 Da (scission of the methyl piperazine ring) and 87 Da [CH2AC—
`(NH2)COOH, from the cysteine residue]. The additional loss of pos-
`sibly hydrogen sulfide resulted in a fragment at m/z 228. A weak ion
`at m/z 401 resulted from the loss of 47 Da from the methyl piperazine
`portion of the molecule that is a characteristic fragmentation pathway
`of N-oxide OLZ (6).
`After 3 hr postdose, plasma contained OLZ, 2-hydroxymethyl,
`N-oxide, N-desmethyl, and the 7-hydroxy metabolites, as well as the
`glucuronide of 7-hydroxy OLZ. After 12 hr, the plasma metabolite
`profile was similar to that obtained at 3 hr, except that the level of
`N-oxide was lower than that of the 7-hydroxy metabolite and no
`7-hydroxy glucuronide was detected.
`The relative amount of each metabolite and parent drug in urine
`was estimated by HPLC with radiochemical detection as detailed in
`the Materials and Methods and is presented in table 3. The amount of
`7-hydroxy OLZ was estimated from the ethyl acetate extract. The
`ethyl acetate extract contained 7-hydroxy OLZ; however, this metab-
`olite was not detectable in the XAD-2 extract. The 7-hydroxy metab-
`
`Compound
`
`TABLE 3
`Estimated amounts of OLZ and its metabolites in urine
`% Urinary Radioactivity
`Mouse
`Dog
`Monkey
`1.1a
`NDb
`39.6
`NDb
`1.1
`3.3
`NDb
`ND
`20.9
`1.7
`ND
`8.2
`ND
`ND
`36.0
`
`7-Hydroxy glucuronide
`7-Hydroxy
`7-Hydroxy-N-oxide
`N-desmethyl-2-hydroxymethyl
`N-desmethyl-2-desmethyl-2-
`carboxy
`2-Hydroxymethyl
`2-Hydroxymethyl glucuronide
`2-Carboxy
`N-oxide
`N-oxide-2-hydroxymethyl
`N-oxide-2-carboxy
`N-desmethyl
`CYSe conjugate
`CYS conjugate of N-oxide
`NACf conjugate
`Olanzapine
`
`14.9
`ND
`6.6
`ND
`ND
`ND
`1.6
`ND
`ND
`ND
`3.6
`
`9.3
`ND
`8.0d
`3.4
`ND
`ND
`0.3
`5.1
`2.3
`ND
`6.1
`
`6.4
`1.7c
`7.0
`NDb
`11.5
`9.0
`NDb
`NDb
`NDb
`2.8
`NDb
`
`82.6
`59.7
`69.1
`Total
`a 7-Hydroxy glucuronide coeluted with the cysteine conjugate of OLZ under
`the HPLC conditions used. An estimate for these metabolites was obtained
`using ms and comparing the ion intensities at m/z 432 and 505.
`b ND, not detected.
`c 2-Hydroxymethyl glucuronide coeluted with 2-carboxy. The level of each
`metabolite was estimated by ms on the basis of ion intensities at m/z 343 and
`505.
`d 2-Carboxy coeluted with the cysteine conjugate of N-oxide. The relative
`amount of each metabolite was estimated by ms on the basis of ion intensities
`at m/z 343 and 448.
`e CYS, cysteine.
`f NAC, N-acetylcysteine.
`
`olite is fairly susceptible to air oxidation and could have decomposed
`during the lengthy XAD-2 extraction procedure.
`Monkeys. An aliquot of the first 24-hr urine sample from each
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`FIG. 7.HPLC radiochromatogram obtained from the ethyl acetate extract of dog urine.
`1, 2-