Simultaneous quantification of loxapine, loxapine N-oxide, amoxapine, 8-hydroxyloxapine and 7-hydroxyloxapine in human plasma using LC-MS/MS.

Loxapine is an antipsychotic medication used for the treatment of schizophrenia. In vivo, loxapine is metabolized to multiple metabolites. A high performance liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of loxapine and 4 of its metabolites, loxapine N-oxide, amoxapine (N-desmethyl loxapine), 8-hydroxyloxapine and 7-hydroxyloxapine, in human plasma to support regulated clinical development. During method development, several technical challenges such as poor chromatography, separation of structural isomers, and inadequate sensitivity were met and overcome. The final method utilized micro-elution solid phase extraction (SPE) to extract plasma samples (100µL), and the resulting extracts were analyzed using reversed phase LC-MS/MS using a turbo-ionspray interface in positive ionization mode with selected reaction monitoring (SRM). The method was fully validated according to the current regulatory guidance for bioanalysis over the calibration curve range 0.0500-50.0ng/mL for all analytes using 1/x2-weighted linear regression analysis. Based on three separate runs, the between-run precision and inter-day precision for all five analytes at all concentrations, including the LLOQ (lower limit of quantitation) quality control at 0.0500ng/mL, varied from 0.0% to 13.8%, while the accuracy ranged from 86.4% to 109.3% of nominal. The extraction recoveries of loxapine and the four metabolites were above 80%. Various forms of short-term and long-term stability were established in both solutions and matrix, including the stability of loxapine and the four metabolites in human plasma for up to 260days of storage at -20°C. This method has been used to support a regulated clinical study, which included the successful execution of incurred sample reanalysis (ISR) testing. To the best of our knowledge, this is the first published methodology in which these five analytes were quantified with a single extraction and injection.

A randomized, placebo-controlled repeat-dose thorough QT study of inhaled loxapine in healthy volunteers.

OBJECTIVE: This randomized, double-blind, active- and placebo-controlled, crossover, thorough QT study assessed the effect of two inhaled loxapine doses on cardiac repolarization as measured by corrected QT (QTc) interval in healthy subjects (ClinicalTrials.gov NCT01854710). METHODS: Subjects received two doses of inhaled loxapine (10 mg) 2 hours apart+oral placebo, two doses of inhaled placebo+oral placebo, or two doses of inhaled placebo+oral moxifloxacin (400 mg; positive control), with ≥3 days washout between treatments. Two-sided 90% confidence intervals (CIs) were calculated around least-squares mean predose placebo-subtracted individually corrected QT durations (ΔΔTcIs) at 12 time points throughout 24 hours after dosing. A ΔΔTcI 95% upper CI exceeding 10 msec was the threshold indicating QTc prolongation (primary endpoint). Secondary endpoints included Fridericia- and Bazett-corrected QT duration and QTcI outliers. Pharmacokinetics and adverse events (AEs) were also assessed. RESULTS: Of 60 subjects enrolled (mean age, 33.8 years; 52% male), 44 completed the study. Post loxapine dosing, no ΔΔTcI 95% upper CI exceeded 10 msec; the largest was 6.31 msec 5 minutes post dose 2. Methodology was validated by ΔΔTcI 95% lower CIs exceeding 5 msec at 9 of 12 time points after moxifloxacin dosing. Loxapine plasma concentrations increased rapidly (mean Cmax, 177 ng/mL; median tmax 2 minutes after dose 2, 2.03 hours after dose 1). There were no deaths, serious AEs, or AEs leading to discontinuation, and one severe AE. CONCLUSIONS: Primary and secondary endpoints indicated two therapeutic doses of inhaled loxapine did not cause threshold QTc prolongation in this study.

Investigation of the disposition of loxapine, amoxapine and their hydroxylated metabolites in different brain regions, CSF and plasma of rat by LC-MS/MS.

Loxapine represents an interesting example of old "new" drug and is recently drawing attention for its novel inhalation formulation for the treatment of both psychiatric and non-psychiatric disorders. It is extensively metabolized to several active metabolites with diverging pharmacological properties. To further pursue the contribution of metabolites to the overall outcome after loxapine administration, quantification of both loxapine and its active metabolites is essential. The current study developed a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of loxapine and its five metabolites (amoxapine, 7-hydroxy-loxapine, 8-hydroxy-loxapine, 7-hydroxy-amoxapine and 8-hydroxy-amoxapine) in rat brain tissues, plasma and cerebrospinal fluid (CSF). By evaluating the effects of perchloric acid and methanol on analyte recovery, the extraction methods were optimized and only small amounts of sample (100 µl for plasma and less than 100mg for brain tissue) were required. The lower limits of quantification (LLOQs) in brain tissue were 3 ng/g for loxapine and amoxapine and 5 ng/g for the four hydroxylated metabolites of loxapine. The LLOQs were 1 ng/ml for loxapine and amoxapine and 2 ng/ml for the four hydroxylated metabolites in plasma, and 10 ng/ml for all analytes in CSF. The developed method was applied to a pharmacokinetic study on rats treated with a low-dose loxapine by oral administration. Four hours after loxapine dosing, high levels of 7-hydroxy-loxapine were found throughout the ten brain regions examined (68-124 ng/g), while only trace amount of loxapine was measured in brain (<5 ng/g) and plasma (<3 ng/ml). The method provides a useful tool for both preclinical and clinical investigations on the dispositions of loxapine and its metabolites, which would help to elucidate their roles in neurotherapeutics.

Validation of HPLC-MS/MS methods for analysis of loxapine, amoxapine, 7-OH-loxapine, 8-OH-loxapine and loxapine N-oxide in human plasma.

BACKGROUND: Two ESI-LC-MS/MS methods were validated for the quantitative analysis of loxapine, amoxapine, 7-OH-loxapine, 8-OH-loxapine and loxapine N-oxide in human K(2)EDTA plasma. Cation-exchange solid-phase extraction (SPE) was used to extract loxapine, amoxapine and the two hydroxylated metabolites, and organic precipitation was used to quantify loxapine N-oxide. RESULTS: Both methods were shown to be accurate (±13%), intra-assay precision was less than 15%, and inter-assay precision was less than 10% in all instances across the entire dynamic range of the assays (0.0500-50.0 ng/ml for the SPE method and 0.100-25.0 ng/ml for the precipitation method). CONCLUSION: The validated methods for loxapine, amoxapine, 7-OH-loxapine, 8-OH-loxapine and loxapine N-oxide have been used to successfully support clinical trials.

Contrasting loxapine to its isomer isoloxapine--the critical role of in vivo D2 blockade in determining atypicality.

BACKGROUND: Loxapine is a typical antipsychotic while isoloxapine, its 8Cl-isomer, shows atypicality in some animal models. The basis for this difference is not well understood. The purpose of this study was to systematically compare the two drugs in in vitro and in vivo animal models, and to understand mechanisms underlying their differential typical/atypical profiles. METHODS: The in vitro and in vivo receptor profiles as well as the action of loxapine and isoloxapine on rat conditioned avoidance response (CAR), catalepsy (CAT), striatal FOS expression and prolactin levels were determined. To understand loxapine's typical profile, we added MDL100,907, to provide loxapine+MDL the same in vivo 5-HT2/D2 ratio as isoloxapine, while holding its D2 component constant. RESULTS: Isoloxapine behaved as an "atypical" antipsychotic demonstrating CAR inhibition, low CAT, no significant prolactin elevation, and minimal FOS expression in the dorsolateral striatum. Loxapine behaved like a typical antipsychotic, showing unexpectedly high in vivo D2 occupancy. Addition of MDL100,907, which resulted in a very high 5-HT2/D2 in vivo ratio, did not alter loxapine + MDL's typical profile. CONCLUSIONS: Loxapine's behaviour as a typical antipsychotic is most likely due to its disproportionately high D2 occupancy. Appropriate action at D2 receptors in vivo, rather than the high 5-HT2/D2 ratio, seems to be critical in determining why isoloxapine behaves like an atypical antipsychotic.

Clozapine reversal of the deficits in coordinated movement induced by D2 receptor blockade does not depend upon antagonism of alpha2 adrenoceptors.

Alpha2 adrenoceptor antagonists have been shown to reverse D2-antagonist-induced catalepsy leading to the hypothesis that the alpha2 antagonistic properties of clozapine underlie the compound's lack of extrapyramidal symptoms in the clinic. The potential for alpha2 antagonists to reverse the motor deficits produced by D2 antagonists (loxapine and haloperidol) was further investigated using a rotating rod (3.5 rpm) test in male Sprague-Dawley rats that requires coordinated movement to perform the task. The effects of loxapine (0.3 mg/kg, s.c.) were dose-dependently and statistically significantly reversed by the administration of clozapine (1,3, 10 mg/kg, i.p., n=10). Isoloxapine (1 mg/kg, i.p.), RX 821002 (2-methoxy-idazoxan; 5.6 mg/kg, i.p.) and yohimbine (5.6 mg/kg, i.p.) did not reverse the effects of loxapine. Furthermore, the motor deficits produced by haloperidol could not be reversed by RX 821002 (5.6 mg/kg, i.p.) or yohimbine (5.6 mg/kg, i.p.). On the other hand, scopolamine (0.03-0.3 mg/kg, i.p.) dose-dependently and statistically significantly antagonised the effects of both loxapine and haloperidol. These results indicate that the anticholinergic rather than the alpha2 antagonistic properties of clozapine may mediate the reversal of the motor deficit induced by D2 antagonism in a rotating rod test.

The role of alpha2-adrenoceptor antagonism in the anti-cataleptic properties of the atypical neuroleptic agent, clozapine, in the rat.

1. The mechanism underlying the anticataleptic properties of the atypical neuroleptic agent, clozapine, has been investigated in the rat. 2.The close structural analogues of clozapine, loxapine (0.1 mg kg(-1) s.c.) and iso-clozapine (1 and 3 mg kg(-1) s.c.) induced catalepsy in rats. In contrast, clozapine and the regio-isomer of loxapine, iso-loxapine (up to 10 mg kg(-1) s.c.) did not produce catalepsy, but at a dose of 1 mg kg(-1) significantly inhibited catalepsy induced by loxapine (0.3 mg kg(-1) s.c.). 3. Radioligand binding assays showed that cataleptogenic potential was most clearly predicted by the D2/5-HT1A, D2/5-HT1B/1D and D2/alpha2-receptor affinity (KD) ratios: i.e. 30-100-fold higher ratios were calculated for loxapine and iso-clozapine, whereas the ratios were less than 1 for clozapine and iso-loxapine. The ratios of affinities for D2 to 5-HT2A, 5-HT2C or D1 did not reflect the grouping of cataleptic and non-cataleptic compounds. 4. Co-treatment with the alpha2-adrenoceptor antagonists, yohimbine (1-10 mg kg(-1) s.c.), RX 821002 (1-10 mg kg(-1) s.c.) and MK-912 (0.3 and 1 mg kg(-1) s.c.) dose-dependently inhibited the cataleptic response to loxapine (0.3 mg kg(-1)). Yohimbine (1-10 mg kg(-1) s.c.) also dose-dependently inhibited the cateleptic response to haloperidol (0.3 mg kg(-1) s.c.). The alpha2-adrenoceptor antagonists had no effect per se. 5. Neither yohimbine (10 mg kg(-1)) nor RX821002 (3 mg kg(-1)) altered the cataleptic response to the D1 receptor antagonist, SCH 23390 (1 mg kg(-1) s.c.), while, like clozapine, both compounds abolished the response to the 5-HT2A receptor antagonist, MDL 100,151 (3 mg kg(-1) s.c.). 6. The present data strongly implicate alpha2-adrenoceptor blockade in the anticataleptic properties of clozapine and suggest that its lack of extrapyramidal side effects in the clinic may also be a consequence of this property.

The role of metabolites in a bioequivalence study 1: loxapine, 7-hydroxyloxapine and 8-hydroxyloxapine.

Loxapine is a drug which is used in the treatment of psychotic disorders. The drug is extensively biotransformed in humans to produce a variety of metabolites, some of which have pharmacological activity. Seven-hydroxyloxapine is 4-5 times more active than the parent drug, although its concentrations in plasma are relatively low. Eight-hydroxyloxapine, on the other hand, is inactive, but is present in higher concentrations in plasma than the parent drug. In the present randomized crossover study to evaluate the bioequivalence of two dosage forms containing loxapine, the parent drug and its 7-hydroxy and 8-hydroxy metabolites were monitored for up to 96 hours after the administration of the test or reference formulations. Estimates of average bioavailability (AUC- infinity, AUC0t and Cmax) were obtained by the difference of the least squares means of log test and log reference. A 90% confidence interval for the log difference was derived from the within-subject error. The test of bioequivalence requires the 90% confidence band so calculated to fall entirely within an imposed regulatory interval of 80-125%. The results showed the two formulations to be bioequivalent in terms of the log mean differences and 90% confidence bands calculated for all three analytes. In fact, the within-subject variabilities of the metabolites were relatively low, so that the 90% confidence intervals for ln AUC0 infinity, ln AUC0t and ln Cmax calculated for the metabolites, were narrower that those for the parent drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous quantitation of loxapine, amoxapine and their 7- and 8-hydroxy metabolites in plasma by high-performance liquid chromatography.

Loxapine, its N-demethylated metabolite amoxapine, and their 7- and 8-hydroxy metabolites were determined simultaneously in plasma by a simple two-step extraction procedure followed by reversed-phase liquid chromatography. Baseline separation was achieved by a 5-microns Spherisorb C6 column. The mobile phase consisted of 5 mM phosphate buffer (with 14 mM orthophosphoric acid)-acetonitrile (with 105 microM nonylamine) (77:23, v/v). Assays of the steady-state plasma samples obtained from seventeen patients on loxapine showed substantial amounts of 8-hydroxy metabolites, lesser amounts of loxapine, amoxapine and 7-hydroxyloxapine and trace amounts of 7-hydroxyamoxapine. As 8-hydroxy metabolites possess only weak dopamine-D2 blocking activity, the final neuroleptic property of loxapine may be affected significantly by metabolic polymorphism.

Ligand binding and platelet uptake studies of loxapine, amoxapine and their 8-hydroxylated derivatives.

Loxapine, amoxapine and their 8-hydroxylated derivatives were studied by means of [3H]imipramine binding to rat cortical membranes, [3H]spiperone binding to rat striatal membranes, and the inhibition of serotonin uptake by human platelets. As inhibitors of [3H]imipramine binding: amoxapine greater than hydroxyamoxapine greater than loxapine = hydroxyloxapine; as inhibitors of platelet serotonin uptake: hydroxyamoxapine greater than amoxapine greater than hydroxyloxapine greater than loxapine; and as inhibitors of [3H]spiperone binding: loxapine greater than amoxapine greater than hydroxyamoxapine greater than hydroxyloxapine. The antipsychotic properties of loxapine and amoxapine were supported by the binding results, which also indicated the probable antipsychotic activities of the metabolites. All 4 compounds may possess dual action of antidepressant effect as well as antipsychotic effect.

Determination of loxapine in human plasma and urine and identification of three urinary metabolites.

1. A g.l.c. method for quantitative determination of loxapine (2-chloro-11-(4-methyl-1-piperazinyl)dibenz(b,f)(1,4)oxazepine), in human plasma and urine is described. 2. Preliminary pharmacokinetic data on plasma concn of loxapine over 12 h from five psychiatric patients who received a total average dose of 80 mg of loxapine succinate per day orally for twelve weeks are presented. 3. In addition to unchanged loxapine, three urinary metabolic products, namely aromatic ring-hydroxy loxapine, desmethyl loxapine and loxapine-N-oxide, were identified using g.l.c.--mass spectrometry.

[Multidimensional diagnostics in pharmacopsychiatry/results of therapy with desmethyl-loxapine (author's transl)]. / Mehrdimensionale Diagnostik in der Pharmakopsychiatrie Ergebnisse eines Therapieversuchs mit Desmethyl-loxapine

In view of the particular problems of psychiatric classification the advantages and disadvantages of standardised rating scales are discussed. The result is that these instruments at present are irreplaceable in psychopathological research on behalf of their reliability in transmitting information and the possibility of quantifying psychopathological syndromes. The practice of 4 rating scales, which are still uncustomary mainly in the area of German language, is demonstrated by means of a psychopharmacological investigation example. These rating scales are: 1. the Hamilton Psychiatric Rating Scale for Depression, 2. the Wittenborn Psychiatric Rating Scale, 3. the Structured clinical Interview (Burdock and Hardesty) and 4. the Befindlichkeitsskala (Condition Scale) (v. Zerssen). Within a duration of treatment of 3 and 4 weeks desmethyl-loxapine [CL 67 772, 2-chloro-11-(1-piperazinyl)-dibenz (b,f)(1,4)-oxazepine] was tested in an open study as an antidepressant on 16 patients with different depressive syndromes. The results of treatment, which can be considered positive, are represented and discussed in an intra-individual pre- and after-treatment comparison in respect of the differences between these scales. The good therapeutic efficacy of desmethyl-loxapine results mainly from a marked anxiolytic effect. All the clinical and chemical examinations performed point out an excellent tolerance of this drug. German versions of standardised rating scales appear urgently indicated.

[The significance of standardized evaluation scales in psychiatric diagnosis as exemplified by the therapeutic intervention with desmethyl-loxapine (author's transl)]. / Die Bedeutung standardisierter Beurteilungsskalen in der psychiatrischen Diagnostik--dargestellt am Beispiel eines Therapieversuchs mit Desmethyl-Loxapin

Taking into account the particular problems of psychiatric classification the advantages and disadvantages of standardised rating scales are discussed. The authors come to the conclusion that these instruments at present are irreplaceable in psychopathological research because of their reliability in transmitting information and the possibility of quantifying psychopathological syndromes. The use of four rating scales which are little known yet within German speaking countries in demonstrated by means of a psychopharmacological investigation on the clinical effects of Desmethyl-Loxapine. These rating scales are: 1. the Hamilton-Psychiatric-Rating-Scale for Depression 2. the Wittenborn-Psychiatric-Rating-Scale 3. the Structured Clinical Interview (Burdock and Hardesty) and 4. the Befindlichkeits-Skala (Condition-Scale). Desmethyl-Loxapine was given in an open study as an antidepressant to 16 patients with different depressive syndromes. The results of treatment which lasted 3 to 4 weeks are discussed. The good therapeutic efficacy of Desmethyl-Loxapine was mainly due to its marked anxiolytic effect. All accomplished clinical examinations showed an excellent tolerance of this drug.