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.

The antipsychotic drug loxapine is an opener of the sodium-activated potassium channel slack (Slo2.2).

Sodium-activated potassium (K(Na)) channels have been suggested to set the resting potential, to modulate slow after-hyperpolarizations, and to control bursting behavior or spike frequency adaptation (Trends Neurosci 28:422-428, 2005). One of the genes that encodes K(Na) channels is called Slack (Kcnt1, Slo2.2). Studies found that Slack channels were highly expressed in nociceptive dorsal root ganglion neurons and modulated their firing frequency (J Neurosci 30:14165-14172, 2010). Therefore, Slack channel openers are of significant interest as putative analgesic drugs. We screened the library of pharmacologically active compounds with recombinant human Slack channels expressed in Chinese hamster ovary cells, by using rubidium efflux measurements with atomic absorption spectrometry. Riluzole at 500 µM was used as a reference agonist. The antipsychotic drug loxapine and the anthelmintic drug niclosamide were both found to activate Slack channels, which was confirmed by using manual patch-clamp analyses (EC(50) = 4.4 µM and EC(50) = 2.9 µM, respectively). Psychotropic drugs structurally related to loxapine were also evaluated in patch-clamp experiments, but none was found to be as active as loxapine. Loxapine properties were confirmed at the single-channel level with recombinant rat Slack channels. In dorsal root ganglion neurons, loxapine was found to behave as an opener of native K(Na) channels and to increase the rheobase of action potential. This study identifies new K(Na) channel pharmacological tools, which will be useful for further Slack channel investigations.

Rapid liquid chromatography/tandem mass spectrometer (LCMS) method for clozapine and its metabolite N-desmethyl clozapine (norclozapine) in human serum.

Clozapine is indicated for the treatment of schizophrenia and related psychotic disorders. Several methods have been developed for monitoring Clozapine levels; however, they possess limited specificity and are often laborious. This study describes a simple liquid chromatography/tandem mass spectrometer (LCMS) method in human serum. The ion transitions monitored were m/z 327, 270, 296 for Clozapine, m/z 313, 192, 227 for Norclozapine and m/z 328, 271 for Loxapine. The assay is linear (25-1000 ng/ml) and showed a good correlation (r=0.98) within the analytical range of 79-1210 ng/ml in human serum. This assay is highly specific and sensitive for the simultaneous measurements of Clozapine and Norclozapine. The simplification of this assay makes it ideal for high throughput analyses of the patient samples in a routine clinical laboratory staffed with general medical technologists.

Pharmacokinetics and Safety of Single-Dose Inhaled Loxapine in Children and Adolescents.

This multisite open-label study sought to characterize the pharmacokinetics and safety of a single dose of inhaled loxapine in children and adolescents. Loxapine powder for oral inhalation was administered via a single-use handheld drug device to children and adolescents (aged 10-17 years) with any condition warranting chronic antipsychotic use. Patients were dosed according to body weight and cohort (<50 kg [n = 15], 2.5 or 5 mg; ≥50 kg [n = 15], 5 or 10 mg); the first 6 patients (cohort 1) enrolled in each weight group received the lower dose. Patients were enrolled in the higher-dose group (cohort 2) after an interim pharmacokinetic and safety analysis of data from cohort 1. Blood samples were collected for 48 hours after dosing to determine the pharmacokinetic profile of loxapine and its metabolites. Safety was assessed using adverse event (AE), laboratory value, physical/neurologic examination, vital sign, electrocardiogram, suicidality, and extrapyramidal symptom assessment. Thirty patients were enrolled and evaluable for pharmacokinetics. Loxapine plasma concentrations peaked by 2 to 5 minutes in most patients; systemic exposure increased with dose in both weight subgroups. Loxapine terminal elimination half-life was ∼13 to 17 hours. The most common AEs were sedation and dysgeusia. Sedation was severe in 1 patient in the <50-kg group (2.5-mg dose) and 1 patient in the ≥50-kg group (5-mg dose). No AEs indicative of bronchospasm or other serious AEs were reported. Inhaled loxapine was rapidly absorbed and generally well tolerated in pediatric patients; no new safety signals were observed.

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.

Multiple dose pharmacokinetics of inhaled loxapine in subjects on chronic, stable antipsychotic regimens.

This randomized, double-blind, placebo-controlled, parallel-group study was to determine the pharmacokinetic characteristics, safety, and tolerability of multiple doses of inhaled loxapine aerosol in subjects on a stable, oral, chronic antipsychotic regimen. Loxapine was delivered by means of a unique thermally generated aerosol comprising drug particles of a size designed for deep lung delivery and absorption. Thirty-two subjects were randomized 1:1:1:1 to receive inhaled loxapine (total doses of 15, 20, or 30 mg) or inhaled placebo administered in 3 divided doses, given 4 hours apart. Following inhalation, the median Tmax was 2 minutes, and concentrations declined to about half Cmax approximately 5 minutes later across the 3 dose levels. The dose proportionality across data from this study combined with data from the single-dose study showed a slope (90%CI) of log AUCinf versus log dose of 0.818 (0.762-0.875) across the 8 doses (n = 60 subjects) studied, indicating reasonable dose proportionality. The most common adverse events were cough (3 of 32, 9%), sedation (3 of 32, 9%), and dysgeusia (2 of 32, 6%). The inhalation of multiple doses of inhaled loxapine were well tolerated in study subjects and provided a safe, well-tolerated means for rapidly and reliably achieving therapeutic plasma concentrations of loxapine. ClinicalTrials.gov identifier: NCT00555412.

Amoxapine: neuroleptic as well as antidepressant?

Amoxapine, a new antidepressant, is the N-desmethyl analog of loxapine, a neuroleptic. There have been reports suggesting that amoxapine itself or its metabolites have neuroleptic as well as antidepressant properties. With in vitro studies using a radioreceptor assay for neuroleptics, the authors found that amoxapine--and one of its metabolites in particular (7-hydroxyamoxapine)--have potent neuroleptic-like activity. Furthermore, blood specimens from patients receiving amoxapine showed the presence of neuroleptic activity in the same assay. The authors note the implications of these findings for gauging the benefits and risks of treatment with amoxapine, including the risk of neurologic effects.

Clinical and plasma level characteristics of intramuscular and oral loxapine.

The intramuscular and oral forms of loxapine succinate were compared in their clinical, side effect, and blood level characteristics in ten hospitalized chronic schizophrenic patients. The first phase of the study determined the single dose that produced moderate sedation (i.e., the sedation threshold), and this dose was essentially the same for the two forms. Continuous administration of the two forms using the individualized sedation threshold dosage also failed to indicate any clinical or side effect differences in the two forms. The blood level characteristics, however, did differ between the two forms. The kinetic studies indicated that there was a larger are under the loxapine curve with the intramuscular form than with the oral form, while the 8-OH loxapine area was larger with the oral form. The steady-state studies also showed that the i.m. form had higher loxapine levels than the oral form. The significance of these findings, both clinically and in terms of the relative activity of loxapine and its metabolites, is discussed.

Improved gas chromatographic analysis of chlorpromazine in blood serum.

Reliable determinations of chlorpromazine levels in blood serum samples obtained from patients were accomplished by electron capture gas chromatography. By using modifications of the procedure to insure stability of the sample, minimal losses during sample preparation and gas chromatography, and by selecting appropriate operating parameters of the electron capture detector, excellent agreement was obtained in replicate analyses with a limit of sensitivity of 1 ng/ml in 1 ml of plasma.

GLC analysis of loxapine, amoxapine, and their metabolites in serum and urine.

A GLC analysis is presented for loxapine, amoxapine, and their major metabolites in serum and urine. Electron-capture detection is employed for serum analysis, and flame ionization is used for urine analysis. The procedure includes trifluoroacetylation of secondary amine functions, followed by trimethylsilylation of phenolic groups after ethyl acetate extraction of the sample. Urine requires prior enzymatic hydrolysis of conjugates. Data indicating the utility of the procedure in hospitalized patients and normal volunteers are presented.

Acute loxapine intoxication in a child.

Thin-layer chromatography (TLC) was used to confirm the alleged ingestion of Loxitane (loxapine succinate) by a 20-month-old child. GC was used to further characterize TLC spots and to quantitate the loxapine concentration of the blood at 0.072 mg/dL, which was consistent with the child's presenting signs of lethargy and ataxia. The appropriate supportive symptomatic therapy, with monitoring for CNS and cardiovascular toxicities, resulted in an uneventful recovery.

GLC/MS assay for loxapine in human biofluids and tissues with deuterium labeled analog as an internal standard.

A quantitative gas liquid chromatographic-mass spectrometric (GLC/MS) assay was developed for the determination of loxapine in human blofluids and tissues. The assay utilizes selected ion monitoring in a GLC effluent of the molecular ion of loxapine generated by electron-impact ionization (EI). Loxapine-d3 was used as the internal standard. The assay can measure 2 ng/mL of loxapine with about 6% precision. The curve relating the amounts of loxapine added in control plasma versus the ion intensity ratio (m/e 327/330) over a large range of loxapine concentrations was linear with essentially zero intercept. The method was used for the analysis of loxapine in human urine, plasma, brain, liver, lung and spleen.

Loxapine delivered as a thermally generated aerosol does not prolong QTc in a thorough QT/QTc study in healthy subjects.

The objective of this study was to establish effects of inhaled loxapine on the QTc interval in this randomized, placebo-controlled, double-blind crossover study. Forty-eight healthy volunteers received a single inhaled placebo or 10 mg loxapine. Plasma concentrations of loxapine increased with a median Tmax of 1 minute and a mean Cmax of 312 ng/mL. After an initial rapid distribution phase, plasma concentrations of loxapine declined with a terminal half-life of 8 hours. Exposure to the active metabolite 7-OH-loxapine was 15% of the parent compound based on mean AUCinf and its terminal half-life was 12 hours. Inhaled loxapine did not increase QT intervals, as demonstrated by the upper bound of the 1-sided 95% CIs placed on the point estimate of the placebo-subtracted change of QTcI (ΔΔQTcI) being less than 10 milliseconds at all 11 post-dose times. The maximum ΔΔQTcI occurred at 1 hour post-dose (LSmean 5.42 milliseconds, upper confidence bound 7.75 milliseconds). The study outcome was validated by the demonstrated assay sensitivity using the positive control moxifloxacin maximum ΔΔQTcI occurred at 3 hour post-dose (LSmean 8.36 milliseconds, lower confidence bound 5.82 milliseconds). The analyses of QTc outliers, and the lack of emergent diagnostic findings for QTcI, QTcB, and QTcF; and simple mean placebo-subtracted changes of QTcI and QTcF supported the primary QT analysis conclusion that this is a negative finding and there is no apparent QT prolongation associated with the therapeutic dose of inhaled loxapine.

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.

Fishing for a drug: solid-phase microextraction for the assay of clozapine in human plasma.

Solid-phase microextraction (SPME) was investigated as a sample preparation method for assaying the neuroleptic drug clozapine in human plasma. A mixture of human plasma, water, loxapine (as internal standard) and aqueous NaOH was extracted with a 100-micron polydimethylsiloxane (PDMS) fiber (Supelco). Desorption of the fiber was performed in the injection port of a gas chromatograph at 260 degrees C (HP 5890; 30 m x 0.53 mm I.D., 1 micron film capillary; nitrogen-phosphorous selective detection). Fibers were used repeatedly in up to about 75 analyses. The recovery was found to be 3% for clozapine from plasma after 30 min of extraction. However, in spite of the low recovery, the analyte was well separated and the calibration was linear between 100 and 1000 ng/ml. The within-day and between-day precision was consistently about 8 to 15% at concentrations of 200 ng/ml to 1000 ng/ml. No interfering drug was found. The limit of detection was 30 ng/ml. The sample volume was 250 microliters. The influence of the concentration of proteins, triglycerides and salt, i.e., changes in the matrix on the peak areas and peak-area ratios was studied. The method is not impaired by physiological changes in the composition of the matrix. Good agreement was found with a liquid-liquid extraction-gas-liquid chromatography (LLE-GLC) standard method and an on-line column-switching high-performance liquid chromatography (HPLC) method for patients' samples and spiked samples, respectively. It is concluded that the method can be used in the therapeutic drug monitoring of clozapine because the therapeutic window of clozapine is from 350 to 600 ng/ml.

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.