Absorption, distribution, metabolism and excretion (ADME) of the ALK inhibitor alectinib: results from an absolute bioavailability and mass balance study in healthy subjects.

1. Alectinib is a highly selective, central nervous system-active small molecule anaplastic lymphoma kinase inhibitor. 2. The absolute bioavailability, metabolism, excretion and pharmacokinetics of alectinib were studied in a two-period single-sequence crossover study. A 50 µg radiolabelled intravenous microdose of alectinib was co-administered with a single 600 mg oral dose of alectinib in the first period, and a single 600 mg/67 µCi oral dose of radiolabelled alectinib was administered in the second period to six healthy male subjects. 3. The absolute bioavailability of alectinib was moderate at 36.9%. Geometric mean clearance was 34.5 L/h, volume of distribution was 475 L and the hepatic extraction ratio was low (0.14). 4. Near-complete recovery of administered radioactivity was achieved within 168 h post-dose (98.2%) with excretion predominantly in faeces (97.8%) and negligible excretion in urine (0.456%). Alectinib and its major active metabolite, M4, were the main components in plasma, accounting for 76% of total plasma radioactivity. In faeces, 84% of dose was excreted as unchanged alectinib with metabolites M4, M1a/b and M6 contributing to 5.8%, 7.2% and 0.2% of dose, respectively. 5. This novel study design characterised the full absorption, distribution, metabolism and excretion properties in each subject, providing insight into alectinib absorption and disposition in humans.

Characterization of anti-drug antibody responses to the T-cell engaging bispecific antibody cibisatamab to understand the impact on exposure.

An appropriately designed pharmacokinetic (PK) assay that is sensitive for anti-drug antibody (ADA) impact on relevant exposure is an alternative strategy to understand the neutralizing potential of ADAs. However, guidance on how to develop such PK assays and how to confirm the functional ADA impact on exposure is missing. Here, the PK assay of a T-cell-engaging bispecific antibody, cibisatamab, was developed based on its mechanism of action (MoA). Using critical monoclonal anti-idiotypic (anti-ID) antibody positive controls as ADA surrogates, the impact on exposure was evaluated pre-clinically. In a phase I clinical trial (NCT02324257), initial data suggest that the combination of ADA and PK assays for correlation of the ADA response with cibisatamab exposure. To understand the neutralizing potential of patient-derived ADAs on drug activity, advanced ADA characterization has been performed. Structural binding analysis of ADAs to antibody domains of the drug and its impact on targeting were assessed. For this purpose, relevant patient ADA binding features were identified and compared with the specific monoclonal anti-ID antibody-positive controls. Comparable results of target binding inhibition and similar impacts on exposure suggest that the observed reduction of Cmax and Ctrough levels in patients is caused by the neutralizing potential of ADAs and allows a correlation between ADA response and loss of exposure. Therefore, the described study provides important functional aspects for the development of an appropriately designed PK assay for bispecific antibodies as an alternative option towards understanding the neutralizing ADA impact on exposure.

CEA-CD3 bispecific antibody cibisatamab with or without atezolizumab in patients with CEA-positive solid tumours: results of two multi-institutional Phase 1 trials.

Cibisatamab is a bispecific antibody-based construct targeting carcinoembryonic antigen (CEA) on tumour cells and CD3 epsilon chain as a T-cell engager. Here we evaluated cibisatamab for advanced CEA-positive solid tumours in two open-label Phase 1 dose-escalation and -expansion studies: as a single agent with or without obinutuzumab in S1 (NCT02324257) and with atezolizumab in S2 (NCT02650713). Primary endpoints were safety, dose finding, and pharmacokinetics in S1; safety and dose finding in S2. Secondary endpoints were anti-tumour activity (including overall response rate, ORR) and pharmacodynamics in S1; anti-tumour activity, pharmacodynamics and pharmacokinetics in S2. S1 and S2 enrolled a total of 149 and 228 patients, respectively. Grade ≥3 cibisatamab-related adverse events occurred in 36% of S1 and 49% of S2 patients. The ORR was 4% in S1 and 7% in S2. In S2, patients with microsatellite stable colorectal carcinoma (MSS-CRC) given flat doses of cibisatamab and atezolizumab demonstrated an ORR of 14%. In S1 and S2, 40% and 52% of patients, respectively, developed persistent anti-drug antibodies (ADAs). ADA appearance could be mitigated by obinutuzumab-pretreatment, with 8% of patients having persistent ADAs. Overall, cibisatamab warrants further exploration in immunotherapy combination strategies for MSS-CRC.

Characterization of [11C]RO5013853, a novel PET tracer for the glycine transporter type 1 (GlyT1) in humans.

We characterize a novel radioligand for the glycine transporter type 1 (GlyT1), [(11)C]RO5013853, in humans. Ten healthy male volunteers, 23-60 years of age, were enrolled in this PET study; seven subjects participated in the evaluation of test-retest reliability and three subjects in whole body dosimetry. Subjects were administered intravenous bolus injections of approximately 1100 MBq (30 mCi) [(11)C]RO5013853 with a high specific activity of about 481 GBq (13 Ci)/µmol. Standard compartmental model analysis with arterial plasma input function, and an alternative noninvasive analysis method which was evaluated and validated by occupancy studies in both baboons and humans, were performed. Mean parameter estimates of the volumes of distribution (VT) obtained by a 2-tissue 5-parameter model were higher in the cerebellum, pons, and thalamus (1.99 to 2.59 mL/mL), and lower in the putamen, caudate, and cortical areas (0.86 to 1.13 mL/mL), with estimates showing less than 10% difference between test and retest scans. Tracer retention was effectively blocked by the specific glycine reuptake inhibitor (GRI), bitopertin (RG1678). [(11)C]RO5013853 was safe and well tolerated. Human dosimetry studies showed that the effective dose was approximately 0.0033 mSv/MBq, with the liver receiving the highest absorbed dose. In conclusion, quantitative dynamic PET of the human brain after intravenous injection of [(11)C]RO5013853 attains reliable measurements of GlyT1 binding in accordance with the expected transporter distribution in the human brain. [(11)C]RO5013853 is a radioligand suitable for further clinical PET studies. Full characterization of a novel radiotracer for GlyT1 in humans is provided. The tracer has subsequently been used to assess receptor occupancy in healthy volunteers and to estimate occupancy at doses associated with best efficacy in a clinical trial with schizophrenic patients with predominantly negative symptoms.

Managing the Impact of Immunogenicity in an Era of Immunotherapy: From Bench to Bedside.

Biotherapeutics have revolutionized our ability to treat life-threatening diseases. Despite clinical success, the use of biotherapeutics has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs). The multifactorial nature of immunogenicity has prevented a standardized approach for assessing this and each of the assessment methods developed so far does not exhibit high enough reliability to be used alone, due to limited predictiveness. This prompted the Roche Pharma Research and Early Development (pRED) Immunogenicity Working Group to establish an internal preclinical immunogenicity toolbox of in vitro/in vivo approaches and accompanying guidelines for a harmonized assessment and management of immunogenicity in early development. In this article, the complex factors influencing immunogenicity and their associated clinical ramifications are discussed to highlight the importance of an end-to-end approach conducted from lead optimization to clinical candidate selection. We then examine the impact of the resulting lead candidate categorization on the design and implementation of a multi-tiered ADA/immunogenicity assay strategy prior to phase I (entry into human) through early clinical development. Ultimately, the Immunogenicity Toolbox ensures that Roche pRED teams are equipped to address immunogenicity in a standardized manner, paving the way for lifesaving products with improved safety and efficacy.

Safety and Tolerability of Multiple Ascending Doses of PRX002/RG7935, an Anti-α-Synuclein Monoclonal Antibody, in Patients With Parkinson Disease: A Randomized Clinical Trial.

Importance: Aggregated α-synuclein is believed to be central to the pathogenesis of Parkinson disease (PD). PRX002/RG7935 (PRX002) is a humanized monoclonal antibody designed to target aggregated forms of α-synuclein, thereby inhibiting neuron-to-neuron transfer of presumed pathogenic forms of α-synuclein, potentially resulting in neuronal protection and slowing disease progression. Objective: To evaluate the safety and tolerability of multiple intravenous infusions of PRX002 in patients with idiopathic PD. Design, Setting, and Participants: Multicenter, randomized, double-blind, placebo-controlled, multiple ascending-dose trial at 8 US study centers from July 2014 to September 2016. Eligible participants were aged 40 to 80 years with mild to moderate idiopathic PD (Hoehn and Yahr stages 1-3). Interventions: Participants were enrolled into 6 ascending-dose cohorts and randomly assigned to receive PRX002 (0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg, or 60 mg/kg) or placebo. Participants received 3 intravenous infusions every 4 weeks of PRX002 or placebo and were monitored during a 24-week observational period. Main Outcomes and Measures: Safety and tolerability assessments included physical and neurological examinations, laboratory tests, vital signs, and adverse events. Pharmacokinetic parameters included maximum PRX002 concentration, area under the curve, and half-life. Results: Of the 80 participants, most were white (97.5%; n = 78) and male (80%; n = 64); median (SD) age was 58 (8.4) years. PRX002 was generally safe and well tolerated; no serious or severe PRX002-related treatment-emergent adverse events (TEAEs) were reported. The TEAEs experienced by at least 5% of patients receiving PRX002, irrespective of relatedness to study drug, were constipation (9.1%; n = 5), infusion reaction (7.3%; n = 4), diarrhea (5.5%; n = 3), headache (5.5%; n = 3), peripheral edema (5.5%; n = 3), post-lumbar puncture syndrome (5.5%; n = 3), and upper respiratory tract infection (5.5%; n = 3). No antidrug antibodies were detected. Serum PRX002 levels increased in an approximately dose-proportional manner; mean terminal elimination half-life was similar across all doses (10.2 days). Rapid dose- and time-dependent mean reductions from baseline vs placebo in free serum α-synuclein levels of up to 97% were seen after a single infusion at the highest dose (F78,284 = 1.66; P = .002), with similar reductions after 2 additional infusions. Mean cerebrospinal fluid PRX002 concentration increased with PRX002 dose and was approximately 0.3% relative to serum across all dose cohorts. Conclusions and Relevance: Single and multiple doses of PRX002 were generally safe and well tolerated and resulted in robust binding of peripheral α-synuclein and dose-dependent increases of PRX002 in cerebrospinal fluid, reaching cerebrospinal fluid concentrations that may be expected to engage extracellular aggregated α-synuclein in the brain. Findings support the design of an ongoing phase 2 clinical study (NCT03100149). Trial Registration: ClinicalTrials.gov Identifier: NCT02157714.

Progress in Prediction and Interpretation of Clinically Relevant Metabolic Drug-Drug Interactions: a Minireview Illustrating Recent Developments and Current Opportunities.

PURPOSE OF REVIEW: This review gives a perspective on the current "state of the art" in metabolic drug-drug interaction (DDI) prediction. We highlight areas of successful prediction and illustrate progress in areas where limits in scientific knowledge or technologies prevent us from having full confidence. RECENT FINDINGS: Several examples of success are highlighted. Work done for bitopertin shows how in vitro and clinical data can be integrated to give a model-based understanding of pharmacokinetics and drug interactions. The use of interpolative predictions to derive explicit dosage recommendations for untested DDIs is discussed using the example of ibrutinib, and the use of DDI predictions in lieu of clinical studies in new drug application packages is exemplified with eliglustat and alectinib. Alectinib is also an interesting case where dose adjustment is unnecessary as the activity of a major metabolite compensates sufficiently for changes in parent drug exposure. Examples where "unusual" cytochrome P450 (CYP) and non-CYP enzymes are responsible for metabolic clearance have shown the importance of continuing to develop our repertoire of in vitro regents and techniques. The time-dependent inhibition assay using human hepatocytes suspended in full plasma allowed improved DDI predictions, illustrating the importance of continued in vitro assay development and refinement. SUMMARY: During the past 10 years, a highly mechanistic understanding has been developed in the area of CYP-mediated metabolic DDIs enabling the prediction of clinical outcome based on preclinical studies. The combination of good quality in vitro data and physiologically based pharmacokinetic modeling may now be used to evaluate DDI risk prospectively and are increasingly accepted in lieu of dedicated clinical studies.

Effect of Food and Esomeprazole on the Pharmacokinetics of Alectinib, a Highly Selective ALK Inhibitor, in Healthy Subjects.

Alectinib, an anaplastic lymphoma kinase (ALK) inhibitor, is approved for treatment of patients with ALK+ non-small cell lung cancer who have progressed, on or are intolerant to, crizotinib. This study assessed the effect of a high-fat meal and the proton pump inhibitor, esomeprazole, on the pharmacokinetics (PK) of alectinib. This was an open-label, 2-group study in healthy subjects. In group 1 (n = 18), subjects were randomly assigned to a 2-treatment (A, fasted conditions; B, following a high-fat meal), 2-sequence (AB or BA) crossover assessment, separated by a 10-day washout. In group 2 (n = 24), subjects were enrolled in a 2-period, fixed-sequence crossover assessment to evaluate the effect of esomeprazole. PK parameters were evaluated for alectinib, its major similarly active metabolite, M4, and the combined exposure of alectinib and M4. Administration of alectinib following a high-fat meal substantially increased the combined exposure of alectinib and M4 to 331% (90%CI, 279%-393%) and 311% (90%CI, 273%-355%) for Cmax and AUC0-∞ , respectively, versus fasted conditions. Coadministration of esomeprazole had no clinically relevant effect on the combined exposure of alectinib and M4. Alectinib should be administered under fed conditions to maximize its bioavailability, whereas no restrictions are required with antisecretory agents.

Clinical Drug-Drug Interactions Through Cytochrome P450 3A (CYP3A) for the Selective ALK Inhibitor Alectinib.

The efficacy and safety of alectinib, a central nervous system-active and selective anaplastic lymphoma kinase (ALK) inhibitor, has been demonstrated in patients with ALK-positive (ALK+) non-small cell lung cancer (NSCLC) progressing on crizotinib. Alectinib is mainly metabolized by cytochrome P450 3A (CYP3A) to a major similarly active metabolite, M4. Alectinib and M4 show evidence of weak time-dependent inhibition and small induction of CYP3A in vitro. We present results from 3 fixed-sequence studies evaluating drug-drug interactions for alectinib through CYP3A. Studies NP28990 and NP29042 enrolled 17 and 24 healthy subjects, respectively, and investigated potent CYP3A inhibition with posaconazole and potent CYP3A induction through rifampin, respectively, on the single oral dose pharmacokinetics (PK) of alectinib. A substudy of the global phase 2 NP28673 study enrolled 15 patients with ALK+ NSCLC to determine the effect of multiple doses of alectinib on the single oral dose PK of midazolam, a sensitive substrate of CYP3A. Potent CYP3A inhibition or induction resulted in only minor effects on the combined exposure of alectinib and M4. Multiple doses of alectinib did not influence midazolam exposure. These results suggest that dose adjustments may not be needed when alectinib is coadministered with CYP3A inhibitors or inducers or for coadministered CYP3A substrates.

Effect of the Wetting Agent Sodium Lauryl Sulfate on the Pharmacokinetics of Alectinib: Results From a Bioequivalence Study in Healthy Subjects.

The anaplastic lymphoma kinase (ALK) inhibitor alectinib is an effective treatment for ALK-positive non-small-cell lung cancer. This bioequivalence study evaluated the in vivo performance of test 3 formulations with the reduced wetting agent sodium lauryl sulfate (SLS) content. This randomized, 4-period, 4-sequence, crossover study compared alectinib (600 mg) as 25%, 12.5%, and 3% SLS hard capsule formulations with the reference 50% SLS clinical formulation in healthy subjects under fasted conditions (n = 49), and following a high-fat meal (n = 48). Geometric mean ratios and 90% confidence intervals (CIs) for Cmax , AUC0-last , and AUC0-∞ of alectinib, its major active metabolite, M4, and alectinib plus M4 were determined for the test formulations versus the reference formulation. Bioequivalence was concluded if the 90%CIs were within the 80% to 125% boundaries. The 25% SLS formulation demonstrated bioequivalence to the reference 50% SLS formulation for Cmax , AUC0-last , and AUC0-∞ of alectinib, M4, and alectinib plus M4 under both fasted and fed conditions. Further reductions in SLS content (12.5% and 3% SLS) did not meet the bioequivalence criteria. Cross-group comparisons showed an approximately 3-fold positive food effect. Reducing SLS to 25% resulted in a formulation that is bioequivalent to the current 50% SLS formulation used in alectinib pivotal trials.

Effects of Cytochrome P450 3A4 Inhibitors-Ketoconazole and Erythromycin-on Bitopertin Pharmacokinetics and Comparison with Physiologically Based Modelling Predictions.

OBJECTIVE: To assess the effect of strong and moderate cytochrome P450 (CYP) 3A4 inhibition on exposure of bitopertin, a glycine reuptake inhibitor primarily metabolized by CYP3A4, and to compare the results with predictions based on physiologically based pharmacokinetic (PBPK) modelling. METHODS: The effects of ketoconazole and erythromycin were assessed in two male volunteer studies with open-label, two-period, fixed-sequence designs. Twelve subjects were enrolled in each of the studies. In period 1, a single dose of bitopertin was administered; in period 2, 400 mg ketoconazole was administered once daily for 17 days or 500 mg erythromycin was administered twice daily for 21 days. A single dose of bitopertin was coadministered on day 5. Pharmacokinetic parameters were derived by non-compartmental methods. Simulated bitopertin profiles using dynamic PBPK modelling for a typical healthy volunteer in GastroPlus(®) were used to predict changes in pharmacokinetic parameters. RESULTS: In healthy volunteers, coadministration of ketoconazole increased the bitopertin area under the plasma concentration-time curve (AUC) from 0 to 312 h (AUC0-312h) 4.2-fold (90 % confidence interval [CI] 3.5-5.0) and erythromycin increased the AUC from time zero to infinity (AUC0-inf) 2.1-fold (90 % CI 1.9-2.3). The peak concentration (C max) increased by <25 % in both studies. Simulated bitopertin profiles using PBPK modelling showed good agreement with the observed AUC ratios in both studies. The predicted AUC0-inf ratios for the interaction with ketoconazole and erythromycin were 7.7 and 1.9, respectively. CONCLUSION: Strong CYP3A4 inhibitors increase AUC0-inf of bitopertin 7- to 8-fold and hence should not be administered concomitantly with bitopertin. Moderate CYP3A4 inhibitors double AUC0-inf.

Combining 'Bottom-Up' and 'Top-Down' Methods to Assess Ethnic Difference in Clearance: Bitopertin as an Example.

BACKGROUND AND OBJECTIVES: We propose a strategy for studying ethnopharmacology by conducting sequential physiologically based pharmacokinetic (PBPK) prediction (a 'bottom-up' approach) and population pharmacokinetic (popPK) confirmation (a 'top-down' approach), or in reverse order, depending on whether the purpose is ethnic effect assessment for a new molecular entity under development or a tool for ethnic sensitivity prediction for a given pathway. The strategy is exemplified with bitopertin. METHODS: A PBPK model was built using Simcyp(®) to simulate the pharmacokinetics of bitopertin and to predict the ethnic sensitivity in clearance, given pharmacokinetic data in just one ethnicity. Subsequently, a popPK model was built using NONMEM(®) to assess the effect of ethnicity on clearance, using human data from multiple ethnic groups. A comparison was made to confirm the PBPK-based ethnic sensitivity prediction, using the results of the popPK analysis. RESULTS: PBPK modelling predicted that the bitopertin geometric mean clearance values after 20 mg oral administration in Caucasians would be 1.32-fold and 1.27-fold higher than the values in Chinese and Japanese, respectively. The ratios of typical clearance in Caucasians to the values in Chinese and Japanese estimated by popPK analysis were 1.20 and 1.17, respectively. The popPK analysis results were similar to the PBPK modelling results. CONCLUSION: As a general framework, we propose that PBPK modelling should be considered to predict ethnic sensitivity of pharmacokinetics prior to any human data and/or with data in only one ethnicity. In some cases, this will be sufficient to guide initial dose selection in different ethnicities. After clinical trials in different ethnicities, popPK analysis can be used to confirm ethnic differences and to support dose justification and labelling. PBPK modelling prediction and popPK analysis confirmation can complement each other to assess ethnic differences in pharmacokinetics at different drug development stages.

Effects of the glycine reuptake inhibitors bitopertin and RG7118 on glycine in cerebrospinal fluid: results of two proofs of mechanism studies in healthy volunteers.

RATIONALE: Hypofunction of NMDA receptors has been implicated in neuropsychiatric disorders including schizophrenia. NMDA receptor neurotransmission can be enhanced through inhibition of glycine reuptake by the glycine transporter type 1 (GlyT1). OBJECTIVES: The primary objective of these studies was to explore the relationship between plasma exposure and glycine cerebrospinal fluid (CSF) concentrations following administration of bitopertin and RG7118 in healthy volunteers. METHODS: The bitopertin study comprised four dose levels (3, 10, 30 and 60 mg) administered once daily for 10 days. In the RG7118 study, placebo, 15 or 30 mg RG7118 was administered once daily for 28 days. CSF samples were taken on day -2 and day 10, and day -1 and day 26 for bitopertin and RG7118, respectively. RESULTS: Twenty-two and 24 subjects participated in the bitopertin and RG7118 study, respectively. In the bitopertin study, CSF glycine concentrations showed a dose-dependent increase from baseline to day 10. The geometric mean ratios (coefficient of variation) of AUC0-12 h on day 10 over baseline were 1.3 (17 %), 1.3 (49 %), 1.7 (18 %) and 2.3 (14 %) after 3, 10, 30 and 60 mg, respectively. In the RG7118 study, the geometric mean ratio of glycine concentration (CV) on day 26 at 6 h post-dose over time-matched baseline was approx. 1.9 (24 and 15 %) for 15 and 30 mg. CONCLUSIONS: The mechanism of action of bitopertin and RG7118, i.e. inhibition of glycine reuptake in the brain, was confirmed. The maximal increase observed in healthy volunteers was similar to the one observed in animals showing the good translatability of this biomarker.

Quality markers of drug information on the Internet: an evaluation of sites about St. John's wort.

PURPOSE: We aimed to evaluate websites about St. John's wort for the quality of their content, including accuracy as reflected by statement of correct indication and mentioning of interacting drugs, the presence of formal criteria as reflected by adherence to published standards for health information on the Internet, and the validity of individual formal criteria as markers of content quality. SUBJECTS AND METHODS: The Internet was searched with the metasearch engine WebFerret for sites about St. John's wort. A cross-sectional survey of a representative sample of randomly selected sites (n = 208) was performed. The main outcomes were the percentage of sites fulfilling the two criteria of content quality, the percentage of sites exhibiting eight formal criteria, and the associations between formal criteria and criteria of content quality, as determined by multivariate logistic regression analysis. RESULTS: Twenty-two percent (n = 45) of the websites correctly listed depression as the only indication for use of St. John's wort, and 22% (n = 46) identified at least one drug interaction with St. John's wort. Citing scientific publications was associated with mentioning the correct indication (odds ratio [OR] = 4.4; 95% confidence interval [CI]: 1.4 to 14) and mentioning of any interacting drug (OR = 6.0; 95% CI: 2.0 to 18). Absence of financial interest was associated with mentioning the correct indication (OR = 5.4; 95% CI: 2.2 to 14) and interacting drugs (OR = 3.1; 95% CI: 1.2 to 7.7). CONCLUSION: The content quality of sites about St. John's wort was generally poor. Our results suggest that Internet users should prefer noncommercial sites that reference the information to scientific publications when searching for drug information.

A drug database model as a central element for computer-supported dose adjustment within a CPOE system.

The incidence of adverse drug reactions may be decreased by computerized physician order entry (CPOE) with decision support. The authors describe the development of a drug database model for computer-supported dose adjustment within a CPOE system. The following two core elements were included: (1) To allow electronic dose and volume calculation, the relation between strength (e.g., 5 mg/1 mL) and prescribed unit (e.g., 1 ampoule containing 2 mL) must be available in coded form. (2) The site of action along with the parent active ingredient, i.e., the pure drug without salt or ester, is necessary for linkage to knowledge bases. All complex examples of drugs that were examined could be described by the data model. With the ultimate goal of increasing prescribing effectiveness and quality the authors developed a drug database model for inclusion in a CPOE system, which allows dose calculations and may be coupled to decision support systems.

Simultaneous determination of hypericin and hyperforin in human plasma with liquid chromatography-tandem mass spectrometry.

A selective and sensitive method for the simultaneous determination of hypericin and hyperforin--the two main active ingredients of St. John's Wort (SJW) extract--in human plasma depending on liquid/liquid-extraction and LC/MS/MS detection has been developed, validated after specifying the stability of the photosensitive hypericin in plasma samples during light exposure and applied to samples of a patient. After extraction with ethyl acetate/n-hexane in the darkness, sample extracts were chromatographed isocratically within 6 min on a Kromasil RP-18 column. The analytes were detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source. The limit of quantification was 0.05 ng/mL for hypericin and 0.035 ng/mL for hyperforin. The accuracy of the method varied between 101.9 and 114.2% and the precision ranged from 4.7 to 15.4% (S.D., batch-to-batch) for both analytes. The method was linear at least between 0.05 and 10 ng/mL for hypericin and between 0.035 and 100 ng/mL for hyperforin. Using this method hypericin and hyperforin were determined successfully in a patient over seven days following discontinuation of exposure with therapeutic doses of St. John's Wort extract.

Highly sensitive determination of saquinavir in biological samples using liquid chromatography-tandem mass spectrometry.

A selective and sensitive method for the determination of the HIV protease inhibitor saquinavir in human plasma, saliva, and urine using liquid-liquid extraction and LC-MS-MS has been developed, validated, and applied to samples of a healthy individual. After extraction with ethyl acetate, sample extracts were chromatographed isocratically within 5 min on Kromasil RP-18. The drug was detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source and 2H(5)-saquinavir as internal standard. The limit of quantification was 0.05 ng/mL. The accuracy of the method varied between -1 and +10% (SD within-batch) and the precision ranged from +4 to +10% (SD batch-to-batch). The method is linear at least within 0.05 and 87.6 ng/mL. After a regular oral dose (600 mg) saquinavir concentrations were detectable for 48 h in plasma and were well correlated with saliva concentrations (r(2)=0.9348, mean saliva/plasma ratio 1:15.1). The method is well suited for low saquinavir concentrations in different matrices.

Physiologically based absorption modelling to predict the impact of drug properties on pharmacokinetics of bitopertin.

Bitopertin (RG1678) is a glycine reuptake inhibitor in phase 3 trials for treatment of schizophrenia. Its clinical oral pharmacokinetics is sensitive to changes in drug substance particle size and dosage form. Physiologically based pharmacokinetic (PBPK) absorption model simulations of the impact of changes in particle size and dosage form (either capsules, tablets, or an aqueous suspension) on oral pharmacokinetics was verified by comparison to measured plasma concentrations. Then, a model parameter sensitivity analysis was applied to set limits on the particle sizes included in tablets for the market. The model was also used to explore the in vitro to in vivo correlation. Simulated changes in oral pharmacokinetics caused by differences in particle size and dosage form were confirmed in two separate relative bioavailability studies. Model parameter sensitivity analyses predicted that AUCinf was hardly reduced as long as particle diameter (D50) remained smaller than 30 µm, and >20% reduced Cmax is anticipated only when particle diameter exceeds 15 µm. An exploration of the sensitivity to the presence of larger particles within a polydisperse distribution showed that simulated Cmax is again more affected than AUC but is less than 20% reduced as long as D50 is less than 8 µm and D90 is smaller than 56 µm. PBPK absorption modelling can contribute to a quality by design (QbD) approach for clinical formulation development and support the setting of biorelevant specifications for release of the product.

Effect of bitopertin, a glycine reuptake inhibitor, on negative symptoms of schizophrenia: a randomized, double-blind, proof-of-concept study.

IMPORTANCE: In schizophrenia, the severity of negative symptoms is a key predictor of long-term disability. Deficient signaling through the N-methyl-D-aspartate receptor is hypothesized to underlie many signs and symptoms associated with schizophrenia in particular negative symptoms. Glycine acts as an N-methyl-D-aspartate receptor coagonist. Blockade of the glycine transporter type 1 to inhibit glycine reuptake and elevate synaptic glycine concentrations represents an effective strategy to enhance N-methyl-D-aspartate receptor transmission. OBJECTIVE: To determine the efficacy and safety of bitopertin (RG1678), a glycine reuptake inhibitor, in patients with schizophrenia and predominant negative symptoms who were stable while taking an antipsychotic treatment. DESIGN, SETTING, AND PARTICIPANTS: This randomized, double-blind, placebo-controlled, phase 2 proof-of-concept trial involved 323 patients with schizophrenia and predominant negative symptoms across 66 sites worldwide. INTERVENTIONS: Bitopertin (10, 30, or 60 mg/d) or placebo added to standard antipsychotic therapy for a treatment duration of 8 weeks. MAIN OUTCOMES AND MEASURES: Change from baseline in the Positive and Negative Syndrome Scale negative factor score. RESULTS: In the per-protocol population, 8 weeks of treatment with bitopertin was associated with a significant reduction of negative symptoms in the 10-mg/d (mean [SE] reduction in negative symptoms score, -25% [2%]; P = .049) and 30-mg/d (mean [SE], -25% [2%]; P = .03) bitopertin groups, a significantly higher response rate and a trend toward improved functioning in the 10-mg/d group when compared with placebo (mean [SE], -19% [2%]). Results reached trend-level significance in the intent-to-treat population. Estimates of bitopertin binding to glycine transporter type 1 showed that low to medium levels of occupancy yielded optimal efficacy in patients, consistent with findings in preclinical assays. CONCLUSIONS AND RELEVANCE: Bitopertin-mediated glycine reuptake inhibition may represent a novel treatment option for schizophrenia, with the potential to address negative symptoms. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00616798.

Physiologically based pharmacokinetic modelling to predict single- and multiple-dose human pharmacokinetics of bitopertin.

BACKGROUND: Bitopertin (RG1678) is a glycine reuptake inhibitor currently in phase 3 trials for treatment of schizophrenia. This paper describes the use of physiologically based pharmacokinetic (PBPK) modelling and preclinical data to gain insights into and predict bitopertin clinical pharmacokinetics. METHODS: Simulations of pharmacokinetics were initiated early in the drug discovery stage by integrating physicochemical properties and in vitro measurements into a PBPK rat model. Comparison of pharmacokinetics predicted by PBPK modelling with those measured after intravenous and oral dosing in rats and monkeys showed a good match and thus increased confidence that a similar approach could be applied for human prediction. After comparison of predicted plasma concentrations with those measured after single oral doses in the first clinical study, the human model was refined and then applied to simulate multiple-dose pharmacokinetics. RESULTS: Clinical plasma concentrations measured were in good agreement with PBPK predictions. Predicted area under the plasma concentration-time curve (AUC) was within twofold of the observed mean values for all dose levels. Maximum plasma concentration (C max) at higher doses was well predicted but approximately twofold below observed values at the lower doses. A slightly less than dose-proportional increase in both AUC and C max was observed, and model simulations indicated that when the dose exceeded 50 mg, solubility limited the fraction of dose absorbed. Refinement of the absorption model with additional solubility and permeability measurements further improved the match of simulations to observed single-dose data. Simulated multiple-dose pharmacokinetics with the refined model were in good agreement with observed data. CONCLUSIONS: Clinical pharmacokinetics of bitopertin can be well simulated with a mechanistic PBPK model. This model supports further clinical development and provides a valuable repository for pharmacokinetic knowledge gained about the molecule.