Maternal phenylketonuria syndrome: studies in mice suggest a potential approach to a continuing problem.

BackgroundUntreated phenylketonuria (PKU), one of the most common human genetic disorders, usually results in mental retardation. Although a protein-restricted artificial diet can prevent retardation, dietary compliance in adults is often poor. In pregnant PKU women, noncompliance can result in maternal PKU syndrome, where high phenylalanine (Phe) levels cause severe fetal complications. Enzyme substitution therapy using Phe ammonia lyase (PAL) corrects PKU in BTBR Phe hydroxylase (Pahenu2) mutant mice, suggesting a potential for maternal PKU syndrome treatment in humans.MethodsWe reviewed clinical data to assess maternal PKU syndrome incidence in pregnant PKU women. We treated female PKU mice (on normal diet) with PAL, stabilizing Phe at physiological levels, and mated them to assess pregnancy outcomes.ResultsPatient records show that, unfortunately, the efficacy of diet to prevent maternal PKU syndrome has not significantly improved since the problem was first noted 40 years ago. PAL treatment of pregnant PKU mice shows that offspring of PAL-treated dams survive to adulthood, in contrast to the complete lethality seen in untreated mice, or limited survival seen in mice on a PKU diet.ConclusionPAL treatment reduced maternal PKU syndrome severity in mice and may have potential for human PKU therapy.

Acetylator Status Impacts Amifampridine Phosphate (Firdapse™) Pharmacokinetics and Exposure to a Greater Extent Than Renal Function.

PURPOSE: The purpose of this study is to evaluate safety, tolerability, and pharmacokinetic (PK) properties of amifampridine phosphate (Firdapse™) and its major inactive 3-N-acetyl metabolite in renally impaired and healthy individuals with slow acetylator (SA) and rapid acetylator (RA) phenotypes. METHODS: This was a Phase I, multicenter, open-label study of the PK properties and safety profile of amifampridine phosphate in individuals with normal, mild, moderate, or severely impaired renal function. Amifampridine phosphate was given as a single 10 mg (base equivalent) dose, and the plasma and urine PK properties of amifampridine and its 3-N-acetyl metabolite were determined. The safety profile was evaluated by monitoring adverse events (AEs), clinical laboratory tests, and physical examinations. FINDINGS: Amifampridine clearance was predominantly metabolic through N-acetylation, regardless of renal function in both acetylator phenotypes. In individuals with normal renal function, mean renal clearance represented approximately 3% and 18% of the total clearance of amifampridine in RA and SA, respectively. Large differences in amifampridine exposure were observed between acetylation phenotypes across renal function levels. Mean amifampridine exposure values of AUC0-∞ and Cmax were up to 8.8-fold higher in the SA group compared with the RA group across renal function levels. By comparison, mean AUC0-∞ was less affected by renal function within an acetylator group, only 2- to 3-fold higher in individuals with severe renal impairment (RI) compared with those with normal renal function. Exposure to amifampridine in the SA group with normal renal function was higher (AUC0-∞, approximately 1.8-fold; Cmax, approximately 4.1-fold) than the RA group with severe RI. Exposure to the inactive 3-N-acetyl metabolite was higher than amifampridine in both acetylator groups, independent of renal function level. The metabolite is cleared by renal excretion, and exposure was clearly dependent on renal function with 4.0- to 6.8-fold increases in AUC0-∞ from normal to severe RI. No new tolerability findings were observed. IMPLICATIONS: A single dose of 10 mg of amifampridine phosphate was well tolerated, independent of renal function and acetylator status. The results indicate that the PK profile of amifampridine is affected by metabolic acetylator phenotype to a greater extent than by renal function level, supporting Firdapse™ administration in individuals with RI in line with current labeling recommendations. Amifampridine should be dosed to effect per the individual patient need, altering administration frequency and dose in normal through severe RI. The therapeutic dose of amifampridine phosphate should be tailored to the individual patient needs by gradual dose titration up to the present maximum recommended dose (60-80 mg/day) or until dose-limiting AEs intervene to avoid overdosing and underdosing. EudraCT identifier: 2013-005349-35.

Effects of Food Intake on the Relative Bioavailability of Amifampridine Phosphate Salt in Healthy Adults.

PURPOSE: Amifampridine (3,4-diaminopyridine) has been approved in the European Union for the treatment of Lambert-Eaton myasthenic syndrome. Amifampridine has a narrow therapeutic index, and supratherapeutic exposure has been associated with dose-dependent adverse events, including an increased risk for seizure. This study assessed the effect of food on the relative bioavailability of amifampridine in healthy subjects and informed on conditions that can alter exposure. METHODS: This randomized, open-labeled, 2-treatment, 2-period crossover study enrolled 47 healthy male and female subjects. Subjects were randomly assigned to receive 2 single oral doses of amifampridine phosphate salt (20 mg base equivalents per dose) under fed or fasted conditions separated by a washout period. Blood and urine samples for pharmacokinetic analyses were taken before and after dosing. Plasma concentrations of amifampridine and an inactive 3-N-acetyl metabolite were determined. The relative bioavailability values of amifampridine and metabolite were assessed based on the plasma PK parameters AUC0-∞, AUC0-t, and Cmax in the fed and fasted states using noncompartmental pharmacokinetic analysis. Parent drug and metabolite excretion were calculated from urinary concentrations. A food effect on bioavailability would be established if the 90% CI of the ratio of population geometric mean value of AUC0-∞, AUC0-t, or Cmax between fed and fasted administration was not within the bioequivalence range of 80% to 125%. Tolerability was assessed based on adverse-event reporting, clinical laboratory assessments, physical examination including vital sign measurements, 12-lead ECG, and concurrent medication use. FINDINGS: Food slowed and somewhat decreased the absorption of amifampridine. There was a decrease in exposure (Cmax, 44%; AUC, 20%) after oral administration of amifampridine phosphate salt in the presence of food, and mean Tmax was 2-fold longer in the fed state. The extent of exposure and plasma elimination half-life of the major metabolite was greater than those of amifampridine in the fed and fasted conditions. Mean AUCs in the fed and fasted states were slightly greater in women than men, with no difference in mean Cmax. Orally administered amifampridine was renally eliminated (>93%) as the parent compound and metabolite within 24 hours. Single oral doses of 20 mg of amifampridine phosphate salt were considered well tolerated in both the fed and fasted conditions. High intersubject variability (%CVs, >30%) in amifampridine pharmacokinetic parameter values was observed. IMPLICATIONS: At the intended dose under fasting conditions, amifampridine exposure may be increased. European Union Drug Regulating Authorities Clinical Trials identifier: 2011-000596-13.

Genetic variation in aryl N-acetyltransferase results in significant differences in the pharmacokinetic and safety profiles of amifampridine (3,4-diaminopyridine) phosphate.

The clinical use of amifampridine phosphate for neuromuscular junction disorders is increasing. The metabolism of amifampridine occurs via polymorphic aryl N-acetyltransferase (NAT), yet its pharmacokinetic (PK) and safety profiles, as influenced by this enzyme system, have not been investigated. The objective of this study was to assess the effect of NAT phenotype and genotype on the PK and safety profiles of amifampridine in healthy volunteers (N = 26). A caffeine challenge test and NAT2 genotyping were used to delineate subjects into slow and fast acetylators for PK and tolerability assessment of single, escalating doses of amifampridine (up to 30 mg) and in multiple daily doses (20 mg QID) of amifampridine. The results showed that fast acetylator phenotypes displayed significantly lower C max, AUC, and shorter t 1/2 for amifampridine than slow acetylators. Plasma concentrations of the N-acetyl metabolite were approximately twofold higher in fast acetylators. Gender differences were not observed. Single doses of amifampridine demonstrated dose linear PKs. Amifampridine achieved steady state plasma levels within 1 day of dosing four times daily. No accumulation or time-dependent changes in amifampridine PK parameters occurred. Overall, slow acetylators reported 73 drug-related treatment-emergent adverse events versus 6 in fast acetylators. Variations in polymorphic NAT corresponding with fast and slow acetylator phenotypes significantly affects the PK and safety profiles of amifampridine.

Nonclinical evaluation of CNS-administered TPP1 enzyme replacement in canine CLN2 neuronal ceroid lipofuscinosis.

The CLN2 form of neuronal ceroid lipofuscinosis, a type of Batten disease, is a lysosomal storage disorder caused by a deficiency of the enzyme tripeptidyl peptidase-1 (TPP1). Patients exhibit progressive neurodegeneration and loss of motor, cognitive, and visual functions, leading to death by the early teenage years. TPP1-null Dachshunds recapitulate human CLN2 disease. To characterize the safety and pharmacology of recombinant human (rh) TPP1 administration to the cerebrospinal fluid (CSF) as a potential enzyme replacement therapy (ERT) for CLN2 disease, TPP1-null and wild-type (WT) Dachshunds were given repeated intracerebroventricular (ICV) infusions and the pharmacokinetic (PK) profile, central nervous system (CNS) distribution, and safety were evaluated. TPP1-null animals and WT controls received 4 or 16mg of rhTPP1 or artificial cerebrospinal fluid (aCSF) vehicle every other week. Elevated CSF TPP1 concentrations were observed for 2-3 days after the first ICV infusion and were approximately 1000-fold higher than plasma levels at the same time points. Anti-rhTPP1 antibodies were detected in CSF and plasma after repeat rhTPP1 administration, with titers generally higher in TPP1-null than in WT animals. Widespread brain distribution of rhTPP1 was observed after chronic administration. Expected histological changes were present due to the CNS delivery catheters and were similar in rhTPP1 and vehicle-treated animals, regardless of genotype. Neuropathological evaluation demonstrated the clearance of lysosomal storage, preservation of neuronal morphology, and reduction in brain inflammation with treatment. This study demonstrates the favorable safety and pharmacology profile of rhTPP1 ERT administered directly to the CNS and supports clinical evaluation in patients with CLN2 disease.

Immunogenicity of Elosulfase Alfa, an Enzyme Replacement Therapy in Patients With Morquio A Syndrome: Results From MOR-004, a Phase III Trial.

PURPOSE: Morquio A syndrome (mucopolysaccharidosis IVA [MPS IVA]) is a lysosomal storage disorder caused by deficiency of the enzyme N-acetylgalactosamine-6-sulfatase, which is required to degrade the glycosaminoglycan keratan sulfate. Morquio A is associated with extensive morbidity and early mortality. Elosulfase alfa is an enzyme replacement therapy that provides a treatment option for patients with Morquio A. We examined the immunogenicity profile of elosulfase alfa, assessing any correlations between antidrug antibodies and the efficacy and safety outcomes in 176 patients with Morquio A from a 24-week international Phase III trial. METHODS: Patients were randomized to placebo (n = 59) or elosulfase alfa 2.0 mg/kg administered weekly (n = 58) or every other week (n = 59) as an ~4-hour infusion. Blood samples were routinely tested to determine drug-specific total antibody titer and neutralizing antibody (NAb) positivity. Drug-specific immunoglobulin E positivity was tested routinely and in response to severe hypersensitivity adverse events (AEs). Antidrug antibody positivity and titer were compared with efficacy and safety metrics to assess possible correlations. FINDINGS: The 176 patients in the trial were 54% female, with a mean age of 11.9 years. In all patients treated with elosulfase alfa antidrug antibodies developed, and in the majority, antibodies capable of interfering with cation-independent mannose-6-phosphate receptor binding in vitro (NAb) developed. Less than 10% of patients tested positive for drug-specific IgE during the study. Despite the high incidence of anti-elosulfase alfa antibodies, no correlations were detected between higher total antibody titers or NAb positivity and worsened 6-minute walk test results, urine keratin sulfate levels, or hypersensitivity AEs. Drug-specific IgE positivity had no apparent association with the occurrence of anaphylaxis, other hypersensitivity AEs, and/or treatment withdrawal. IMPLICATIONS: Despite the universal development of antidrug antibodies, elosulfase alfa treatment was both safe and well tolerated and immunogenicity was not associated with reduced treatment effect. ClinicalTrials.gov identifier: NCT01275066. (Clin Ther.

A prospective population pharmacokinetic analysis of sapropterin dihydrochloride in infants and young children with phenylketonuria.

BACKGROUND AND OBJECTIVES: Untreated phenylketonuria (PKU), a hereditary metabolic disorder caused by a genetic mutation in phenylalanine hydroxylase (PAH), is characterized by elevated blood phenylalanine (Phe) and severe neurologic disease. Sapropterin dihydrochloride, a synthetic preparation of naturally occurring PAH cofactor tetrahydrobiopterin (BH4), activates residual PAH in a subset of patients, resulting in decreased blood Phe and increased Phe tolerance. The objective of this study was to determine the appropriate dose of sapropterin in pediatric patients (0-6 years). The study design used D-optimization and was prospectively powered to achieve precise estimates of clearance and volume of distribution. METHODS: Oral sapropterin (5 or 20 mg/kg) was administered once daily. Sapropterin plasma concentrations were measured by a validated method. Population pharmacokinetic analysis was performed with NONMEM(®) version 7.2 on pooled data from 156 pediatric and adult PKU patients in two phase III clinical studies. RESULTS: The best pharmacokinetic model was a one-compartment model with an absorption lag, first-order input, and linear elimination, with a factor describing endogenous BH4 levels. Body weight was the only covariate significantly affecting sapropterin pharmacokinetics. Based on recommended dosing, exposure across age groups was comparable. The absorption rate and terminal half-life suggest flip-flop pharmacokinetic behavior where absorption is rate limiting. CONCLUSION: The effect of weight on sapropterin pharmacokinetics was significant and exposure was comparable across age groups; thus, weight-based dosing is appropriate. The doses selected for pediatric patients provided similar exposure as in adults. Given the slow absorption and elimination half-life, once-daily dosing is justified.

Pharmacokinetic and pharmacodynamic evaluation of elosulfase alfa, an enzyme replacement therapy in patients with Morquio A syndrome.

BACKGROUND AND OBJECTIVES: Morquio A syndrome (mucopolysaccharidosis IVA; MPS IVA) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase, an enzyme required for degradation of the glycosaminoglycan keratan sulfate. Enzyme replacement therapy with elosulfase alfa provides a potential therapy for Morquio A syndrome. We analyzed the pharmacokinetics and pharmacodynamics of elosulfase alfa in Morquio A patients from a phase III clinical trial. METHODS: In a randomized double-blind study, elosulfase alfa at 2.0 mg/kg was administrated weekly or every other week for 24 weeks. Pharmacokinetic parameters of elosulfase alfa were determined at weeks 0 and 22 by non-compartmental analysis. Safety was assessed throughout the study. The relationship of pharmacokinetic parameters to patient demographics, pharmacodynamic assessments, immunogenicity, and efficacy and safety outcomes were assessed graphically by treatment group. RESULTS: Elosulfase alfa exposure and half-life (t(½)) increased for both dose regimens during the study. There appeared to be no consistent trend between drug clearance (CL) and patient's sex, race, body weight, or age. All patients developed anti-drug antibodies, but no association was noted between total antibody titer and CL. In contrast, positive neutralizing antibody (NAb) status appeared to associate with decreased CL and prolonged t(½) for patients in the cohort dosed weekly. NAb may interfere with receptor-mediated cellular uptake and lead to increased circulation time of elosulfase alfa. CONCLUSION: Despite the association between NAb and decreased drug clearance, neither dosing cohort showed associations between drug exposure and change in urinary keratan sulfate, 6-min walk test distances, or the occurrence of adverse events.

Single-dose, subcutaneous recombinant phenylalanine ammonia lyase conjugated with polyethylene glycol in adult patients with phenylketonuria: an open-label, multicentre, phase 1 dose-escalation trial.

BACKGROUND: Phenylketonuria is an inherited disease caused by impaired activity of phenylalanine hydroxylase, the enzyme that converts phenylalanine to tyrosine, leading to accumulation of phenylalanine and subsequent neurocognitive dysfunction. Phenylalanine ammonia lyase is a prokaryotic enzyme that converts phenylalanine to ammonia and trans-cinnamic acid. We aimed to assess the safety, tolerability, pharmacokinetic characteristics, and efficacy of recombinant Anabaena variabilis phenylalanine ammonia lyase (produced in Escherichia coli) conjugated with polyethylene glycol (rAvPAL-PEG) in reducing phenylalanine concentrations in adult patients with phenylketonuria. METHODS: In this open-label, phase 1, multicentre trial, single subcutaneous injections of rAvPAL-PEG were given in escalating doses (0·001, 0·003, 0·010, 0·030, and 0·100 mg/kg) to adults with phenylketonuria. Participants aged 18 years or older with blood phenylalanine concentrations of 600 µmol/L or higher were recruited from among patients attending metabolic disease clinics in the USA. The primary endpoints were safety and tolerability of rAvPAL-PEG. Secondary endpoints were the pharmacokinetic characteristics of the drug and its effect on concentrations of phenylalanine. Participants and investigators were not masked to assigned dose group. This study is registered with ClinicalTrials.gov, number NCT00925054. FINDINGS: 25 participants were recruited from seven centres between May 6, 2008, and April 15, 2009, with five participants assigned to each escalating dose group. All participants were included in the safety population. The most frequently reported adverse events were injection-site reactions and dizziness, which were self-limited and without sequelae. Two participants had serious adverse reactions to intramuscular medroxyprogesterone acetate, a drug that contains polyethylene glycol as an excipient. Three of five participants given the highest dose of rAvPAL-PEG (0·100 mg/kg) developed a generalised skin rash. By the end of the study, all participants had developed antibodies against polyethylene glycol, and some against phenylalanine ammonia lyase as well. Drug concentrations peaked about 89-106 h after administration of the highest dose. Treatment seemed to be effective at reducing blood phenylalanine in all five participants who received the highest dose (mean reduction of 54·2% from baseline), with a nadir about 6 days after injection and an inverse correlation between drug and phenylalanine concentrations in plasma. Phenylalanine returned to near-baseline concentrations about 21 days after the injection. INTERPRETATION: Subcutaneous administration of rAvPAL-PEG in a single dose of up to 0·100 mg/kg was fairly safe and well tolerated in adult patients with phenylketonuria. At the highest dose tested, rAvPAL-PEG reduced blood phenylalanine concentrations. In view of the development of antibodies against polyethylene glycol (and in some cases against phenylalanine ammonia lyase), future studies are needed to assess the effect of repeat dosing. FUNDING: BioMarin Pharmaceutical.

Recombinant human tripeptidyl peptidase-1 infusion to the monkey CNS: safety, pharmacokinetics, and distribution.

CLN2 disease is caused by deficiency in tripeptidyl peptidase-1 (TPP1), leading to neurodegeneration and death. The safety, pharmacokinetics (PK), and CNS distribution of recombinant human TPP1 (rhTPP1) were characterized following a single intracerebroventricular (ICV) or intrathecal-lumbar (IT-L) infusion to cynomolgus monkeys. Animals received 0, 5, 14, or 20mg rhTPP1, ICV, or 14 mg IT-L, in artificial cerebrospinal fluid (aCSF) vehicle. Plasma and CSF were collected for PK analysis. Necropsies occurred at 3, 7, and 14 days post-infusion. CNS tissues were sampled for rhTPP1 distribution. TPP1 infusion was well tolerated and without effect on clinical observations or ECG. A mild increase in CSF white blood cells (WBCs) was detected transiently after ICV infusion. Isolated histological changes related to catheter placement and infusion were observed in ICV treated animals, including vehicle controls. The CSF and plasma exposure profiles were equivalent between animals that received an ICV or IT-L infusion. TPP1 levels peaked at the end of infusion, at which point the enzyme was present in plasma at 0.3% to 0.5% of CSF levels. TPP1 was detected in brain tissues with half-lives of 3-14 days. CNS distribution between ICV and IT-L administration was similar, although ICV resulted in distribution to deep brain structures including the thalamus, midbrain, and striatum. Direct CNS infusion of rhTPP1 was well tolerated with no drug related safety findings. The favorable nonclinical profile of ICV rhTPP1 supports the treatment of CLN2 by direct administration to the CNS.

Relative bioavailability of sapropterin from intact and dissolved sapropterin dihydrochloride tablets and the effects of food: a randomized, open-label, crossover study in healthy adults.

BACKGROUND: Phenylketonuria (PKU) is an autosomal recessive metabolic disorder characterized by hyperphenylalaninemia in association with neurocognitive and neuromotor impairment. Sapropterin dihydrochloride (hereafter referred to as sapropterin) administered orally as dissolved tablets is approved by the US Food and Drug Administration for hyperphenylalaninemia in patients with tetrahydrobiopterin responsive PKU. OBJECTIVES: This study compared the relative oral bioavailability of sapropterin when administered as intact and dissolved tablets. It also assessed the effect of food on the oral bioavailability of sapropterin administered as intact tablets. METHODS: This was a randomized, open-label, 3-treatment, 6-sequence, 3-period crossover study in healthy male and female subjects. Subjects were randomized to receive single oral 10-mg/kg doses of sapropterin administered as dissolved tablets after a fast; as intact tablets after a fast; and as intact tablets with a high-calorie, high-fat meal. The 3 dosing periods were separated by a washout period of at least 7 days. In each dosing period, blood samples were obtained within 40 minutes before and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 18, and 24 hours after dosing. A follow-up assessment was performed 5 to 7 days after the last dosing period. The relative bioavailability of sapropterin from the 3 dosing regimens was assessed based on C(max), AUC(0-t), and AUC(0-infinity), estimated from calculated plasma tetrahydrobiopterin concentrations using a noncompartmental model. Safety assessments included physical examinations, clinical laboratory tests, and ECGs at the beginning and end of the study. Vital signs were monitored periodically during each treatment period. RESULTS: The study enrolled 32 healthy subjects (16 men, 16 women) with a mean (SD) age of 29.2 (9.0) years, height of 172.7 (10.0) cm, weight of 73.0 (13.9) kg, and body mass index ranging from 18 to 30 kg/m(2). Twenty-three were white, 5 African American, 2 Asian/Pacific Islander, 1 Hispanic, and 1 Native American. The estimated geometric mean ratio of AUC(0-t) for intact compared with dissolved tablets under fasting conditions was 141.24% (90% CI, 122.05-163.43), and the geometric mean ratio of AUC(0-t) for intact tablets under fed compared with fasting conditions was 143.46% (90% CI, 124.22-165.69). Nine subjects (28.1%) reported a total of 20 treatment-emergent adverse events (AEs). The most frequently reported AEs were gastrointestinal disorders (6 subjects [18.8%]) and central nervous system disorders (4 [12.5%]). Eight AEs considered possibly or probably related to sapropterin were reported by 4 subjects (12.5%); these were of mild severity and gastrointestinal in nature. No severe or serious AEs or discontinuations due to AEs occurred during the study. CONCLUSIONS: Administration of sapropterin as intact tablets and with a high-calorie, high-fat meal was associated with increased drug exposure. Oral administration of sapropterin 10 mg/kg as intact tablets with or without food was generally well tolerated.

In vitro and in vivo properties of 3-tert-butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d]-[1,2,4]triazine (MRK-016), a GABAA receptor alpha5 subtype-selective inverse agonist.

3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d][1,2,4]triazine (MRK-016) is a pyrazolotriazine with an affinity of between 0.8 and 1.5 nM for the benzodiazepine binding site of native rat brain and recombinant human alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors. It has inverse agonist efficacy selective for the alpha5 subtype, and this alpha5 inverse agonism is greater than that of the prototypic alpha5-selective compound 3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-hdyl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine (alpha5IA). Consistent with its greater alpha5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than alpha5IA. MRK-016 gave good receptor occupancy after oral dosing in rats, with the dose required to produce 50% occupancy being 0.39 mg/kg and a corresponding rat plasma EC(50) value of 15 ng/ml that was similar to the rhesus monkey plasma EC(50) value of 21 ng/ml obtained using [(11)C]flumazenil positron emission tomography. In normal rats, MRK-016 enhanced cognitive performance in the delayed matching-to-position version of the Morris water maze but was not anxiogenic, and in mice it was not proconvulsant and did not produce kindling. MRK-016 had a short half-life in rat, dog, and rhesus monkey (0.3-0.5 h) but had a much lower rate of turnover in human compared with rat, dog, or rhesus monkey hepatocytes. Accordingly, in human, MRK-016 had a longer half-life than in preclinical species ( approximately 3.5 h). Although it was well tolerated in young males, with a maximal tolerated single dose of 5 mg corresponding to an estimated occupancy in the region of 75%, MRK-016 was poorly tolerated in elderly subjects, even at a dose of 0.5 mg, which, along with its variable human pharmacokinetics, precluded its further development.

Elimination of diastereomer interference to determine Telcagepant (MK-0974) in human plasma using on-line turbulent-flow technology and off-line solid-phase extraction coupled with liquid chromatography/tandem mass spectrometry.

To eliminate the diastereomer interference on Telcagepant (MK-0974) determination during clinical study support, on-line high turbulent-flow liquid chromatography (HTLC) methods, HTLC-A and HTLC-B that covered dynamic range of 0.5-500 nM and 5-5000 nM, respectively, were developed. To meet the requirement of rapid assay transfer among multiple laboratories and analysts, a solid-phase extraction (SPE) assay was derived from the existing HTLC-B assay under the same dynamic range. The on-line HTLC assays were achieved through direct injection of plasma samples, extraction of analyte with a Cohesive C18 column (50 mm x 0.5 mm, 50 microm), followed by HPLC separation on a FluoPhase RP column (100 mm x 2.1 mm, 5 microm) and MS/MS detection. The off-line SPE assay used Waters Oasis HLB microElution plate to extract the analytes from plasma matrix before injecting on a FluoPhase RP column (150 mm x 2.1 mm, 5 microm) for LC-MS/MS analysis. Under both on-line and off-line assay conditions, the diastereomer 1c was chromatographically separated from MK-0974. Cross-validation with the pooled samples demonstrated that both on-line and off-line assays provided comparable data with a difference of < 2.6%. The assays were proved to be specific, accurate and reliable, and have been used to support multiple clinical studies. The pros and cons of on-line and off-line assays with regard to man power involved in sample preparation, total analysis time, carryover, cost efficiency, and the requirement for assay transfer are discussed.

Reversed-phase chiral liquid chromatography on polysaccharide-based stationary phase coupled with tandem mass spectrometry for simultaneous determination of four stereoisomers of MK-0974 in human plasma.

MK-0974 (1a), N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifuoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo-[4,5-B] pyridine-1-yl)piperidine-1-carboxamide, is a novel calcitonin gene-related peptide (CGRP) receptor antagonist with two chiral centers. Direct separation of its four stereoisomers (1a-d) was achieved using a cellulose chiral stationary phase, a Chiralcel OJ-RH column (150 mm x 4.6 mm), under reversed-phase condition, following the extraction of 0.2 mL plasma on Oasis muElution HLB 96-well solid-phase-extraction (SPE) plate. The tandem mass spectrometric detection was conducted in the positive-ion mode with a turbo-ion-spray (TIS) interface using multiple-reaction-monitoring on a Sciex API3000. Addition of ammonium trifluoroacetate to low-organic mobile phase improved detection sensitivity by more than 30-fold. The simultaneous quantification of the four stereoisomers in human plasma was validated over the ranges of 0.5-5000 nM for 1a and 0.5-500 nM for its three isomers (1b-d). Intraday validation, conducted with five lots of human control plasma, resulted in <12.4% (% coefficient of variation, CV) precision and 96.3-105.4% accuracy for all four stereoisomers. Further evaluation indicated that the assay was specific, the samples were stable after three freeze/thaw cycles, the recovery was reasonable (above 65%) and no matrix effect was observed for all four isomers. Investigation on the chiral integrity of 1a indicated that the diastereomer 1c, inversion at azepinone-3 carbon, was the only isomer observed in the post-dose clinical samples and accounted for 2.4-5.2% of MK-0974 exposure in the circulatory system. The possibility of inversion during blood collection, plasma storage and sample preparation was ruled out, while inversion was observed in the clinical formulation accounting for approximately 0.12% of 1a in a 100-mg capsule.

An in vivo microdialysis coupled with liquid chromatography/tandem mass spectrometry study of cortisol metabolism in monkey adipose tissue.

It is postulated that elevated tissue concentrations of cortisol may be associated with the development of metabolic syndrome, obesity, and type 2 diabetes. The 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) enzyme regenerates cortisol from inactive cortisone in tissues such as liver and adipose. To better understand the pivotal role of 11beta-HSD1 in disease development, an in vivo microdialysis assay coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis using stable isotope-labeled (SIL) cortisone as a substrate was developed. This assay overcomes the limitations of existing methodologies that suffer from radioactivity exposure and analytical assay sensitivity and specificity concerns. Analyte extraction efficiencies (E(d)) were evaluated by retrodialysis. The conversion of SIL-cortisone to SIL-cortisol in rhesus monkey adipose tissue was studied. Solutions containing 100, 500, and 1000 ng/mL SIL-cortisone were locally delivered through an implanted 30-mm microdialysis probe in adipose tissue. At the delivery rate of 1.0 and 0.5 microL/min, E(d) values for SIL-cortisone were between 58.7+/-5.6% (n=4) and 72.7+/-1.3% (n=4), whereas at 0.3 microL/min E(d) reached nearly 100%. The presence of 11beta-HSD1 activities in adipose tissue was demonstrated by production of SIL-cortisol during SIL-cortisone infusion. This methodology could be applied to cortisol metabolism studies in tissues of other mammalian species.

Strategies on efficient method development of on-line extraction assays for determination of MK-0974 in human plasma and urine using turbulent-flow chromatography and tandem mass spectrometry.

On-line extraction assays using cohesive high-turbulence liquid chromatography (HTLC) coupled with tandem mass spectrometer (MS/MS) have been developed for the determination of MK-0974 in human plasma and urine. In this report, a four-step strategy for efficient method development of an on-line extraction assay was discussed. Several challenges - namely extraction recovery, carryover and analyte loss to urine container - were addressed. The assay procedures included sample preparation on a Packard MultiPROBE II liquid-handling system, direct injection on a Cohesive Flux 2300 system, on-line extraction with a Cohesive C(18) column (0.5mmx50mm, 50microm), HPLC separation on a FluophasePFP column (50mmx3mm, 5microm) under cohesive quick-elution mode and MS detection on a Sciex API4000 in multiple-reaction monitoring (MRM) mode using positive ionization and turbo ion-spray. Since 37-80% analyte loss was observed in urine QCs, 1% BSA was added to bring urine QC accuracy back to approximately 100% of nominal. Because of the nature of BSA, the urine assay was established by adapting the plasma method. Thus, the two assays were able to be validated side-by-side, which reduced the validation time by approximately two-fold. The linear dynamic ranges were 0.5-500 and 1-1000nM for the plasma and urine assays, respectively. Obtained from five standard curves constructed with five lots of human plasma or urine, the intra-day precision (%CV) was <3.14 and <2.62%, and the accuracy was 98.3-101.0 and 99.13-100.64% of nominal for plasma and urine assays, respectively. Both plasma and urine QC samples were stable when kept at room temperature for 4h, at -70 degrees C for 3 weeks, or after three freeze-thaw cycles. Both assays gave reasonable relative recovery (>88.8%) and acceptable matrix effect (<15%). The carryover from the upper limit of quantification (ULOQ) was able to be controlled at <20% of lower limit of quantification (LLOQ).

Determination of sitagliptin in human urine and hemodialysate using turbulent flow online extraction and tandem mass spectrometry.

High turbulence liquid chromatography (HTLC, or turbulent flow online extraction) and tandem mass spectrometry (MS/MS) methods for the determination of sitagliptin in human urine and hemodialysate were developed and validated to support clinical studies. A narrow bore large particle size reversed-phase column (Cyclone, 50 mm x 1.0 mm, 60 microm) and a BDS Hypersil C18 column (30 mm x 2.1 mm, 3 microm) were used as extraction and analytical columns, respectively. For the urine assay, the LLOQ was 0.1 microg/ml, the linear calibration range was 0.1 to 50 microg/ml, the interday precision (R.S.D.%, n=5) was 2.3-6.5%, and the accuracy was 96.9-106% of the nominal value. For the urine quality control samples (QCs), the intraday precision (R.S.D.%, n=5) and accuracy were 1.8-2.6% and 96.2-106% of the nominal value, respectively. The interday precision (R.S.D.%) for 56 sets of urine QCs over a 6-month period varied from 3.8% to 5.5% and the accuracy from 102% to 105% of the nominal value. For the hemodialysate assay, the LLOQ was 0.01 ng/ml, the linear dynamic range was 0.01-5.0 ng/ml, the interday precision was 1.6-4.1%, and the accuracy was 89.8-104% of the nominal value. For hemodialysate QCs, the intraday precision and accuracy varied from 2.3% to 8.9% and from 99.8% to 111% of the nominal value, respectively. These results demonstrated that both methods are selective, accurate, precise, reproducible, and suitable for quantifying sitagliptin in hemodialysate and human urine samples.

Automated enzyme inhibition assay method for the determination of atorvastatin-derived HMG-CoA reductase inhibitors in human plasma using radioactivity detection.

INTRODUCTION: A Tecan-based enzyme inhibition assay has been developed for the determination of atorvastatin-derived 'active' and 'total' (active inhibitors plus atorvastatin lactone and other potential inhibitors following base hydrolysis) 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitor concentrations in human plasma. Atorvastatin is an inhibitor of HMG-CoA reductase, which is a key rate-limiting enzyme in the cholesterol biosynthesis. Previously, atorvastatin-derived HMG-CoA reductase inhibitors were measured via enzyme inhibition assays by manual operation. METHODS: In this work, an enzyme assay procedure based on 8-tip Tecan robotics and set-up in a 96-well plate format with customized hardware is presented. Following protein precipitation of the plasma sample, an aliquot of the resulting supernatant is mixed with HMG-CoA reductase and (14)C-labeled HMG-CoA prior to incubation. The product, (14)C-mevalonic acid, is lactonized, separated from unreacted (14)C-substrate, and counted in a liquid scintillation counter. Plasma HMG-CoA reductase inhibitor concentrations are measured against atorvastatin as the standard. Tecan Genesis 150 and 200 robotic workstations were used for the protein precipitation, enzyme incubation, and product separation. RESULTS: The standard calibration range for the assay was 0.4-20 ng eq/mL. Intra-day precision (%CV) data for the calibration standard and quality control (QC) samples (n=5 replicates) were both

An in vitro microdialysis methodology to study 11beta-hydroxysteroid dehydrogenase type 1 enzyme activity in liver microsomes.

Microdialysis sampling coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS) was used to observe in vitro 11beta-hydroxysteroid dehydrogenase type 1 (HSD1) enzyme-catalyzed conversion of stable-isotope-labeled cortisone to cortisol in liver microsomes from dog, monkey, and human. Experimental conditions that would affect the microdialysis sampling approach including probe length, perfusion fluid flow rate, extraction efficiency (E(d)), substrate concentration, and enzyme reaction conditions were evaluated. Dialysates containing high salt concentrations (>150 mM) were directly assayed using LC/MS/MS without additional sample cleanup. The sensitivity (with lower level of quantitation at 0.1 ng/mL) and selectivity of this assay allowed detection of the enzyme reactants at physiologically relevant levels. The interconversion from M+4 cortisone to M+4 cortisol was detected in dog, human, and monkey liver microsomes. Results show species-specific reaction profiles, with a five times higher conversion rate in dog liver microsomes than in human and monkey liver microsomes. Based on M+4 cortisol production rate obtained using a microdialysis infusion of M+4 cortisone to the microsomes coincubated with a proprietary 11beta-HSD1 inhibitor of different concentrations, the degrees of enzyme inhibition were found to be 40 and 85%, consistent with values obtained by a traditional in vitro incubation method. The microdialysis sampling methodology with LC/MS/MS provided extensive information about 11beta-HSD1 activities in microsomes from different mammalian species.

Investigation of high-throughput ultrafiltration for the determination of an unbound compound in human plasma using liquid chromatography and tandem mass spectrometry with electrospray ionization.

A high-throughput ultrafiltration method with a direct injection assay has been developed to determine unbound concentrations of a high-protein binding compound, an alpha(v)beta(3) bone integrin antagonist (I), in human plasma for a clinical pharmacokinetic study. The 96-well MultiScreen filter plate with Ultracel-PPB membrane was evaluated for the separation of unbound from protein-bound compound I by ultrafiltration. The sample preparation was automated using a Packard MultiPROBE II EX liquid handling system to transfer the plasma samples to the 96-well PPB plate for centrifugation and to prepare ultrafiltrate samples for analysis. Using on-line extraction with a column-switching setup for sample clean-up and separation, the ultrafiltrate samples were directly injected onto a reversed-phase HPLC system and analyzed using a mass spectrometer interfaced with an electrospray ionization (ESI) source in the positive ionization mode (LC/ESI-MS/MS). The performance of the ultrafiltration using Ultracel-PPB 96-well plate for unbound I analysis was evaluated and optimized with respect to sample volume, centrifugation temperature, speed and time, and the relationship of the well positions of the PPB plate versus filtrate volumes and concentrations. The assay intraday accuracy and precision were between 93.9 and 104.8 and <7.3% (CV), respectively. The linear range of the calibration curve for the assay was 0.1-500 ng/mL on a Finnigan TSQ Quantum LC/ESI-MS/MS system. Evaluation and validation of the unbound plasma assay demonstrated it to be rapid, sensitive and reproducible.