Asymmetric inner wedge group sequential tests with applications to verifying whether effective drug concentrations are similar in adults and children.

Extrapolating from information available on one patient group to support conclusions about another is common in clinical research. For example, the findings of clinical trials, often conducted in highly selective patient cohorts, are routinely extrapolated to wider populations by policy makers. Meanwhile, the results of adult trials may be used to support conclusions about the effects of a medicine in children. For example, if the effective concentration of a drug can be assumed to be similar in adults and children, an appropriate paediatric dosing rule may be found by 'bridging', that is, by matching the adult effective concentration. However, this strategy may result in children receiving an ineffective or hazardous dose if, in fact, effective concentrations differ between adults and children. When there is uncertainty about the equality of effective concentrations, some pharmacokinetic-pharmacodynamic data may be needed in children to verify that differences are small. In this paper, we derive optimal group sequential tests that can be used to verify this assumption efficiently. Asymmetric inner wedge tests are constructed that permit early stopping to accept or reject an assumption of similar effective drug concentrations in adults and children. Asymmetry arises because the consequences of under- and over-dosing may differ. We show how confidence intervals can be obtained on termination of these tests and illustrate the small sample operating characteristics of designs using simulation. Copyright © 2016 John Wiley & Sons, Ltd.

Pharmacokinetics and Pharmacodynamics of Canakinumab in Patients With Systemic Juvenile Idiopathic Arthritis.

The characterization of the pharmacokinetic (PK) and pharmacodynamic (PD) properties in pediatric patients is essential in supporting the recommended dosage of canakinumab in the relevant population. Here the PK and PD properties of canakinumab-a monoclonal antibody-in pediatric patients with systemic juvenile idiopathic arthritis (SJIA) are presented. Blood samples were obtained from 4 phase 2/3 clinical studies in patients with SJIA. Canakinumab PK properties and total interleukin (IL)-1ß kinetic properties were characterized by a population-based PK-binding model. On administration, canakinumab increased total IL-1ß complex in SJIA patients. Canakinumab clearance and volume of distribution were not impacted by age in pediatric patients after correction for the patient's body weight. The estimated serum clearance of canakinumab was 0.106 ± 0.00689 L/day, with a corresponding volume of distribution at steady state of 3.2 L and an estimated half-life of 22 days, based on a model typical body weight of 33 kg. Body-weight-based dosing provided comparable canakinumab exposure across the age groups in patients 2 to <20 years with SJIA. In younger children, a modest increase in the turnover rate of IL-1ß was observed. Compared to other indications, IL-1ß production rate was higher and clearance was slower in patients with SJIA. Low immunogenicity incidence of 3.1% was observed, and none of the patients had neutralizing antibodies. In conclusion, the PK/PD findings further support dose selection of canakinumab in patients with SJIA.

Pharmacokinetic and pharmacodynamic properties of canakinumab in patients with gouty arthritis.

Pharmacokinetics and pharmacodynamics of the anti-interleukin (IL)-1ß monoclonal antibody, canakinumab, in gouty arthritis patients from three studies are reported. Canakinumab has low serum clearance (0.214 L/day), low steady-state volume of distribution (7.44 L), a 25.8-day half-life, and approximately 60% subcutaneous absolute bioavailability in a typical 93-kg patient. Creatinine clearance had a small positive impact on serum canakinumab clearance that is not likely to be clinically relevant. Binding to circulating IL-1ß was demonstrated by increases in total serum IL-1ß following canakinumab dosing. Total IL-1ß kinetics and canakinumab pharmacokinetics were characterized by a population-based pharmacokinetic-binding model, where the estimated apparent in vivo dissociation constant (signifying binding affinity of canakinumab to circulating IL-1ß) was 0.99 nmol/L in gouty arthritis patients. Canakinumab treatment provided rapid, sustained decreases in C-reactive protein and serum amyloid A, provided superior pain relief to triamcinolone acetonide, and increased time to first recurrent attack (P ≤ 0.01 favoring all canakinumab doses vs. triamcinolone acetonide).

Metabolism of dextrorphan by CYP2D6 in different recombinantly expressed systems and its implications for the in vitro assessment of dextromethorphan metabolism.

Cytochrome P450 2D6 (CYP2D6) mediated formation of dextrorphan (DOR) from dextromethorphan (DEX) is widely used as a marker to assess the activity of this enzyme both in vitro and in vivo. The sequential metabolism of DOR during in vitro studies, particularly using recombinant systems (rCYPs) expressing human CYP2D6, is assumed to be negligible. The extent of metabolism was investigated for a range of DEX and DOR concentrations in microsomal preparations from three different rCYPs expressing human CYP2D6 (yeast, Supersomes and Bactosomes) containing 10 pmol of the enzyme. Bactosomes and Supersomes, but not yeast rCYP microsomes, were capable of metabolising DOR to 3-hydroxymorphinan (HYM). Two novel CYP2D6 related metabolites were identified in Bactosomes, and assigned as single hydroxylations in the phenyl rings of DOR and HYM using ion-trap mass spectrometry. Therefore, in rCYP systems with high turn over rate (e.g. Bactosomes) DOR may not be considered as an end product particularly at low concentrations of DEX; leading to an underestimation of true metabolic rate. The results also put further emphasis on the necessity of optimising study conditions when switching between rCYP sources.

Inactivation of CYP2D6 by methylenedioxymethamphetamine in different recombinant expression systems.

Recombinantly expressed CYP450 systems (rCYPs) are often used to screen for irreversible/quasi-irreversible enzyme inhibitors during drug development. The concentration- and time-dependent inactivation of CYP2D6 by methylenedioxymethamphetamine (MDMA) was compared in three different rCYP2D6 systems (yeast microsomes, Supersomestrade mark and Bactosomestrade mark) under the conditions of the most commonly used protocols in assessing mechanism-based inactivation (MBI). MDMA (2-20microM) was pre-incubated with enzyme for 0, 2.5 and 5min followed by a five-fold dilution and further incubation with dextromethorpan (DEX) (50microM). The formation of dextrorphan (DOR) from DEX was used as a specific marker of CYP2D6 activity. Concentration- and time-dependent inactivation of CYP2D6 by MDMA was observed with each rCYP system. However, the apparent kinetic parameters for MBI (k(inact), the maximum inactivation rate constant and K(I), the inhibitor concentration associated with half maximal rate of inactivation) were significantly greater (p<0.05) for Bactosomestrade mark (0.95+/-0.33min(-1), 42.9+/-20.1microM) than those found using yeast microsomes (0.28+/-0.04min(-1), 2.86+/-1.18microM) and Supersomestrade mark (0.38+/-0.05min(-1), 3.66+/-0.10microM). After correction for depletion of MDMA during pre-incubation, k(inact) and K(I) values determined using Bactosomestrade mark decreased significantly but remained higher than for the other rCYP systems (p<0.05). Substantial metabolism of DOR after its formation from DEX was also observed using Supersomestrade mark and Bactosomestrade mark. Sub-optimal study design when investigating MBI may compromise the quantitative characterization of inhibitory characteristics using some rCYP systems.

Kinetics of the time-dependent inactivation of CYP2D6 in cryopreserved human hepatocytes by methylenedioxymethamphetamine (MDMA).

Methylenedioxymethamphetamine (MDMA) was investigated in cryopreserved human hepatocytes as a time-dependent inactivator (TDI) of CYP2D6 using dextromethorphan (DEX) as a probe substrate. Inhibition kinetic parameters k(inact), the maximal rate of inactivation, and K(I), the inhibitor concentration at half the maximal activation rate, were determined. Time- and concentration-dependent inhibition were confirmed, and the influence of different elements of study design (e.g. cell number, stability of hepatocytes, dilution after preincubation) on estimated kinetic parameters were evaluated. Dilution factors (DF) of 1.2, 5 or total removal of inhibitor (by washing cells after preincubation, WR) resulted in k(inact) and K(I) (+/-S.E.) values of 0.02+/-0.002 min(-1) and 0.88+/-0.31 microM, 0.01+/-0.001 min(-1) and 1.23+/-0.70 microM, and 0.01+/-0.001 min(-1) and 2.10+/-1.32 microM, respectively; indicating that insufficient dilution may lead to overestimation of CYP2D6 inactivation. Accounting for MDMA depletion during the preincubation, corrected K(I) values were significantly lower (0.11+/-0.05 microM, 0.15+/-0.09 microM, 0.24+/-0.16 microM for DF of 1.2, 5, and WR, respectively). Inactivation efficiency in hepatocytes, as measured by k(inact)/K(I), was 10-fold less than that previously reported in human liver microsomes or recombinantly expressed systems. Possible causes for the observed differences between in vitro systems warrant further investigation. These may include differences in metabolic consumption of MDMA in each system, non-specific binding and presence of active efflux in hepatocytes.

The impact of experimental design on assessing mechanism-based inactivation of CYP2D6 by MDMA (Ecstasy).

MDMA (3-4-methylenedioxymethamphetamine, commonly known as Ecstasy) is a potent mechanism-based inhibitor (MBI) of cytochrome P450 2D6 (CYP2D6), causing quasi-irreversible inhibition of the enzyme in vitro. An evaluation of the in vivo implications of this phenomenon depends on the accuracy of the estimates of the parameters that define the inhibition in vitro, namely k(inact) (the maximal inhibition rate) and KI (the inactivation constant). These values are determined in two steps, pre-incubation of the enzyme with the inhibitor (enzyme inactivation), followed by dilution and further incubation to measure residual enzyme activity with a probe substrate. The aim of this study was to assess the impact of different dilutions and probe substrate concentrations on the estimates of k(inact) and KI using recombinantly expressed CYP2D6. Enzyme activity was measured by the conversion of dextromethorphan (DEX) to dextrorphan (DOR). Dilution factors of 1.25, 2, 5, 10, 25 and 50 (DEX at 30 microM) gave mean (+/-SE) values of k(inact) (min-1) of 0.20+/-0.06, 0.21+/-0.05, 0.31+/-0.06, 0.37+/-0.11, 0.51+/-0.10 and 0.58+/-0.08, respectively, and KI (microM) values (after correction for non-specific microsomal binding) of 2.22+/-1.90, 2.80+/-1.34, 5.78+/-2.07, 6.36+/-2.93, 3.99+/-1.57 and 4.86+/-1.37, respectively. Accordingly, high (e.g. 50 fold) and low (e.g. 1.25 fold) dilutions were associated with statistically significant differences in kinetic values (p <0.05). Varying DEX concentration (10-100 microM) was not associated with significant changes in k(inact) and KI values when a five-fold dilution was used (with the exception of a lower KI at 10 microM DEX). High dilution was also shown to reduce non-specific microsomal binding of MDMA. The changes in the two kinetic parameters were dependent on the experimental procedure and shown to be unlikely to have a material influence on the maximum inhibition of CYP2D6 expected in vivo after typical recreational doses of MDMA (50-100 mg), since the potency of inhibition was high. The different values of the kinetic parameters were predicted to have a marginal influence on the time for recovery of enzyme activity following re-synthesis of CYP2D6.