The preclinical pharmacology of the high affinity anti-IL-6R Nanobody® ALX-0061 supports its clinical development in rheumatoid arthritis.

INTRODUCTION: The pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology. METHODS: ALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control. RESULTS: ALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration. CONCLUSIONS: ALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA.

In vitro studies on the metabolism of trabectedin (YONDELIS) in monkey and man, including human CYP reaction phenotyping.

Trabectedin (YONDELIS) is a potent anticancer agent which was recently approved in Europe for the treatment of soft tissue sarcoma. The drug is currently also in clinical development for the treatment of ovarian carcinoma. In vitro experiments were conducted to investigate the hepatic metabolism of [(14)C]trabectedin in Cynomolgus monkey and human liver subcellular fractions. The biotransformation of trabectedin was qualitatively similar in 12,000 x g supernatants of both species, and all human metabolites were also produced by the monkey. The trabectedin metabolites were identified by QTOF mass spectrometry, and HPLC co-chromatography with reference compounds. Trabectedin was metabolized via different biotransformation pathways. Most of the metabolic conversions occurred at the trabectedin A domain including mono-oxidation and di-oxidation, carboxylic acid formation with and without additional oxidation, and demethylation either without (N-demethylation to ET-729) or with additional mono-, di- or tri-oxidation. Another metabolite resulted from O-demethylation at the trabectedin C subunit, and in addition, aliphatic ring opening of the methylene dioxybridge at the B domain was detected. Overall, demethylation and oxidation played a major role in phase I metabolism of the drug. Human cDNA expressed CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, 3A4 and 3A5 in E. coli membranes, but not CYP1B1, 2C19, and 4A11 were able to metabolize [(14)C]trabectedin. Experiments with chemical inhibitors and CYP inhibitory antibodies indicated that, at therapeutic levels, CYP3A4 is the main human CYP isoform involved in trabectedin's hepatic metabolism. In monkey and human liver microsomes, trabectedin was not substantially metabolized by glucuronidation.

Tissue distribution and depletion kinetics of bortezomib and bortezomib-related radioactivity in male rats after single and repeated intravenous injection of 14 C-bortezomib.

PURPOSE: The body distribution of total radioactivity (TR) and bortezomib was investigated in male Sprague-Dawley rats after single and repeated i.v. (bolus) administration with (14)C-labelled bortezomib (VELCADE) (0.2 mg/kg; 0.28 MBq./kg). METHODS: Bortezomib was dosed on days 1, 4, 8, and 11 (i.e. a clinical dosing cycle) and the animals were sacrificed at selected time points following single and repeated dose administration for the quantification of TR in blood, plasma, and various tissues by liquid scintillation counting following organ dissection or by quantitative whole body autoradiography. In selected tissues, bortezomib levels were determined by LC-MS/MS. RESULTS: In general, plasma TR levels were less than 10% of the corresponding blood concentrations. TR was rapidly and widely distributed to the tissues with only limited penetration into the central nervous system (CNS). In the tissues, highest levels of TR were measured in bortezomib-eliminating organs (liver and kidney), lymphoid tissues, and regions of rapidly dividing cells (e.g. the bone marrow, intestinal mucosa). Low TR concentrations were found in the CNS (tissue-to-blood ratio of approximately 0.05 after repeated dosing). With the exception of the liver, TR consisted almost exclusively of the parent drug. Tissue concentrations of TR and bortezomib increased up to about threefold from the first to the third dose administration, after which they remained constant. CONCLUSION: No undue tissue accumulation of TR and of bortezomib was observed in rats following a full clinical dosing cycle of bortezomib.

Pharmacokinetic considerations and efficacy of levofloxacin in an inhalational anthrax (postexposure) rhesus monkey model.

Because the treatment of inhalational anthrax cannot be studied in human clinical trials, it is necessary to conduct efficacy studies using a rhesus monkey model. However, the half-life of levofloxacin was approximately three times shorter in rhesus monkeys than in humans. Computer simulations to match plasma concentration profile, area under the concentration-time curve (AUC), and time above MIC for a human oral dose of 500 mg levofloxacin once a day identified a dosing regimen in rhesus monkeys that would most closely match human exposure: 15 mg/kg followed by 4 mg/kg administered 12 h later. Approximately 24 h following inhalational exposure to approximately 49 times the 50% lethal doses of Bacillus anthracis (Ames strain), monkeys were treated daily with vehicle, levofloxacin, or ciprofloxacin for 30 days. Ciprofloxacin was administered at 16 mg/kg twice a day. Following the 30-day treatment, monkeys were observed for 70 days. Nine of 10 control monkeys died within 9 days of exposure. No clinical signs were observed in fluoroquinolone-treated monkeys during the 30 treatment days. One monkey died 8 days after levofloxacin treatment, and two monkeys from the ciprofloxacin group died 27 and 36 days posttreatment, respectively. These deaths were probably related to the germination of residual spores. B. anthracis was positively cultured from several tissues from the three fluoroquinolone-treated monkeys that died. MICs of levofloxacin and ciprofloxacin from these cultures were comparable to those from the inoculating strain. These data demonstrate that a humanized dosing regimen of levofloxacin was effective in preventing morbidity and mortality from inhalational anthrax in rhesus monkeys and did not select for resistance.

Mixed-effects modelling of the interspecies pharmacokinetic scaling of pegylated human erythropoietin.

The aim of this study was to develop a population pharmacokinetic model for interspecies allometric scaling of pegylated r-HuEPO (PEG-EPO) pharmacokinetics to man. A total of 927 serum concentrations from 193 rats, 6 rabbits, 34 monkeys, and 9 dogs obtained after a single dose of PEG-EPO, administered by the i.v. (dose range: 12.5-550 microg/kg) and s.c. (dose range: 12.5-500 microg/kg) routes, were pooled in this analysis. An open two-compartment model with first-order absorption and lag time (Tlag) and linear elimination from the central compartment was fitted to the data using the NONMEM V software. Body weight (WT) was used as a scaling factor and the effect of brain weight (BW), sex, and pregnancy status on the pharmacokinetic parameters was investigated. The final model was evaluated by means of a non-parametric bootstrap analysis and used to predict the PEG-EPO pharmacokinetic parameters in healthy male subjects. The systemic clearance (CL) in males was estimated to be 4.08WT1.030xBW-0.345 ml/h. In females, the CL was 90.7% of the CL in males. The volumes of the central (Vc) and the peripheral (Vp) compartment were characterized as 57.8WT0.959 ml, and 48.1WT1.150 ml, respectively. Intercompartmental flow was estimated at 2.32WT0.930 ml/h. Absorption rate constant (Ka) was estimated at 0.0538WT-0.149. The absolute s.c. bioavailability F was calculated at 52.5, 80.2, and 49.4% in rat, monkey, and dog, respectively. The interindividual variability in the population pharmacokinetic parameters was fairly low (<35%). Non-parametric bootstrap confirmed the accuracy of the NONMEM estimates. The mean model predicted pharmacokinetic parameters in healthy male subjects of 70 kg were estimated at: CL: 26.2 ml/h; Vc: 3.6l; Q: 286 l/h; Vp: 6.9l, and Ka: 0.031 h-1. The population pharmacokinetic model developed was appropriate to describe the time course of PEG-EPO serum concentrations and their variability in different species. The model predicted pharmacokinetics of PEG-EPO in humans suggest a less frequent dosing regimen relative to erythropoietin and darbepoetin, potentially leading to a simplification of anemia management.

Population pharmacokinetic analysis of pegylated human erythropoietin in rats.

The purpose of this study was to model the pharmacokinetics of the pegylated human erythropoietin (PEG-EPO) after single-dose administration in rats, and to evaluate the influence of weight, sex, and pregnancy status on the pharmacokinetic parameters. A total of 436 serum concentrations from 193 Sprague-Dawley rats were obtained from four pharmacokinetic/toxicokinetic studies, in which a single dose of PEG-EPO was administered by the intravenous (i.v.; dose range: 2.5 to 500 microg/kg) and subcutaneous (s.c.; dose range: 12.5 to 500 microg/kg) route. Pharmacokinetic analysis was performed using nonlinear mixed effect modeling (NONMEM V software) to determine the population mean of pharmacokinetic parameters and the variances of the interindividual random effects. The effect of weight, sex, and pregnancy status on the pharmacokinetic parameters was evaluated by forward inclusion and backward elimination process, using the likelihood ratio test. Nonparametric bootstrap analysis was employed as an internal model evaluation technique to qualify the model developed. An open two-compartment model with linear elimination from the central compartment, a first-order absorption with lag time characterized the serum concentration-time profiles of PEG-EPO after i.v. and s.c. administration. For a male rat of 0.24 kg, the average CL, Vc, Q, Vp, Ka, Tlag, and F was estimated to be 0.728 mL/h, 15.8 mL, 0.373 mL/h, 6.99 mL, 0.0618 h(-1), 3.13 h, and 48.8%, respectively. A twofold increase in weight corresponded with a 170 and 238% increase in CL and Vc, respectively. In female rats, Vp was reduced by 11%, whereas F was increased by 15%. No effect of pregnancy status on any of the parameters could be identified. The interindividual variability in CL, Vc, Vp, Ka, and F was estimated at 10.7, 14.7, 16.6, 11.0, and 13.6%, respectively. Nonparametric bootstrap analysis confirmed the accuracy and the precision of the NONMEM parameter estimates. A population pharmacokinetic approach was used to integrate the knowledge gathered from several pharmacokinetic/toxicokinetic studies in rats. The pharmacokinetics of PEG-EPO in the rat was successfully modeled using a two-compartmental model with a linear elimination from the central compartment and a first-order absorption process with lag time. Weight and sex, but not pregnancy status, were identified as covariates of interest during preclinical development. The population pharmacokinetic model developed will be further used for the purpose of interspecies scaling and PK/PD modeling.

Selective serotonin reuptake inhibitors and cytochrome P-450 mediated drug-drug interactions: an update.

The selective serotonin reuptake inhibitors (SSRIs) have become the most prescribed antidepressants in many countries. Although the SSRIs share a common mechanism of action, they differ substantially in their chemical structure, metabolism, and pharmacokinetics. Perhaps the most important difference between the SSRIs is their potential to cause drug-drug interactions through inhibition of cytochrome-P450 (CYP) isoforms. This paper provides an update on both the in vitro and in vivo evidence with respect to CYP-mediated drug-drug interactions with this class of antidepressants. The available evidence clearly indicates that the individual SSRIs display a distinct profile of cytochrome P450 inhibition. Fluvoxamine is a potent CYP1A2 and CYP2C19 inhibitor, and a moderate CYP2C9, CYP2D6, and CYP3A4 inhibitor. Fluoxetine and paroxetine are potent CYP2D6 inhibitors, whereas fluoxetine's main metabolite, norfluoxetine, has a moderate inhibitory effect on CYP3A4. Sertraline is a moderate CYP2D6 inhibitor; citalopram appears to have little effect on the major CYP isoforms. Fluoxetine deserves special attention as inhibitory effects on CYP-activity can persist for several weeks after fluoxetine discontinuation because of the long half-life of fluoxetine and its metabolite norfluoxetine. Drug combinations with SSRIs should be assessed on an individual basis. Knowledge regarding the CYP-isoforms involved in the metabolism of the co-administered drug may help clinicians to anticipate and avoid potentially dangerous drug-drug interactions. Anticipated interactions can usually be managed by appropriate dose adjustment and titration of the object drug. In some cases, therapeutic drug monitoring can be useful. Equally well, an SSRI with limited interaction potential may be selected to treat depression in patients that receive other medications.