Utility of in silico prediction of target suppression for antibodies against soluble targets: static versus dynamic models.

PURPOSE: Antibodies that bind soluble targets such as cytokines belong to an important class of immunotherapies. Target levels can significantly accumulate after antibody administration due to formation of antibody-target complex, accompanied with suppression in free target which is often difficult to measure. Being a surrogate for pharmacodynamic activity, free target suppression is often predicted using in silico tools. The objective of this work is to illustrate the utility of modelling and to compare static versus dynamic models in the prediction of free target suppression. METHODS: Using binding principles, we have derived a static equation to predict free target suppression at steady state (FTSS). This equation operates with five input parameters and accounts for target accumulation over time. Its predictivity was compared to a dynamic model and to other existing metrics in literature via simulations and assumptions were illustrated. RESULTS: We demonstrated the utility of in silico tools in prediction of free target suppression using static and dynamic models and clarified the assumptions in key input parameters and their limitations. Predicted values using the FTSS equation correlate very well with those from the dynamic model at level > 20% target suppression, relevant for antagonistic antibodies. CONCLUSION: In silico tools are needed to predict target suppression by antibody drugs. Static or dynamic models can be used dependant on the scope, available data and undertaken assumptions. These tools can be used to guide discovery and development of antibodies and has the potential to reduce clinical failure.

Prediction of Half-Life Extension of Peptides via Serum Albumin Binding: Current Challenges.

The development of peptide therapeutics has increased enormously in recent decades. Many of the peptide drugs and antibody fragments that lack Fc backbone have a short half-life in circulation. In general, the half-life supports the design of the dosing regimen and frequency of administration, which are key aspects in the discovery of peptide drugs intended for long duration of action. Less frequent administration such as weekly or monthly can improve compliance and adherence to therapy. Serum albumin binding is a key approach to extend the half-life of peptide drugs. Despite the evidence of half-life prolongation of a variety of peptide drugs via albumin, quantitative prediction for humans is still a key question. Challenges in the measurement of albumin binding and in understanding the clearance mechanisms can limit quantitative prediction. We integrated pharmacokinetic concepts and albumin binding across species in a quantitative model to be used as a tool for prediction of half-life. Preliminary validation on a limited dataset indicated a good correlation between predicted and observed values. Further development of more quantitative models is warranted.

Receptor occupancy of dual glucagon-like peptide 1/glucagon receptor agonist SAR425899 in individuals with type 2 diabetes.

Unimolecular dual agonists for the glucagon-like peptide 1 receptor (GLP1R) and glucagon receptor (GCGR) are emerging as a potential new class of important therapeutics in type 2 diabetes (T2D). Reliable and quantitative assessments of in vivo occupancy on each receptor would improve the understanding of the efficacy of this class of drugs. In this study we investigated the target occupancy of the dual agonist SAR425899 at the GLP1R in pancreas and GCGR in liver by Positron Emission Tomography/Computed Tomography (PET/CT). Patients with T2D were examined by [68Ga]Ga-DO3A-Tuna-2 and [68Ga]Ga-DO3A-Exendin4 by PET, to assess the GCGR in liver and GLP1R in pancreas, respectively. Follow up PET examinations were performed after 17 (GCGR) and 20 (GLP-1R) days of treatment with SAR425899, to assess the occupancy at each receptor. Six out of 13 included patients prematurely discontinued the study due to adverse events. SAR425899 at a dose of 0.2 mg daily demonstrated an average GCGR occupancy of 11.2 ± 14.4% (SD) in N = 5 patients and a GLP1R occupancy of 49.9 ± 13.3%. Fasting Plasma Glucose levels (- 3.30 ± 1.14 mmol/L) and body weight (- 3.87 ± 0.87%) were lowered under treatment with SAR425899. In conclusion, SAR425899 demonstrated strong interactions at the GLP1R, but no clear occupancy at the GCGR. The study demonstrates that quantitative target engagement of dual agonists can be assessed by PET.

A novel dual glucagon-like peptide and glucagon receptor agonist SAR425899: Results of randomized, placebo-controlled first-in-human and first-in-patient trials.

AIMS: To evaluate the safety, pharmacokinetics and pharmacodynamics of SAR425899, a novel polypeptide, active as an agonist at both the glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCR), in healthy volunteers and in overweight/obese patients with type 2 diabetes (T2D). METHODS: Subcutaneous administrations of SAR425899 were tested in two randomized, placebo-controlled, double-blind clinical trials. In the first trial, healthy overweight volunteers (body mass index [BMI] 25-30 kg/m2 ; n = 32) received single-ascending doses (0.01-0.1 mg) of SAR425899 or placebo. In the second, a multiple-ascending-dose trial (NCT02411825), healthy normal- to overweight volunteers (BMI 20-30 kg/m2 ; n = 40) and overweight/obese patients with T2D (BMI 28-42 kg/m2 ; n = 36) received daily doses of SAR425899 or placebo over 21 or 28 days, respectively. RESULTS: The most frequently reported adverse events were gastrointestinal; gastrointestinal side effects were less pronounced in patients with T2D compared with healthy volunteers. SAR425899 significantly reduced levels of fasting plasma glucose (P < 0.05 vs. placebo) and glycated haemoglobin (P < 0.001 versus placebo) in patients with T2D. Additionally, SAR425899 led to reductions in body weight, with a maximal reduction of 5.32 kg in healthy volunteers and 5.46 kg in patients with T2D (P < 0.001 vs. placebo) at end of treatment. CONCLUSIONS: SAR425899 was well tolerated and led to favourable glycaemic effects in patients with T2D and weight reduction in both healthy volunteers and patients. Whether dual GLP-1R/GCR agonism represents a treatment method that is superior to pure GLP-1R agonists for obesity and diabetes treatment remains to be confirmed.

Pharmacokinetic and Pharmacodynamic Relationship of Blinatumomab in Patients with Non-Hodgkin Lymphoma.

BACKGROUND: Blinatumomab is a bispecific T-cell engager (BiTE®) antibody construct targeting CD3ε on T cells and CD19 on B cells. We describe the relationship between pharmacokinetics (PK) of blinatumomab and pharmacodynamic (PD) changes in peripheral lymphocytes, serum cytokines, and tumor size in patients with non-Hodgkin lymphoma (NHL). METHODS: In a phase 1 study, 76 patients with relapsed/refractory NHL received blinatumomab by continuous intravenous infusion at various doses (0.5 to 90 µg/m2/day). PD changes were analyzed with respect to dose, blinatumomab concentration at steady state (Css), and cumulative area under the concentration-versus-time curve (AUCcum). RESULTS: B-cell depletion occurred within 48 hours at doses ≥5 µg/m2/day, followed first-order kinetics, and was blinatumomab exposure-dependent. Change in tumor size depended on systemic blinatumomab exposure and treatment duration and could be fitted to an Emax model, which predicted a 50% reduction in tumor size at AUCcum of ≥1,340 h×µg/L and Css of ≥1,830 pg/mL, corresponding to a blinatumomab dose of 47 µg/m2/day for 28 days. The magnitude of transient cytokine elevation, observed within 1-2 days of infusion start, was dose-dependent, with less pronounced elevation at low starting doses. CONCLUSION: B-lymphocyte depletion following blinatumomab infusion was exposure-dependent. Transient cytokine elevation increased with dose; it was less pronounced at low starting doses. Tumor response was a function of exposure, suggesting utility for the PK/PD relationship in dose selection for future studies, including NHL and other malignant settings.

Changes in clinical laboratory parameters and pharmacodynamic markers in response to blinatumomab treatment of patients with relapsed/refractory ALL.

BACKGROUND: Blinatumomab has shown a remission rate of 69% in an exploratory single-arm, phase II dose-escalation study in adult patients with relapsed/refractory B-precursor acute lymphoblastic leukemia (ALL). We evaluated changes in laboratory parameters and immunopharmacodynamic markers in patients who received blinatumomab in the exploratory phase II study. METHODS: Data from 36 adults with relapsed/refractory ALL receiving blinatumomab as 4-week continuous IV infusions in various dose cohorts were analyzed for changes in liver enzymes, first-dose parameters, peripheral blood cell subpopulations, and cytokine/granzyme B release. Associations with clinical response were evaluated. RESULTS: Liver enzymes and inflammatory parameters transiently increased primarily during the first treatment week without clinical symptoms and reversed to baseline levels thereafter. B and T cells showed expected depletion and redistribution kinetics, respectively. Similarly, thrombocytes and T cells displayed an initial decline in cell counts, whereas neutrophils peaked during the first days after infusion start. T-cell redistribution coincided with upregulation of LFA-1 and CD69. Patients who responded to blinatumomab had more pronounced T-cell expansion, which was associated with proliferation of CD4+ and CD8+ T cells and memory subsets. Release of cytokines and granzyme B primarily occurred during the first week of cycle 1, except for IL-10, which was released in subsequent cycles. Blinatumomab step-dosing was associated with lower cytokine release and lower body temperature. CONCLUSIONS: In this study of relapsed/refractory ALL, blinatumomab-induced changes in laboratory parameters were transient and reversible. The evaluated PD markers demonstrated blinatumomab activity, and the analysis of cytokines supported the rationale for stepwise dosing. (ClinicalTrials.gov Identifier NCT01209286.).

Immunopharmacologic response of patients with B-lineage acute lymphoblastic leukemia to continuous infusion of T cell-engaging CD19/CD3-bispecific BiTE antibody blinatumomab.

T cell-engaging CD19/CD3-bispecific BiTE Ab blinatumomab has shown an 80% complete molecular response rate and prolonged leukemia-free survival in patients with minimal residual B-lineage acute lymphoblastic leukemia (MRD(+) B-ALL). Here, we report that lymphocytes in all patients of a phase 2 study responded to continuous infusion of blinatumomab in a strikingly similar fashion. After start of infusion, B-cell counts dropped to < 1 B cell/µL within an average of 2 days and remained essentially undetectable for the entire treatment period. By contrast, T-cell counts in all patients declined to a nadir within < 1 day and recovered to baseline within a few days. T cells then expanded and on average more than doubled over baseline within 2-3 weeks under continued infusion of blinatumomab. A significant percentage of reappearing CD8(+) and CD4(+) T cells newly expressed activation marker CD69. Shortly after start of infusion, a transient release of cytokines dominated by IL-10, IL-6, and IFN-γ was observed, which no longer occurred on start of a second treatment cycle. The response of lymphocytes in leukemic patients to continuous infusion of blinatumomab helps to better understand the mode of action of this and other globally T cell-engaging Abs. The trial is registered with www.clinicaltrials.gov identifier NCT00560794.

The PKC inhibitor AEB071 may be a therapeutic option for psoriasis.

PKC isoforms tau, alpha, and beta play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.

Pharmacokinetics, safety, and tolerability of R411, a dual alpha4beta1-alpha4beta7 integrin antagonist after oral administration at single and multiple once-daily ascending doses in healthy volunteers.

R411 is a dual alpha4beta1-alpha4beta7 integrin antagonist under development for the treatment of chronic asthma. The objective of this study was to investigate the pharmacokinetics and safety of R411 and its active metabolite, RO0270608, in humans. A 3-part phase I trial was conducted in 132 healthy volunteers: (1) 12 subjects received 200 mg R411 as a single oral dose or 100 mg RO0270608 as an intravenous infusion in a 1-sequence crossover design; (2) 7 groups of 10 subjects received 1 of 7 single oral doses of R411 (10-1200 mg) in a parallel, placebo-controlled, ascending adaptive dose design; and (3) 5 groups of 10 subjects each received repeated oral qd doses of R411 (50-900 mg) for up to 3 weeks in a parallel, placebo-controlled, ascending adaptive dose design. The absolute bioavailability of RO0270608 (mean +/- standard deviation) after oral administration of R411 was 27% +/- 4%, and the terminal half-life was 7.33 +/- 2.29 hours. After IV infusion of RO0270608, total clearance (mean +/- standard deviation) was 19.4 +/- 7.1 L/h, and the volume of distribution was 93.1 +/- 36.1 L. After single ascending oral doses of R411, area under the concentration-time curve from 0 to infinity of active metabolite RO0270608 increased proportionally from 150 to 1200 mg (P > .05). Following repeated administration, the oral clearance was independent of time. No drug accumulation was observed, and no safety concerns were revealed up to a dose of 900 mg after up to 3 weeks of treatment.

Contribution of CYP3A4, CYP2B6, and CYP2C9 isoforms to N-demethylation of ketamine in human liver microsomes.

Ketamine is a widely used drug for its anesthetic and analgesic properties; it is also considered as a drug of abuse, as many cases of ketamine illegal consumption were reported. Ketamine is N-demethylated by liver microsomal cytochrome P450 into norketamine. The identification of the enzymes responsible for ketamine metabolism is of great importance in clinical practice. In the present study, we investigated the metabolism of ketamine in human liver microsomes at clinically relevant concentrations. Liver to plasma concentration ratio of ketamine was taken into consideration. Pooled human liver microsomes and human lymphoblast-expressed P450 isoforms were used. N-demethylation of ketamine was correlated with nifedipine oxidase activity (CYP3A4-specific marker reaction), and it was also correlated with S-mephenytoin N-demethylase activity (CYP2B6-specific marker reaction). Orphenadrine, a specific inhibitor to CYP2B6, and ketoconazole, a specific inhibitor to CYP3A4, inhibited the N-demethylation of ketamine in human liver microsomes. In human lymphoblast-expressed P450, the activities of CYP2B6 were higher than those of CYP3A4 and CYP2C9 at three concentrations of ketamine, 0.005, 0.05, and 0.5 mM. When these results were extrapolated using the average relative content of these P450 isoforms in human liver, CYP3A4 was the major enzyme involved in ketamine N-demethylation. The present study demonstrates that CYP3A4 is the principal enzyme responsible for ketamine N-demethylation in human liver microsomes and that CYP2B6 and CYP2C9 have a minor contribution to ketamine N-demethylation at therapeutic concentrations of the drug.

Protein binding of ketamine and its active metabolites to human serum.

Ketamine is an anaesthetic agent extensively used in intensive care patients, and it has proved its efficacy in the management of burned patients. In these patients, alterations in serum protein binding occur that may have significant clinical implications. Scarce data were observed in the literature about the binding of ketamine to human plasma proteins, and no data about the binding of its active metabolites, norketamine (NK) and dehydronorketamine (DHNK) were found. In this study, protein binding of ketamine, NK and DHNK in human serum were determined using the ultrafiltration technique. The percentage of drug bound to serum proteins at 30 degrees C was found to be 69%, 60% and 50% for DHNK, ketamine and NK, respectively, while these percentages were 75%, 64% and 54% for DHNK, ketamine and NK respectively at 20 degrees C. The binding of ketamine and its metabolites was independent of drug concentration.