Dose-related differences in the pharmacodynamic and toxicologic response to a novel hyperglycosylated analog of recombinant human erythropoietin in Sprague-Dawley rats with similarly high hematocrit.

We recently reported results that erythropoiesis-stimulating agent (ESA)-related thrombotic toxicities in preclinical species were not solely dependent on a high hematocrit (HCT) but also associated with increased ESA dose level, dose frequency, and dosing duration. In this article, we conclude that sequelae of an increased magnitude of ESA-stimulated erythropoiesis potentially contributed to thrombosis in the highest ESA dose groups. The results were obtained from two investigative studies we conducted in Sprague-Dawley rats administered a low (no thrombotic toxicities) or high (with thrombotic toxicities) dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114), 3 times weekly for up to 9 days or for 1 month. Despite similarly increased HCT at both dose levels, animals in the high-dose group had an increased magnitude of erythropoiesis measured by spleen weights, splenic erythropoiesis, and circulating reticulocytes. Resulting prothrombotic risk factors identified predominantly or uniquely in the high-dose group were higher numbers of immature reticulocytes and nucleated red blood cells in circulation, severe functional iron deficiency, and increased intravascular destruction of iron-deficient reticulocyte/red blood cells. No thrombotic events were detected in rats dosed up to 9 days suggesting a sustained high HCT is a requisite cofactor for development of ESA-related thrombotic toxicities.

Cytokines associated with increased erythropoiesis in Sprague-Dawley rats administered a novel hyperglycosylated analog of recombinant human erythropoietin.

We previously reported an increased incidence of thrombotic toxicities in Sprague-Dawley rats administered the highest dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114) for 1 month as not solely dependent on high hematocrit (HCT). Thereafter, we identified increased erythropoiesis as a prothrombotic risk factor increased in the AMG 114 high-dose group with thrombotic toxicities, compared to a low-dose group with no toxicities but similar HCT. Here, we identified pleiotropic cytokines as prothrombotic factors associated with AMG 114 dose level. Before a high HCT was achieved, rats in the AMG 114 high, but not the low-dose group, had imbalanced hemostasis (increased von Willebrand factor and prothrombin time, decreased antithrombin III) coexistent with cytokines implicated in thrombosis: monocyte chemotactic protein 1 (MCP-1), MCP-3, tissue inhibitor of metalloproteinases 1, macrophage inhibitory protein-2, oncostatin M, T-cell-specific protein, stem cell factor, vascular endothelial growth factor, and interleukin-11. While no unique pathway to erythropoiesis stimulating agent-related thrombosis was identified, cytokines associated with increased erythropoiesis contributed to a prothrombotic intravascular environment in the AMG 114 high-dose group, but not in lower dose groups with a similar high HCT.

Pharmacokinetic and pharmacodynamic modeling of romiplostim in animals.

PURPOSE: Romiplostim is a novel thrombopoiesis-stimulating peptibody that targets the thrombopoietin c-Mpl receptor, resulting in increased platelet production. The pharmacodynamic-mediated disposition (PDMDD) and its stimulatory effect on platelet production in Sprague-Dawley rats, rhesus monkeys, and cynomolgus monkeys following IV bolus and SC administration at various dose levels were determined. METHODS: The pharmacokinetic (PK) profile was described by a PDMDD model that accounts for romiplostim binding to the c-Mpl receptor. The PD model contained a series of aging compartments for precursor cells in bone marrow and platelets. The stimulatory function was described by an on-and-off function operating on the fractional receptor occupancy (RO). The threshold effect, RO(thr), and K(D) parameters were determinants of drug potency, whereas S(max) reflected drug efficacy. RESULTS: The model implicated that receptor-mediated clearance was negligible. RO(thr) estimated occupancies were 0.288, 0.385, 0.771 for rats, rhesus, and cynomolgus monkeys, respectively. The analogous estimated values of K(D) were 4.05, 2320, and 429 ng/mL, implying that romiplostim was much more potent in rats, which was confirmed by a dose-response (ratio of peak platelet count to baseline) relationship. CONCLUSIONS: The model adequately described romiplostim serum concentrations and platelet counts in rats, rhesus monkeys, and cynomolgus monkeys, and quantified linear clearance, PDMDD, and potency of romiplostim.

Investigation of the pharmacokinetics of romiplostim in rodents with a focus on the clearance mechanism.

PURPOSE: Romiplostim, a treatment for adults with immune thrombocytopenia (ITP), is a novel thrombopoietin mimetic agent with a similar mechanism of action as thrombopoietin with no sequence homology. Structurally, it is a peptibody containing thrombopoietin mimetic peptides and the Fc portion of human IgG(1). We investigated romiplostim pharmacokinetics in rodents with a focus on the clearance mechanism. METHODS: Studies with appropriate controls were conducted in four models: FcRn knockout mice, thrombocytopenic mice, splenectomized rats, and bilateral nephrectomized rats. Catabolic breakdown of romiplostim was investigated in normal rats. The primary analytical method determines the intact/active romiplostim concentration, and the secondary method determines the sum of romiplostim and its catabolic degradants. RESULTS: FcRn interaction results in prolonged exposure. Platelets are involved in the target-mediated elimination, a saturable process and more prominent at low dose. Splenectomy does not affect the romiplostim pharmacokinetics in rats, an observation not unexpected. Nephrectomy in rats results in a greater increase of romiplostim exposure at a higher romiplostim dose, a nonlinearity likely due to saturation of competing pathway. Catabolism plays a major role in romiplostim elimination. CONCLUSION: Romiplostim clearance involves multiple mechanisms, including a nonlinear pathway. Consequently, the relative contribution of different mechanisms appears to be dose dependent.

Clinical Pharmacokinetics and Pharmacodynamics of Etelcalcetide, a Novel Calcimimetic for Treatment of Secondary Hyperparathyroidism in Patients With Chronic Kidney Disease on Hemodialysis.

Etelcalcetide, a d-amino acid peptide, is an intravenous calcimimetic approved for the treatment of secondary hyperparathyroidism. Etelcalcetide binds the calcium-sensing receptor and increases its sensitivity to extracellular calcium, thereby decreasing secretion of parathyroid hormone (PTH) by chief cells. Etelcalcetide and its low-molecular-weight transformation products are rapidly cleared by renal excretion in healthy subjects, but clearance is substantially reduced and dependent on hemodialysis in end-stage renal disease. The effective half-life is 3-5 days in patients undergoing hemodialysis 3 times a week. A clinical study using a single microtracer intravenous dose of [14 C]etelcalcetide indicated that 60% of the administered dose was eliminated in dialysate. Etelcalcetide undergoes reversible disulfide exchange with serum albumin to form a serum albumin peptide conjugate that is too large (67 kDa) to be dialyzed, until a subsequent exchange forms etelcalcetide or a low-molecular-weight transformation product. This exchange from albumin is apparent after hemodialysis, when it partially restores etelcalcetide concentrations in plasma. Etelcalcetide has no known risks for drug-drug interactions. In phase 3 studies, 74%-75% of hemodialysis patients with secondary hyperparathyroidism who received etelcalcetide achieved a >30% PTH reduction from baseline versus 8%-10% of patients who received placebo. The pharmacokinetics and pharmacodynamics of etelcalcetide in hemodialysis patients supports a 5-mg starting dose administered after hemodialysis and uptitration in 2.5- or 5-mg increments every 4 weeks to a maximum dose of 15 mg 3 times a week.

Clinical immunogenicity of the d-amino acid peptide therapeutic etelcalcetide: Method development challenges and anti-drug antibody clinical impact assessments.

The immunogenicity risk assessment and bioanalytical strategy for novel therapeutics should account for both unique biophysical properties and potential consequences of immunogenicity. When assessing the immunogenicity risk of etelcalcetide, a peptide agonist of the calcium-sensing receptor, we considered the potential that the d-amino acid 'backbone' and biotransformation of etelcalcetide could allow the drug to act as a hapten. As a consequence, we validated and implemented a surface plasmon resonance immunoassay platform with both etelcalcetide and etelcalcetide-'carrier' surfaces to detect anti-drug antibodies (ADA). No evidence of in-vitro neutralizing activity with surrogate controls was detected despite multiple immunization approaches and a sensitive cell-based activity assay. Therefore, a neutralizing assay was not implemented for clinical support. We conducted an integrated analysis of immunogenicity data pooled from two pivotal placebo-controlled trials to define the clinical impact of anti-etelcalcetide antibodies. While both pre-existing and developing anti-etelcalcetide antibodies were detected, we show here that they have no consequences for clinical exposure, efficacy, or safety of etelcalcetide.

Pharmacokinetics, Biotransformation, and Excretion of [14C]Etelcalcetide (AMG 416) Following a Single Microtracer Intravenous Dose in Patients with Chronic Kidney Disease on Hemodialysis.

Etelcalcetide (AMG 416) is a novel synthetic peptide calcium-sensing receptor activator in clinical development as an intravenous calcimimetic for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease (CKD) on hemodialysis. Etelcalcetide is composed of seven D-aminoacids with an L-cysteine linked to a D-cysteine by a disulfide bond. A single intravenous dose of [14C]etelcalcetide (10 mg; 26.3 kBq; 710 nCi) was administered to patients with CKD on hemodialysis to elucidate the pharmacokinetics, biotransformation, and excretion of etelcalcetide in this setting. Blood, dialysate, urine, and feces were collected to characterize the pharmacokinetics, biotransformation product profiles, mass balance, and formation of anti-etelcalcetide antibodies. Accelerator mass spectrometry was necessary to measure the microtracer quantities of C-14 excreted in the large volumes of dialysate and other biomatrices. An estimated 67 % of the [14C]etelcalcetide dose was recovered in dialysate, urine, and feces 176 days after dose administration. Etelcalcetide was primarily cleared by hemodialysis, with approximately 60 % of the administered dose eliminated in dialysate. Minor excretion was observed in urine and feces. Biotransformation resulted from disulfide exchange with endogenous thiols, and preserved the etelcalcetide D-amino acid backbone. Drug-related radioactivity circulated primarily as serum albumin peptide conjugate (SAPC). Following removal of plasma etelcalcetide by hemodialysis, re-equilibration occurred between SAPC and L-cysteine present in blood to partially restore the etelcalcetide plasma concentrations between dialysis sessions. No unanticipated safety signals or anti-etelcalcetide or anti-SAPC antibodies were detected.

Experimental and statistical approaches in method cross-validation to support pharmacokinetic decisions.

A case study of experimental and statistical approaches for cross-validating and examining the equivalence of two ligand binding assay (LBA) methods that were employed in pharmacokinetic (PK) studies is presented. The impact of changes in methodology based on the intended use of the methods was assessed. The cross-validation processes included an experimental plan, sample size selection, and statistical analysis with a predefined criterion of method equivalence. The two methods were deemed equivalent if the ratio of mean concentration fell within the 90% confidence interval (0.80-1.25). Statistical consideration of method imprecision was used to choose the number of incurred samples (collected from study animals) and conformance samples (spiked controls) for equivalence tests. The difference of log-transformed mean concentration and the 90% confidence interval for two methods were computed using analysis of variance. The mean concentration ratios of the two methods for the incurred and spiked conformance samples were 1.63 and 1.57, respectively. The 90% confidence limit was 1.55-1.72 for the incurred samples and 1.54-1.60 for the spiked conformance samples; therefore, the 90% confidence interval was not contained within the (0.80-1.25) equivalence interval. When the PK parameters of two studies using each of these two methods were compared, we determined that the therapeutic exposure, AUC((0-168)) and C(max), from Study A/Method 1 was approximately twice that of Study B/Method 2. We concluded that the two methods were not statistically equivalent and that the magnitude of the difference was reflected in the PK parameters in the studies using each method. This paper demonstrates the need for method cross-validation whenever there is a switch in bioanalytical methods, statistical approaches in designing the cross-validation experiments and assessing results, or interpretation of the impact of PK data.