Model-Based Characterization of the Pharmacokinetics, Target Engagement Biomarkers, and Immunomodulatory Activity of PF-06342674, a Humanized mAb Against IL-7 Receptor-α, in Adults with Type 1 Diabetes.

IL-7 receptor-α (IL-7Rα) blockade has been shown to reverse autoimmune diabetes in the non-obese diabetic mouse by promoting inhibition of effector T cells and consequently altering the balance of regulatory T (Treg) and effector memory (TEM) cells. PF-06342674 is a humanized monoclonal antibody that binds to and inhibits the function of IL-7Rα. In the current phase 1b study, subjects with type 1 diabetes (T1D) received subcutaneous doses of either placebo or PF-06342674 (1, 3, 8 mg/kg/q2w or 6 mg/kg/q1w) for 10 weeks and were followed up to 18 weeks. Nonlinear mixed effects models were developed to characterize the pharmacokinetics (PK), target engagement biomarkers, and immunomodulatory activity. PF-06342674 was estimated to have 20-fold more potent inhibitory effect on TEM cells relative to Treg cells resulting in a non-monotonic dose-response relationship for the Treg:TEM ratio, reaching maximum at ~ 3 mg/kg/q2w dose. Target-mediated elimination led to nonlinear PK with accelerated clearance at lower doses due to high affinity binding and rapid clearance of the drug-target complex. Doses ≥ 3 mg/kg q2w result in sustained PF-06342674 concentrations higher than the concentration of cellular IL-7 receptor and, in turn, maintain near maximal receptor occupancy over the dosing interval. The results provide important insight into the mechanism of IL-7Rα blockade and immunomodulatory activity of PF-06342674 and establish a rational framework for dose selection for subsequent clinical trials of PF-06342674. Furthermore, this analysis serves as an example of mechanistic modeling to support dose selection of a drug candidate in the early phases of development.

Molecular Characterization of Familial Hypercholesterolemia in a North American Cohort.

BACKGROUND: Familial hypercholesterolemia (FH) confers a very high risk of premature cardiovascular disease and is commonly caused by mutations in low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin/kexin type 9 (PCSK9) and very rarely in LDLR adaptor protein 1 (LDLRAP1) genes. OBJECTIVE: To determine the prevalence of pathogenic mutations in the LDLR, APOB, and PCSK9 in a cohort of subjects who met Simon Broome criteria for FH and compare the clinical characteristics of mutation-positive and mutation-negative subjects. METHODS: Ninety-three men and 107 women aged 19 to 80 years from lipid clinics in the United States and Canada participated. Demographic and historical data were collected, physical examination performed, and serum lipids/lipoproteins analyzed. Targeted sequencing analyses of LDLR and PCSK9 coding regions and exon 26 of APOB were performed followed by detection of LDLR deletions and duplications. RESULTS: Disease-causing LDLR and APOB variants were identified in 114 and 6 subjects, respectively. Of the 58 LDLR variants, 8 were novel mutations. Compared with mutation-positive subjects, mutation-negative subjects were older (mean 49 years vs 57 years, respectively) and had a higher proportion of African Americans (1% vs 12.5%), higher prevalence of hypertension (21% vs 46%), and higher serum triglycerides (median 86 mg/dL vs 122 mg/dL) levels. CONCLUSIONS: LDLR mutations were the most common cause of heterozygous FH in this North American cohort. A strikingly high proportion of FH subjects (40%) lacked mutations in known culprit genes. Identification of underlying genetic and environmental factors in mutation-negative patients is important to further our understanding of the metabolic basis of FH and other forms of severe hypercholesterolemia.

Immunomodulatory activity of humanized anti-IL-7R monoclonal antibody RN168 in subjects with type 1 diabetes.

BACKGROUND: The cytokine IL-7 is critical for T cell development and function. We performed a Phase Ib study in patients with type 1 diabetes (T1D) to evaluate how blockade of IL-7 would affect immune cells and relevant clinical responses. METHODS: Thirty-seven subjects with T1D received s.c. RN168, a monoclonal antibody that blocks the IL -7 receptor α (IL7Rα) in a dose-escalating study. RESULTS: Between 90% and 100% IL-7R occupancy and near-complete inhibition of pSTAT5 was observed at doses of RN168 1 mg/kg every other week (Q2wk) and greater. There was a significant decline in CD4+ and CD8+ effector and central memory T cells and CD4+ naive cells, but there were fewer effects on CD8+ naive T cells. The ratios of Tregs to CD4+ or CD8+ effector and central memory T cells versus baseline were increased. RNA sequencing analysis showed downmodulation of genes associated with activation, survival, and differentiation of T cells. Expression of the antiapoptotic protein Bcl-2 was reduced. The majority of treatment-emergent adverse events (TEAEs) were mild and not treatment related. Four subjects became anti-EBV IgG+ after RN168, and 2 had symptoms of active infection. The immunologic response to tetanus toxoid was preserved at doses of 1 and 3 mg/kg Q2wk but reduced at higher doses. CONCLUSIONS: This trial shows that, at dosages of 1-3 mg/kg, RN168 selectively inhibits the survival and activity of memory T cells while preserving naive T cells and Tregs. These immunologic effects may serve to eliminate pathologic T cells in autoimmune diseases. TRIAL REGISTRATION: NCT02038764. FUNDING: Pfizer Inc.

Single and multiple ascending-dose study of glucagon-receptor antagonist RN909 in type 2 diabetes: a phase 1, randomized, double-blind, placebo-controlled trial.

PURPOSE: This first-in-human study assessed safety, immunogenicity, pharmacokinetics, and pharmacodynamics of RN909, a monoclonal antibody antagonist of the glucagon receptor, in type 2 diabetes (T2DM) subjects. METHODS: This study enrolled 84 T2DM subjects receiving stable metformin regimens. Forty-four subjects were randomized to receive single escalating doses of RN909 (0.3 to 6 mg/kg subcutaneously (SC), or 1 mg/kg intravenously (IV)), or placebo; 40 subjects were randomized to receive multiple escalating doses (50 to 150 mg SC) or placebo every 4 weeks for 12 weeks. RESULTS: RN909 was well tolerated; treatment-related elevated liver function tests (LFTs) were observed in 4/33 (12.1%) and 5/32 (15.6%) subjects treated with single and multiple doses, respectively, versus 1/10 (10%) and 0 in the respective placebo groups. RN909 dose-normalized AUCinf increased more than dose-proportionally following single SC doses, and after multiple doses, accumulation ratios ranged from 1.3 to 3.4. The incidence of antidrug antibodies (ADA) was 33% after single doses and 50% after multiple doses. RN909 produced dose-dependent, durable fasting plasma glucose (FPG)-lowering at day 29 (mean change -20.6 to -97.5 mg/dL) and day 85 (mean change; -27.2 to -43.5 mg/dL) after single and multiple doses, respectively. HbA1c also was reduced after single (mean change -0.30% to -1.44%), and multiple doses (-0.83% to -1.56%). CONCLUSION: RN909 was well tolerated after single and multiple doses in T2DM subjects, with diarrhea and elevated LFTs the most frequent adverse events. The appearance of ADA did not affect pharmacokinetics or efficacy. Robust lowering of FPG and HbA1c was observed.

The effects of single- and multiple-dose administration of bococizumab (RN316/PF-04950615), a humanized IgG2Δa monoclonal antibody binding proprotein convertase subtilisin/kexin type 9, in hypercholesterolemic subjects treated with and without atorvastatin: Results from four phase I studies.

AIMS: Three single-dose and one multiple-dose phase I studies were conducted in subjects with primary hypercholesterolemia to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of bococizumab, a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. METHODS: The dosing schedules for hypercholesterolemic subjects randomized in the four phase I studies were (1) ascending, single, intravenous (IV) bococizumab (0.3, 1, 3, 6, 12, or 18 mg/kg), or placebo (N = 48; baseline low-density lipoprotein cholesterol [LDL-C] ≥130 mg/dL); (2) single, IV bococizumab (0.5 or 4 mg/kg; no placebo) added to ongoing atorvastatin 40 mg/day (N = 24); (3) single, fixed, subcutaneous (SC) bococizumab (100 or 200 mg), or IV bococizumab (200 mg; no placebo; N = 49; baseline LDL-C ≥130 mg/dL); and (4) weekly IV bococizumab (0.25, 0.5, 1, or 1.5 mg/kg) or placebo for 4 weeks (N = 67; baseline LDL-C ≥130 mg/dL). RESULTS: Bococizumab pharmacokinetics were well characterized following single IV or SC doses and following multiple IV doses. Exposure to single-dose bococizumab increased slightly greater than dose-proportionally and clearance decreased with increasing dose. In the single-dose studies, maximal mean percent reductions from baseline in LDL-C ranged from 43% (0.3 mg/kg) to 84% (18 mg/kg) in bococizumab-treated subjects, compared with 2% for placebo. For the multiple-dose study, maximal reductions in LDL-C ranged from 55% (0.25 mg/kg) to 66% (1 mg/kg) in bococizumab-treated subjects, compared with 9% for placebo. In all studies, adverse events were infrequent, transient, and not dose-related. CONCLUSIONS: Bococizumab was generally safe and well tolerated. Bococizumab lowered LDL-C levels substantially in all four studies.

Effects of 12 weeks of treatment with intravenously administered bococizumab, a humanized monoclonal antibody blocking proprotein convertase subtilisin/kexin type 9, in hypercholesterolemic subjects on high-dose statin.

AIMS: Two multiple-dose phase II studies were conducted in subjects with primary hypercholesterolemia to evaluate the LDL-C lowering efficacy, safety, and tolerability of bococizumab, a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. METHODS: The results from the two phase II, double-blinded, randomized, placebo-controlled, multicenter studies conducted in the USA and Canada were combined. In Study 1, 90 subjects with LDL-C ≥100 mg/dL received intravenous (IV) placebo or bococizumab 0.25, 1, 3, or 6 mg/kg. In Study 2, 45 subjects with LDL-C ≥80 mg/dL received IV placebo or bococizumab 1 or 3 mg/kg. Subjects were treated every 4 weeks for 12 weeks. Dosing was interrupted if LDL-C dipped to ≤25 mg/dL and resumed if LDL-C returned to ≥40 mg/dL. The primary endpoint was percent LDL-C reduction from baseline at Week 12. RESULTS: At Week 12, the reductions from baseline in LDL-C vs placebo in the bococizumab 0.25, 1, 3, and 6 mg/kg groups were 9.3%, 10.2%, 41.6%, and 52.0%, respectively (P < .001 vs placebo for all). LDL-C reductions were greater (69.9%) in subjects who received all three doses of bococizumab 6 mg/kg (P < .001 vs placebo). Pharmacogenomic analysis revealed that 15% of hyperlipidemic subjects carried polymorphisms associated with familial hypercholesterolemia, with maximal LDL-C reductions being similar between carriers and noncarriers. Adverse events were mild, unrelated to bococizumab, and resolved by Week 12. CONCLUSIONS: These studies demonstrated that bococizumab safely and effectively lowered LDL-C in hypercholesterolemic subjects on high doses of statin.

Effects of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibition with Bococizumab on Lipoprotein Particles in Hypercholesterolemic Subjects.

PURPOSE: Monoclonal antibody inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) elicit significant reductions in serum LDL-C levels. However, little is known about their effects on lipoprotein particles. The purpose of this analysis was to evaluate the effect of PCSK9 inhibition with bococizumab (RN316/PF-04950615), a humanized monoclonal antibody to PCSK9, on LDL, VLDL, and HDL particle concentration and size in hypercholesterolemic subjects. METHODS: Data from 3 double-blind, placebo-controlled, randomized studies were analyzed. In study 1, a total of 67 hypercholesterolemic subjects received IV placebo or bococizumab 0.25, 0.5, 1, or 1.5 mg/kg weekly for 4 weeks. In studies 2 and 3, a total of 135 hypercholesterolemic subjects taking statins received IV placebo or bococizumab 0.25, 1, 3, or 6 mg/kg monthly for 12 weeks. Lipoprotein particle concentration and size were measured by using nuclear magnetic resonance spectroscopy. FINDINGS: Overall, the majority of subjects were men (51.9%) aged >50 years of age and of white ethnic origin. In total, 189 subjects with both baseline and 2-week posttreatment data were included in the analysis. After PCSK9 inhibition with bococizumab 0.5, 1, 1.5, 3, and 6 mg/kg, concentrations of total LDL, total small LDL, and small VLDL particles decreased significantly versus baseline and placebo (P < 0.05), whereas concentrations of HDL particles increased (P < 0.05). The size of the LDL, VLDL, and HDL particles increased after PCSK9 inhibition. Reductions in LDL-C and total LDL particle concentrations were highly correlated. IMPLICATIONS: The effect of inhibiting PCSK9 with bococizumab on lipoprotein particle concentration and size are consistent with the general mechanism of PCSK9 inhibitors in blocking PCSK9-mediated downregulation of LDL receptors. PCSK9 inhibition has the potential to provide a clinical benefit through the modulation of atherogenic lipoprotein particles in addition to LDL-C lowering, and this effect will likely be assessed in future analyses of data from cardiovascular outcomes trials of PCSK9 monoclonal antibodies that are currently being conducted. ClinicalTrials.gov identifiers: NCT01243151, NCT01342211, and NCT01350141.

A Mechanism-Based Pharmacokinetic/Pharmacodynamic Model for Bococizumab, a Humanized Monoclonal Antibody Against Proprotein Convertase Subtilisin/Kexin Type 9, and Its Application in Early Clinical Development.

Bococizumab (RN316/PF-04950615), a humanized monoclonal antibody, binds to secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) and prevents its downregulation of low-density lipoprotein receptor, leading to improved clearance and reduction of low-density lipoprotein cholesterol (LDL-C) in plasma. A mechanism-based drug-target binding model was developed, accounting for bococizumab, PCSK9, and LDL-C concentrations and the effects of concomitant administration of statins. This model was utilized to better understand the pharmacokinetic/pharmacodynamic (PK/PD) data obtained from 3 phase 1 and 2 phase 2a clinical studies. First, simulations performed with this model demonstrated that the conventional method of the area-under-the-curve ratio for bioavailability determination underestimated the subcutaneous bioavailability of bococizumab due to its target-mediated disposition. Second, a covariate model component for statin effects on bococizumab PK/PD was characterized, including a description of the decreased baseline LDL-C, increased baseline PCSK9, and increased LDL-C lowering with concomitant use of statins. Last, the impact of the dosing regimens with and without a dose holiday on bococizumab's LDL-C-lowering effectiveness was shown to be predictable due to the well-characterized PK-PD relationship.

Safety and tolerability of LBR-101, a humanized monoclonal antibody that blocks the binding of CGRP to its receptor: Results of the Phase 1 program.

BACKGROUND: LBR-101 is a fully humanized monoclonal antibody that binds to calcitonin gene-related peptide. OBJECTIVE: The objective of this article is to characterize the safety and tolerability of LBR-101 when administered intravenously to healthy volunteers, by presenting the pooled results of the Phase 1 program. METHODS: LBR-101 was administered to 94 subjects, while 45 received placebo. Doses ranged from 0.2 mg to 2000 mg given once (Day 1), as a single IV infusion, or up to 300 mg given twice (Day 1 and Day 14). RESULTS: Subjects receiving placebo reported an average of 1.3 treatment-emerging adverse events vs 1.4 per subject among those receiving any dose of LBR-101, and 1.6 in those receiving 1000 mg or higher. Treatment-related adverse events occurred in 21.2% of subjects receiving LBR-101, compared to 17.7% in those receiving placebo. LBR-101 was not associated with any clinically relevant patterns of change in vital signs, ECG parameters, or laboratory findings. The only serious adverse event consisted of "thoracic aortic aneurysm" in a participant later found to have an unreported history of Ehlers-Danlos syndrome. CONCLUSION: Single IV doses of LBR-101 ranging from 0.2 mg up to 2000 mg and multiple IV doses up to 300 mg were well tolerated. Overt safety concerns have not emerged. A maximally tolerated dose has not been identified.

Pharmacokinetic Profile of Orally Administered Scyllo-Inositol (Elnd005) in Plasma, Cerebrospinal Fluid and Brain, and Corresponding Effect on Amyloid-Beta in Healthy Subjects.

ELND005 (scyllo-inositol), an endogenous inositol stereoisomer, is being investigated as an oral treatment for Alzheimer's disease (AD). Pharmacokinetics of ELND005 in plasma, cerebrospinal fluid (CSF), and brain was characterized in healthy young subjects. Eight men received 2000 mg ELND005 every 12 hours for 10 days. Plasma and CSF samples were collected at predetermined time points; ELND005 and amyloid-beta (Aß) fragments were measured by validated bioanalytical methods. Brain ELND005 levels, estimated by (1) H Magnetic Resonance Spectroscopy (MRS) scans were obtained from gray/white matter voxels at baseline and Day 8. ELND005 was well-tolerated during the study. During the apparent steady state, ELND005 plasma levels rapidly peaked at 39.8 µg/mL and decreased to an average trough concentration of 10.6 µg/mL at the end of the 12-hour dosing regimen. In contrast, CSF drug levels slowly peaked at 13.7 µg/mL and remained near the same level with average trough concentrations of 12.4 µg/mL. At Day 8, Brain ELND005 concentrations increased by 58-76% compared to baseline levels. The CSF concentrations achieved in this study were similar to those associated with efficacy in transgenic models of AD. No changes were detected in plasma and CSF levels of Aß fragments.

Increasing serum half-life and extending cholesterol lowering in vivo by engineering antibody with pH-sensitive binding to PCSK9.

Target-mediated clearance and high antigen load can hamper the efficacy and dosage of many antibodies. We show for the first time that the mouse, cynomolgus, and human cross-reactive, antagonistic anti-proprotein convertase substilisin kexin type 9 (PCSK9) antibodies J10 and the affinity-matured and humanized J16 exhibit target-mediated clearance, resulting in dose-dependent pharmacokinetic profiles. These antibodies prevent the degradation of low density lipoprotein receptor, thus lowering serum levels of LDL-cholesterol and potently reducing serum cholesterol in mice, and selectively reduce LDL-cholesterol in cynomolgus monkeys. In order to increase the pharmacokinetic and efficacy of this promising therapeutic for hypercholesterolemia, we engineered pH-sensitive binding to mouse, cynomolgus, and human PCSK9 into J16, resulting in J17. This antibody shows prolonged half-life and increased duration of cholesterol lowering in two species in vivo by binding to endogenous PCSK9 in mice and cynomolgus monkeys, respectively. The proposed mechanism of this pH-sensitive antibody is that it binds with high affinity to PCSK9 in the plasma at pH 7.4, whereas the antibody-antigen complex dissociates at the endosomal pH of 5.5-6.0 in order to escape from target-mediated degradation. Additionally, this enables the antibody to bind to another PCSK9 and therefore increase the antigen-binding cycles. Furthermore, we show that this effect is dependent on the neonatal Fc receptor, which rescues the dissociated antibody in the endosome from degradation. Engineered pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load.

Enhancement of the efficacy of an antagonist of an extracellular receptor by attachment to the surface of a biocompatible carrier.

PURPOSE: In order to improve the in vitro and in vivo efficacy of an integrin antagonist (IA) of the extracellular domain of the alphavbeta3 integrin, a receptor upregulated on tumor neovasculature, the IA was attached to the surface of a dextran-coated liposome (DCL). IA-DCLs were characterized in vitro, and the pharmacokinetic and antitumor properties were assessed in vivo. METHODS: The in vitro binding properties were measured with purified integrin, endothelial cells, and melanoma cells. The pharmacokinetic parameters were measured in healthy mice with 14C-labeled IA-DCLs and anti-tumor efficacy was assessed with the M21 human melanoma xenograft mouse model. RESULTS: In vitro, IC50 values for IA-DCLs and IA are similar, and IA-DCLs inhibit cell proliferation relative to controls. IA-DCLs are stable in serum, and the pharmacokinetic half-life in mice is 23 h. In the M21/mouse model, statistically significant inhibition of tumor growth was observed for mice treated with IA-DCLs, whereas controls including saline, DCLs lacking IA, and cyclo(RGDfV) were ineffective. Increased apoptosis and a reduction in vessel counts relative to controls were present in tumors from animals treated with IA-DCLs. CONCLUSIONS: These results demonstrate that IA-DCLs are potent anti-angiogenic therapeutic agents with superior in vivo activity and pharmacology compared to unmodified IA.