Nonclinical Pharmacokinetics Disposition, and Drug-Drug Interaction Potential of a Novel D-Amino ...

55 Pages·2016·6.34 MB·English

DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.115.068007
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Nonclinical Pharmacokinetics Disposition, and Drug-Drug
Interaction Potential of a Novel D-Amino Acid Peptide Agonist of
the Calcium Sensing Receptor AMG 416 (Etelcalcetide)

Raju Subramanian, Xiaochun Zhu, Savannah J. Kerr, Joel D. Esmay, Steven W. Louie,
Katheryne Z. Edson, Sarah Walter, Michael Fitzsimmons, Mylo Wagner, Marcus Soto, Roger
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Pham, Sarah F. Wilson, and Gary L. Skiles nlo
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Pharmacokinetics and Drug Metabolism, Amgen Inc., Thousand Oaks, California (R.S., X.Z, d
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S.J.K., J.D.E., S.W.L., K.Z.E, S.W., M.W., M.S., R.P., S.F.W, G.L.S); and Covance tjo
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Laboratories, Madison, Wisconsin (M.F.) .o
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DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.115.068007
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Running Title: Nonclinical Pharmacokinetics and Disposition of Etelcalcetide
Corresponding Author:
Raju Subramanian
Amgen Inc.
One Amgen Center Drive
Thousand Oaks, CA 91320
Phone: [phone]
Email: [email protected]

Text pages: 29 D
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Tables: 4 lo
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Figures: 8 d
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References: 41 d
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Word Count: 10, 315 (abstract to reference) .a
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Abstract: 246 tjo
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Introduction: 498 als
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Discussion: 1527 arg
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Nonstandard Abbreviations: AUC, area under the curve; ABT, 1-aminobenzotriazole; BCRP, J
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breast cancer resistance protein; BSEP, bile salt export pump; BDC, bile duct cannulated; BN, ls
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bilaterally nephrectomized; CID, collision-induced dissociation; CKD, chronic kidney disease; b
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CYP450s, cytochromes P450; DDI, drug-drug interaction; FA, formic acid; HPT, 12
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hyperparathyroidism; IS, internal standard (13C D 15N-AMG 416); IV, intravenous; JVC, jugular 3
3 3
vein cannulated; LC, liquid chromatography; LC-14C-HRMS, liquid chromatography-14C-high
resolution mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry;
β
LE, Long Evans; LSC, liquid scintillation counter; NADPH, reduced -nicotinamide adenine
dinucleotide phosphate; NCA, non-compartmental analysis; OAT, organic anion transporter;
OATP, organic anion transporter polypeptide; OCT, organic cation transporter; P, phosphorus;
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DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.115.068007
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Pgp, P-glycoprotein; PBS, phosphate buffered saline; PK, pharmacokinetics; PTH, parathyroid
hormone; QWBA, quantitative whole body autoradiography; RBC, red blood cell; SAPC, serum
albumin peptide conjugate; SD, Sprague-Dawley; SRM, selected reaction monitoring; TCEP,
tris(2-carboxyethyl phosphine); TFA, trifluoroacetic acid; Total M11, also referred to as TM11,
M11 liberated by the chemical reduction of all AMG 416-related homo- and hetero-
disulfides with an intact D-amino acid backbone; Vss, volume of distribution at steady state.
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DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.115.068007
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ABSTRACT
AMG 416 (Etelcalcetide) is a novel synthetic peptide agonist of the calcium-sensing receptor
composed of a linear chain of seven D-amino acids (referred to as the “D-amino acid backbone”)
with a D-cysteine linked to an L-cysteine via a disulfide bond. AMG 416 contains four basic D-
arginine residues and is a +4 charged peptide at physiological pH with a molecular weight of
1048.3 Da. The pharmacokinetics (PK), disposition and potential of AMG 416 to cause drug-
drug interaction were investigated in nonclinical studies with two single 14C-labels placed either
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at a potentially metabolically labile acetyl position or on the D-alanine next to D-cysteine in the nlo
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interior of the D-amino acid backbone. Following intravenous dosing, the PK and disposition of fro
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AMG 416 were similar in male and female rats. Radioactivity rapidly distributed to most tissues m
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in rats with intact kidneys and renal elimination was the predominant clearance pathway. No tjo
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strain-dependent differences were observed. In bilaterally nephrectomized rats, minimal ls
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radioactivity (1.2%) was excreted via non-renal pathways. Biotransformation occurred primarily t A
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via disulfide exchange with endogenous thiol-containing molecules in whole blood rather than J
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metabolism by enzymes such as proteases or CYP450s; the D-amino acid backbone remained ls
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unaltered. A substantial proportion of the plasma radioactivity was covalently conjugated to b
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albumin. AMG 416 presents a low risk for CYP450 or transporter-mediated drug-drug 1
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interactions because it showed no interactions in vitro. These studies demonstrated a 14C label on 3
either the acetyl or the D-alanine in the D-amino acid backbone would be appropriate for clinical
studies.
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DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.115.068007
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INTRODUCTION
Secondary hyperparathyroidism (HPT), a consequence of chronic kidney disease (CKD) in
humans, is a chronic and progressive disease characterized by elevated levels of parathyroid
hormone (PTH), disturbances in the homeostasis of calcium (Ca), phosphorus (P), vitamin D,
and fibroblast growth factor 23 (FGF-23) levels, and parathyroid gland hyperplasia (Rodriguez et
al., 2005; Tfelt-Hansen and Brown, 2005; Goodman and Quarles, 2008; Cunningham et al.,
2011). Left untreated, secondary HPT leads to disturbances in mineral metabolism, bone
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disease, cardiovascular complications, and increased mortality (Block et al., 2004; Moe et al., n
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2005). Treatment options include the calcimimetic, cinacalcet (Sensipar®/Mimpara®, Amgen Inc, d fro
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Thousand Oaks, CA), vitamin D, and phosphate binders (Nemeth et al., 1998; Block et al., m
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2004). etjo
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AMG 416 (etelcalcetide, Amgen Inc, Thousand Oaks, CA; Figure 1) is a novel calcimimetic .o
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currently in clinical development for the treatment of secondary HPT. Studies have shown that
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AMG 416 lowers parathyroid hormone (PTH) levels and normalizes mineral metabolism in Jo
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uremic animal models (Walter et al., 2013; Walter et al., 2014). AMG 416, administered ls o
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intravenously thrice weekly at the end of each hemodialysis session in CKD patients, has ru
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demonstrated PTH lowering effect and improved treatment adherence with reduced adverse 2
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events (Martin et al., 2014; Bell et al., 2015; Cozzolino et al., 2015).
AMG 416 is a novel synthetic peptide and is composed of a linear chain of seven D-amino
acids, referred herein as the “D-amino acid backbone” of the molecule, and an L-cysteine linked
to an N-terminal D-cysteine through a disulfide bond. The N-terminal D-cysteine and the C-
terminal arginine in AMG 416 are capped with acetyl and amide groups, respectively. AMG 416
contains four basic D-arginine residues and is a positively charged peptide (+4) at physiological
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DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.115.068007
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pH (7.4) with a molecular weight of 1048.3 Da. Given the structure of AMG 416, it lead us to
hypothesize that the molecule may be expected to be: (a) poorly permeable unless assisted by
active transport; (b) unlikely to interact with CYP450s because it bears little resemblance to their
known substrates; (c) resistant to protease mediated hydrolysis due to the presence of a D-amino
acid backbone; (d) biotransformed by disulfide exchange, specifically by endogenous thiols in
blood and this pathway will likely be a significant metabolic pathway for this molecule; and (e)
to be mainly cleared by renal elimination. However, AMG 416 is administered clinically to CKD
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patients with minimal kidney function and on maintenance hemodialysis. nlo
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There is no approved drug that consists primarily of D-amino acids and minimal literature is fro
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available on the fate of predominantly D-amino acid containing peptides. The objective of the d
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present study was to test the hypotheses stated above and overall, to characterize the nonclinical tjo
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pharmacokinetics and disposition of AMG 416 using in vitro methods and in vivo in rats with .o
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normal and no kidney function. Also, the potential of AMG 416 to cause CYP450 or transported S
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mediated drug-drug interactions was determined in vitro. The findings from these studies helped Jo
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in the selection of a radiolabel site in AMG 416 for clinical studies. o
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DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.115.068007
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MATERIALS AND METHODS
Test Articles and Reagents The structures and nomenclature for AMG 416 are shown in Figure
1. AMG 416 was synthesized by Bachem (Torrance, CA) and was stored in 0.1% trifluoroacetic
acid (TFA) in water at pH 4.0. Three lots of [14C]AMG 416, with each incorporating a single 14C
label, were used in the in vivo studies. Of those, two lots designated [14C]Ac-AMG 416,
incorporated 14C into the carbonyl carbon of the acetyl moiety. The first, [14C]Ac-AMG 416 lot
(specific activity 28.2 mCi/mmol; 95.1% radioactive purity) was synthesized by PolyPeptide
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Laboratories (San Diego, CA). The second, [14C]Ac-AMG 416 lot (specific activity 60.3 nlo
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mCi/mmol, 98.8% radioactive purity) was synthesized at Amgen (Thousand Oaks, CA). The fro
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third lot, designated [14C]Ala-AMG416 (specific activity 56.3 mCi/mmol; 97.0% radioactive m
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purity), contained the 14C-label on the carbonyl carbon of the D-alanine adjacent to D-cysteine etjo
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moiety and was synthesized by PolyPeptide Laboratories. The first and third lots of radiolabeled ls
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AMG 416 contained a single degradant (M5, a deamidation product of AMG 416) present at t A
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approximately 2% of the total 14C content. AMG 416 internal standard (IS; 13C3D315N-AMG T Jo
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416), M10, M11, and M12 were supplied by Amgen. (Waltham, MA). Tris(2-carboxyethyl ls
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phosphine) (TCEP) hydrochloride was obtained from Sigma-Aldrich (St. Louis, MO). Additional b
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information on other materials used in the study is provided in Supplemental Information. 1
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In Vitro Methods 3
Blood to Plasma Ratio and RBC Uptake Pooled whole blood from rat, healthy human
volunteers, and CKD patients was incubated with [14C]Ac-AMG 416 at three concentrations
(0.1, 1.0 and 10 µM) at 37 °C for 4 hr. An aliquot of blood (100 µL) was removed and the
remainder of the mixture was centrifuged at 14,000 × g for 5 min at 4 °C to isolate the plasma.
The radioactivity in blood and plasma aliquots was determined by 14C analysis.
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DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.115.068007
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Fresh human whole blood was initially diluted with one volume of cold RBC experiment
buffer (see supplemental materials) and gently mixed. Samples were centrifuged at 150 × g for 5
min at 5°C. The supernatants were then removed and replaced with one volume of fresh cold
RBC buffer. The previous step was repeated four times to obtain washed red blood cells. After
the final wash, the RBC pellet was suspended in an equal volume of the cold RBC experiment
buffer and used for direct uptake experiments. [14C]Ac-AMG 416 (10 µM) was incubated in the
washed RBC-buffer mixture at 37°C for 2 hr. After centrifugation, the supernatant was separated
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and the pelleted RBCs were lysed with cold ammonium-chloride-potassium (ACK) lysing buffer. lo
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The radioactivity of both supernatant and the lysate was determined by 14C analysis. fro
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Non-Covalent Plasma Protein Binding An ultra-filtration method was used to determine the d
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non-covalent protein binding of AMG 416, while minimizing its covalent binding to plasma tjo
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proteins. An ultra-filtration method was used for this purpose because the unbound fraction ls.o
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could be separated from the plasma within 12 min, while other methods, including equilibrium t A
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dialysis and ultra-centrifugation, require hours to obtain the unbound fraction. J
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Whole blood from rat, dog, healthy human volunteers and CKD patients was incubated with ls
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AMG 416 (50, 1000 or 10,000 ng/ml) on an orbital shaker at 100 rpm for 4 hr at 37 ºC in 5% b
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CO and 90% relative humidity and then immediately centrifuged at 5,250 × g for 10 min to 12
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obtain plasma. The plasma was divided into two aliquots. One aliquot of the plasma was
acidified with the addition of formic acid (FA; 1% final concentration, v/v) and subsequently
prepared and analyzed as plasma control representing the total starting concentration of AMG
416 in plasma. The other aliquot of plasma from whole blood incubations was placed in 30,000
Da cutoff ultra-filtration devices (500 µL each in quadruplicate) and centrifuged at 2000 × g in a
fixed angle rotor for 12 min. At the end of centrifugation, the residual filtered plasma and the
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DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.115.068007
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ultra-filtrate were added to FA to achieve 1% final acid (v/v). The AMG 416 in plasma and
filtrate was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
method described in supplemental methods.
CYP450 and Transporter Interactions The CYP450 metabolic stability of AMG 416 (5
µM) was determined in HLM and human liver S9 fractions (HLS9) at 1 mg/mL protein
concentration in pH 7.4 phosphate buffer (100 mM) with or without NADPH and with or without
1% FA at 37 °C. In addition, HLM and HLS9 were pre-incubated with 1-aminobenzotriazole
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(ABT; 5 mM) and NADPH to inactivate CYP450 enzymes prior to AMG 416 addition. nlo
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The reversible inhibition potential of AMG 416 (0.2, 1, and 5 µg/mL) toward human CYP450 fro
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isozymes 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, 2B6, and 3A4 was evaluated following a m
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published method (Walsky and Obach, 2004). The time dependent inhibition potential of AMG tjo
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416 (5 µM) toward human CYP450 isozymes 1A2, 2C8, 2C9, 2C19, 2D6 and 3A was evaluated ls
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following published methods (Atkinson et al., 2005; Obach et al., 2007). Induction of human t A
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CYP450 isozymes 1A2, 2B6, and 3A4 by AMG 416 was evaluated in cryopreserved primary J
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human hepatocytes following the previously published method (Hewitt et al., 2007). The ls
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hepatocytes were incubated with AMG 416 at 0.2, 1, and 5 µg/mL for the first day; higher b
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concentrations of 0.4, 2, and 10 µg/mL were used on the second and third days. 1
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The permeability and transporter assays measured vectorial transport or accumulation of 3
[14C]AMG 416 or the radiolabeled probe substrates in cell line monolayers using published
methods with slight modifications. Appropriate positive and negative controls were used in each
assessment. The transcellular permeability with LLC-PK1 and efflux transport by P-glycoprotein
(Pgp) and breast cancer resistance protein (BCRP) was assessed in MDR1-LLC-PK1, and
BCRP-MDCKII cell monolayers (Schinkel et al., 1995; Booth-Genthe et al., 2006). The [14C]
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DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.115.068007
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AMG 416 direct uptake assay was conducted in HEK293 cells transfected with organic anion
transporter (OAT) 1, OAT3, organic anion transporter polypeptide (OATP) 1B1, OATP1B3,
peptide transporter (PEPT) 1, PEPT2, or organic cation transporter (OCT) 2 transporter
(Yamazaki et al., 2005; Chu et al., 2007). The effect of AMG 416 on human bile salt export
pump (BSEP) transporter was evaluated following a published method (van Staden et al., 2012).
Whole Blood Incubation for Plasma Analysis Whole blood from rat, healthy human
volunteers, and CKD patients was incubated with AMG 416 (200 µM), [14C]Ac-AMG 416 (1,
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5,10, or 200 µM), or [14C]Ala-AMG 416 (200 µM) at 37 °C. Mixtures were shaken at 300 rpm nlo
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for 4 hr and then immediately centrifuged at 14,000 × g for 5 min at 4 °C. Plasma was fro
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transferred to a clean vial containing an appropriate amount of aqueous FA (final concentration, d
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1% FA v/v), and then stored at -80°C until further analysis. tjo
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S9 Fraction and Hepatocyte Incubations Rat and human liver and kidney S9 fraction (2 ls.o
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mg/mL final concentration) incubations were conducted in a final volume of 1 mL buffer (100 t A
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mM phosphate buffer with 3 mM MgCl , pH 7.4) with either [14C]Ac-AMG 416 or [14C]Ala- J
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AMG 416 (7.5 µM) and AMG 416 (2.5 µM) at 37 ºC. Reaction mixtures were terminated after 6 ls
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hr by the addition of FA (final concentration, 10% v/v). For the 0 hr incubations, 10 µM of the b
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corresponding 14C-labeled AMG 416 was added to 0.5 mL of S9 suspended buffer and 12
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immediately quenched. The quenched reaction mixture was vortex-mixed, centrifuged at 20,000
× g for 20 min at 4 °C and then stored immediately at -80°C until further analysis.
Pooled cryopreserved human hepatocytes (two separate lots) were suspended in 1 mL of
Krebs-Henseleit buffer at a final concentration of 2 million cells/mL. To these cell suspensions,
either [14C]Ac-AMG 416 or [14C]Ala-AMG 416 (10 µM) was added. Cell mixtures were
incubated at 37 ºC in a water bath shaken at 75 rpm in a humidified (95% relative humidity)
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