Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2016/02/19/dmd.115.068007.DC1

1521-009X/44/8/1319–1331$25.00 http://dx.doi.org/10.1124/dmd.115.068007 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:1319–1331, August 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Special Section on Emerging Novel Enzyme Pathways in Drug Metabolism

Nonclinical Pharmacokinetics, Disposition, and Drug-Drug Interaction Potential of a Novel D-Amino Acid Peptide Agonist of the Calcium-Sensing Receptor AMG 416 (Etelcalcetide) s

Raju Subramanian, Xiaochun Zhu, Savannah J. Kerr,1 Joel D. Esmay, Steven W. Louie, Katheryne Z. Edson, Sarah Walter,2 Michael Fitzsimmons, Mylo Wagner, Marcus Soto, Roger Pham, Sarah F. Wilson, and Gary L. Skiles Downloaded from Pharmacokinetics and Drug Metabolism, Amgen Inc., Thousand Oaks, California (R.S., X.Z, 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 Laboratories, Madison, Wisconsin (M.F.)

Received October 20, 2015; accepted February 8, 2016

ABSTRACT dmd.aspetjournals.org AMG 416 (etelcalcetide) is a novel synthetic peptide agonist of the elimination was the predominant clearance pathway. No strain- calcium-sensing receptor composed of a linear chain of seven dependent differences were observed. In bilaterally nephrectomized D-amino acids (referred to as the D-amino acid backbone) with a rats, minimal radioactivity (1.2%) was excreted via nonrenal path- D-cysteine linked to an L-cysteine via a disulfide bond. AMG 416 ways. Biotransformation occurred primarily via disulfide exchange contains four basic D-arginine residues and is a +4 charged peptide at with endogenous thiol-containing molecules in whole blood rather physiologic pH with a mol. wt. of 1048.3 Da. The pharmacokinetics than metabolism by enzymes, such as proteases or cytochrome at ASPET Journals on September 30, 2021 (PK), disposition, and potential of AMG 416 to cause drug-drug P450s; the D-amino acid backbone remained unaltered. A substantial interaction were investigated in nonclinical studies with two single proportion of the plasma radioactivity was covalently conjugated to 14C-labels placed either at a potentially metabolically labile acetyl albumin. AMG 416 presents a low risk for P450 or transporter- position or on the D-alanine next to D-cysteine in the interior of the mediated drug-drug interactions because it showed no interactions 14 D-amino acid backbone. After i.v. dosing, the PK and disposition of in vitro. These studies demonstrated a C label on either the acetyl or AMG 416 were similar in male and female rats. Radioactivity rapidly the D-alanine in the D-amino acid backbone would be appropriate for distributed to most tissues in rats with intact kidneys, and renal clinical studies.

Introduction and fibroblast growth factor 23 (FGF-23) levels and parathyroid gland Secondary hyperparathyroidism (HPT), a consequence of chronic hyperplasia (Rodriguez et al., 2005; Tfelt-Hansen and Brown, 2005; kidney disease (CKD) in humans, is a chronic and progressive dis- Goodman and Quarles, 2008; Cunningham et al., 2011). Left untreated, ease characterized by elevated levels of parathyroid hormone (PTH), secondary HPT leads to disturbances in mineral metabolism, bone disease, disturbances in the homeostasis of calcium, phosphorus, vitamin D, cardiovascular complications, and increased mortality (Block et al., 2004; Moe et al., 2005). Treatment options include the calcimimetic, cinacalcet (Sensipar/Mimpara, Amgen Inc, Thousand Oaks, CA), vitamin D, and phosphate binders (Nemeth et al., 1998; Block et al., 2004). This study was funded by Amgen Inc., Thousand Oaks, California. AMG 416 (etelcalcetide, Amgen Inc, Thousand Oaks, CA) (Fig. 1) is a 1Current affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington. novel calcimimetic currently in clinical development for the treatment of 2Current affiliation: Antiva Biosciences, South San Francisco, California. secondary HPT. Studies have shown that AMG 416 lowers PTH levels and dx.doi.org/10.1124/dmd.115.068007. normalizes mineral metabolism in uremic animal models (Walter et al., s This article has supplemental material available at dmd.aspetjournals.org. 2013, 2014). AMG 416, administered i.v. three times weekly at the end of

ABBREVIATIONS: AUC, area under the curve; BCRP, breast cancer resistance protein; BSEP, bile salt export pump; BN, bilaterally 13 15 nephrectomized; CKD, chronic kidney disease; FA, formic acid; HPT, hyperparathyroidism; IS, internal standard ( C3D3 N-AMG 416); LC, liquid chromatography; LC-14C-HRMS, liquid chromatography-14C-high resolution mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LE, Long-Evans; OAT, organic anion transporter; OATP, organic anion transporter polypeptide; OCT, organic cation transporter; P450, cytochrome P450; Pgp, P-glycoprotein; PK, pharmacokinetics; PTH, parathyroid hormone; QWBA, quantitative whole-body autoradiography; RBC, red blood cell; SAPC, serum albumin peptide conjugate; SD, Sprague-Dawley; TCEP, tris(2-carboxyethyl phosphine); TFA, trifluoroacetic acid;

Vss, volume of distribution at steady state. 1319 1320 Subramanian et al.

Additional information on other materials used in the study is provided in Supplemental Material.

In Vitro Methods Blood-to-Plasma Ratio and Red Blood Cell Uptake. Pooled whole blood Fig. 1. Structure of AMG 416 (etelcalcetide). Ac, the acetyl group on the from rats, healthy human volunteers, and CKD patients was incubated with N terminus of the D-cysteine; NH2, denotes amidation on the C terminus of the 14 14 [ C]Ac-AMG 416 at three concentrations (0.1, 1.0, and 10 mM) at 37C for D-arginine. C-label was placed on the carbonyl carbon on either the acetyl moiety. 14 14 m ([ C]Ac-AMG 416,denoted by *) or the D-alanine moiety ([ C]Ala-AMG 416, 4 hours. An aliquot of blood (100 l) was removed, and the remainder of the denoted by #). mixture was centrifuged at 14,000g for 5 minutes at 4C to isolate the plasma. The radioactivity in blood and plasma aliquots was determined by 14C analysis. Fresh human whole blood was initially diluted with one volume of cold red each hemodialysis session in CKD patients, has demonstrated PTH- blood cell (RBC) experiment buffer (see Supplemental Material) and gently lowering effect and improved treatment adherence with reduced adverse mixed. Samples were centrifuged at 150g for 5 minutes at 5 C. The supernatants were then removed and replaced with one volume of fresh cold RBC buffer. The events (Martin et al., 2014; Bell et al., 2015; Cozzolino et al., 2015). previous step was repeated four times to obtain washed RBSs. After the final AMG 416 is a novel synthetic peptide and is composed of a linear wash, the RBC pellet was suspended in an equal volume of the cold RBC “ chain of seven D-amino acids, referred herein as the D-amino acid experiment buffer and used for direct uptake experiments. [14C]Ac-AMG 416 backbone” of the molecule, and an L-cysteine linked to an N-terminal (10 mM) was incubated in the washed RBC-buffer mixture at 37C for 2 hours. D-cysteine through a disulfide bond. The N-terminal D-cysteine and the After centrifugation, the supernatant was separated, and the pelleted RBCs were Downloaded from C-terminal arginine in AMG 416 are capped with acetyl and amide lysed with cold ammonium-chloride-potassium (ACK) lysing buffer. The 14 groups, respectively. AMG 416 contains four basic D-arginine residues radioactivity of both supernatant and the lysate was determined by C analysis. and is a positively charged peptide (+4) at physiologic pH (7.4) with Noncovalent Plasma Protein Binding. An ultrafiltration method was used a mol. wt. of 1048.3 Da. Given the structure of AMG 416, it led us to to determine the noncovalent protein binding of AMG 416 while minimizing its hypothesize that the molecule may be expected to be: 1) poorly covalent binding to plasma proteins. An ultrafiltration method was used for this permeable unless assisted by active transport; 2) unlikely to interact purpose because the unbound fraction could be separated from the plasma within dmd.aspetjournals.org with cytochrome P450s (P450s) because it bears little resemblance to 12 minutes, whereas other methods, including equilibrium dialysis and their known substrates; 3) resistant to protease-mediated hydrolysis ultracentrifugation, require hours to obtain the unbound fraction. Whole blood from rats, dogs, healthy human volunteers, and CKD patients owing to the presence of a D-amino acid backbone; 4) biotransformed was incubated with AMG 416 (50, 1000 or 10,000 ng/ml) on an orbital shaker at by disulfide exchange, specifically by endogenous thiols in blood, and 100 rpm for 4 hours at 37Cin5%CO2 and 90% relative humidity and then this pathway will likely be a significant metabolic pathway for this immediately centrifuged at 5250g for 10 minutes to obtain plasma. The plasma molecule; and 5) mainly cleared by renal elimination. However, AMG was divided into two aliquots. One aliquot of the plasma was acidified with the

416 is administered clinically to CKD patients with minimal kidney addition of formic acid (FA; 1% final concentration, v/v) and subsequently at ASPET Journals on September 30, 2021 function and on maintenance hemodialysis. prepared and analyzed as plasma control representing the total starting There is no approved drug that consists primarily of D-amino acids and concentration of AMG 416 in plasma. The other aliquot of plasma from whole minimal literature is available on the fate of predominantly D-amino acid– blood incubations was placed in 30,000 Da cutoff ultrafiltration devices (500 ml containing peptides. The objective of the present study was to test the each in quadruplicate) and centrifuged at 2000g in a fixed angle rotor for hypotheses stated above and overall, to characterize the nonclinical 12 minutes. At the end of centrifugation, the residual filtered plasma and the pharmacokinetics and disposition of AMG 416 using in vitro methods and ultrafiltrate 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 in vivo in rats with normal and no kidney function. Also, the potential of spectrometry (LC-MS/MS) method described in Supplemental Material. AMG 416 to cause P450 or transported mediated drug-drug interactions P450 and Transporter Interactions. The P450 metabolic stability of AMG was determined in vitro. The findings from these studies helped in the 416 (5 mM) was determined in HLM and human liver S9 fractions (HLS9) at selection of a radiolabel site in AMG 416 for clinical studies. 1 mg/ml protein concentration in pH 7.4 phosphate buffer (100 mM) with or without NADPH and with or without 1% FA at 37C. In addition, HLM and HLS9 were preincubated with 1-aminobenzotriazole (5 mM) and NADPH to Materials and Methods inactivate P450 enzymes before the addition of AMG 416. The reversible inhibition potential of AMG 416 (0.2, 1, and 5 mg/ml) toward Test Articles and Reagents human P450 isozymes 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, 2B6, and 3A4 was The structures and nomenclature for AMG 416 are shown in Fig. 1. AMG evaluated following a published method (Walsky and Obach, 2004). The time- 416 was synthesized by Bachem (Torrance, CA) and stored in 0.1% dependent inhibition potential of AMG 416 (5 mM) toward human P450 isozymes trifluoroacetic acid (TFA) in water at pH 4.0. Three lots of [14C]AMG 416, 1A2, 2C8, 2C9, 2C19, 2D6, and 3A was evaluated according to published methods each incorporating a single 14C label, were used in the in vivo studies. Of (Atkinson et al., 2005; Obach et al., 2007). Induction of human P450 isozymes those, two lots designated [14C]Ac-AMG 416, incorporated 14Cintothe 1A2, 2B6, and 3A4 by AMG 416 was evaluated in cryopreserved primary human carbonyl carbon of the acetyl moiety. The first, [14C]Ac-AMG 416 lot hepatocytes following a previously published method (Hewitt et al., 2007). The (specific activity 28.2 mCi/mmol; 95.1% radioactive purity), was synthesized hepatocytes were incubated with AMG 416 at 0.2, 1, and 5 mg/ml for the first day; byPolyPeptideLaboratories(San Diego, CA). The second, [14C]Ac-AMG 416 higher concentrations of 0.4, 2, and 10 mg/ml were used on the second and third days. lot (specific activity 60.3 mCi/mmol, 98.8% radioactive purity), was The permeability and transporter assays measured vectorial transport or ac- synthesized at Amgen (Thousand Oaks, CA). The third lot, designated cumulation of [14C]AMG 416 or the radiolabeled probe substrates in cell-line [14C]Ala-AMG416 (specific activity 56.3 mCi/mmol; 97.0% radioactive purity), monolayers using published methods with slight modifications. Appropriate 14 contained the C-label on the carbonyl carbon of the D-alanine adjacent to positive and negative controls were used in each assessment. The transcellular D-cysteine moiety and was synthesized by PolyPeptide Laboratories. The first permeability with LLC-PK1 and efflux transport by P-glycoprotein (Pgp) and and third lots of radiolabeled AMG 416 contained a single degradant (M5, a breast cancer resistance protein (BCRP) was assessed in MDR1-LLC-PK1 and deamidation product of AMG 416) present at approximately 2% of the total 14C BCRP-MDCKII cell monolayers (Schinkel et al., 1995; Booth-Genthe et al., 13 15 14 content. AMG 416 internal standard (IS; C3D3 N-AMG 416), M10, M11, and 2006). The [ C] AMG 416 direct uptake assay was conducted in HEK293 cells M12 were supplied by Amgen. (Waltham, MA). Tris(2-carboxyethyl phosphine) transfected with organic anion transporter (OAT) 1, OAT3, OAT polypeptide (TCEP) hydrochloride was obtained from Sigma-Aldrich (St. Louis, MO). (OATP) 1B1, OATP1B3, peptide transporter 1, 2, or organic cation transporter Nonclinical Pharmacokinetics and Disposition of Etelcalcetide 1321

(OCT) 2 transporter (Yamazaki et al., 2005; Chu et al., 2007). The effect of AMG In Vivo Methods 416 on human bile salt export pump (BSEP) transporter was evaluated according Study Design. The study overview is outlined in Table 1, and the individual to a published method (van Staden et al., 2012). experiment details are provided as follows under the respective group identifier. Whole-Blood Incubation for Plasma Analysis. Whole blood from rats, All animals were acclimated to study conditions for 4 to 5 days before dosing. healthy human volunteers, and CKD patients was incubated with AMG 416 During acclimation and the test periods, animals were housed in individual, (200 mM), [14C]Ac-AMG 416 (1, 5,10, or 200 mM), or [14C]Ala-AMG 416 suspended, stainless steel wire-mesh cages. All studies were performed in 7- to (200 mM) at 37C. Mixtures were shaken at 300 rpm for 4 hours and then 12-week old male and female Sprague-Dawley (SD) rats except group 11 with immediately centrifuged at 14,000g for 5 minutes at 4C. Plasma was male Long-Evans (LE) rats, weighing 190–350 g, which were obtained from transferred to a clean vial containing an appropriate amount of aqueous FA Charles River Laboratories (Hollister, CA) and were cared for in accordance to (final concentration, 1% FA v/v), and then stored at 280C until further the Guide for the Care and Use of Laboratory Animals, 8th edition (National analysis. Research Council, 2011). Animals were single-housed at an Association for S9 Fraction and Hepatocyte Incubations. Rat and human liver and kidney Assessment and Accreditation of Laboratory Animal Care, international S9 fraction (2 mg/ml final concentration) incubations were conducted in a final accredited facility, and all research protocols were approved by the Institutional volume of 1 ml of buffer (100 mM phosphate buffer with 3 mM MgCl , pH 7.4) 2 Animal Care and Use Committee. All animals were maintained on a 12-hour with either [14C]Ac-AMG 416 or [14C]Ala-AMG 416 (7.5 mM) and AMG 416 light/dark cycle in rooms at 18–26C and 10%–70% humidity. Animals in all (2.5 mM) at 37C. Reaction mixtures were terminated after 6 hours by the groups had access to water ad libitum for the duration of each study. Bilaterally addition of FA (final concentration, 10% v/v). For the 0-hour incubations, 10 mM nephrectomized (BN) rats received sugar cubes as food, and animals in all the of the corresponding 14C-labeled AMG 416 was added to 0.5 ml of S9 suspended remaining groups received the Harlan Laboratory diet (no. 2020X or no. 2016C). buffer and immediately quenched. The quenched reaction mixture was vortex- Downloaded from All radioanalysis data were generated with Debra (versions 5.7 or 6.0; LabLogic mixed, centrifuged at 20,000g for 20 minutes at 4C, and then stored immediately Systems Ltd., Sheffield, UK). All studies were conducted at Amgen except the at 280C until further analysis. quantitative whole-body autoradiography (QWBA; groups 11–13), which were Pooled cryopreserved human hepatocytes (two separate lots) were suspended performed at Covance Laboratories Inc (Madison, WI). All animals were in 1 ml of Krebs-Henseleit buffer at a final concentration of two million cells per determined free of specific pathogens (http://www.criver.com/files/pdfs/app/ milliliter. To these cell suspensions, either [14C]Ac-AMG 416 or [14C]Ala-AMG direct-animal-sampling-rat-chart.aspx). All collected samples were stored 416 (10 mM) was added. Cell mixtures were incubated at 37C in a water bath immediately after collection at 280C until further analysis. shaken at 75 rpm in a humidified (95% relative humidity) atmosphere of 95% dmd.aspetjournals.org oxygen and 5% CO2 and terminated after 2 hours by the addition of FA (final Dose Solution Preparation. For all studies, AMG 416 was formulated in concentration, 10% v/v). The quenched reaction mixture was vortex-mixed, phosphate-buffered saline to achieve the target dose volume of 0.5 ml/kg. An centrifuged at 14000g for 6 minutes at 4C, and then stored at 280C until aliquot of each dose-solution was analyzed for both total 14C and the radio-purity LC-14C-HRMS analysis. For the 0-hour incubations, 10 mMof[14C]AMG 416 before and after dose administration. All rats received a single i.v. bolus dose, was added to 1 ml of hepatocyte suspension, quenched immediately, and which was administered via a surgically implanted jugular vein catheter (groups processed as described herein. 1–10) or tail vein (groups 11–13). at ASPET Journals on September 30, 2021

TABLE 1 Design and purpose of the in vivo rat [14C]AMG 416 studies

Group ID Kidney Functiona 14C- Site Sex (No. of Animals) Dose mg/kg (mCi) Purpose and Time Points 1 Normal Ac M (4) 1.84 (25) Mass balance, excreta met ID F (4) Bile: 0–4, 4–8, 8 24, 24–48, 48–72 h 2 Urine: 0–8, 8–24, 24–48, 48–72 h Feces: 0–24, 24–48, 48–72 h F (4) B/P, plasma met ID 3 Blood: 0.25, 1, 4, 8, 24 h M (10) 2.5 (50) B/P, plasma AMG 416 and total M11 bioanalysis, and plasma met ID 4 Blood: 0.083, 0.25, 0.5, 1, 2, 4, 8, 24 h Ala M (4) 2.5 (38) Mass balance, excreta met ID Bile: 0–4, 4–8, 8–24, 24–48, 48–72 h 5 Urine: 0–8, 8–24, 24–48, 48–72 h Feces: 0–24, 24–48, 48–72 h M (4) plasma met ID 6 Blood: 0.25, 1, 4, 8 h M (2) B/P 7 Blood: 0.25, 1, 4, 8 h None Ac M (6) 2.5 (50) Mass balance, B/P, plasma AMG 416 and total M11 bioanalysis, and plasma met ID 8 Feces: 0–24, 24–48 Blood: 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48 h 9 Normal Ac M (1) 2.5 (50) 14C in expired air 10 Ala M (1) 2.5 (38) Expired air: 0–8, 8–24 11b Ac M (16) 2.5 (50) B/P, QWBA 12 M (10) Blood and carcass: group 11: 0.083, 0.25, 0.5, 1, 4, 12, 24, 48, 72, 96, 168, 336, 1008, 1512, and 2016 h; group 12: 0.083, 0.25, 0.5, 1, 4, 12, 24, 48, 96, and 168 h 13 F (10)

Ac, [14C]Ac-AMG 416; Ala, [14C]Ala-AMG 416; B/P, blood to plasma 14C concentration ratio; F, female; M, male; QWBA, quantitative whole-body autoradiography. aKidney function: Normal = rat with intact kidneys. None = bilaterally nephrectomized rat. bLong-Evans strain; all other groups used Sprague-Dawley strain. 1322 Subramanian et al.

Groups 1, 2, and 5. Both groups consisted of the jugular vein cannulated and radioactivity were interpolated from each standard curve and then converted to bile duct cannulated from male and female rats, respectively. Each rat underwent ng-equivalents/g (ng-eq/g) using the [14C]AMG 416 specific activity. surgery 7–10 days before study initiation to implant two catheters: one into the AMG 416 and total M11 bioanalysis. AMG 416 and total M11 were jugular vein for i.v. dose administration, and the other into the bile duct with a measured in plasma by LC-MS/MS as described in Supplemental Material.To duodenum return. Before drug administration, flow from the bile duct catheter determine the proportion of biotransformation products in plasma that retained was diverted to collect bile and a solution of taurocholic acid (0.0134 g/ml) in the intact D-amino acid backbone, all AMG 416-related homo- and hetero- saline (9 mg/ml NaCl, 0.5 mg/ml KCl) was continuously infused into the disulfides were reduced with TCEP, a disulfide bond reducing agent. The M11 duodenum at a rate of 1 ml/hour for the duration of the study. Each rat was formed upon TCEP reduction is referred to as “Total M11” because it represents 14 administered [ C]AMG 416 (see details in Table 1) and placed in a metabolic all AMG 416–related material present in incubations. Total M11 (also called cage. Following dosing, bile and urine were collected into prefrozen (280C) TM11 in biotransformation profiles) is distinct from M11 present in the in vitro BASi I-Cup devices (Bioanalytical Systems, Inc., West Lafayette, IN), and feces incubates due to biotransformation (described in Results). were collected at room temperature. Sample preparation for metabolite analysis. Urine: Urine was first pooled in Group 3, 4, 6, and 7. Rats were implanted with jugular and femoral vein proportion to the volume collected at each time period (0–72 hour) from each cannulae 7–10 days before study initiation. After dose administration, blood animal in the same group. A grand pool was then prepared by combining equal (200–1000 ml) was collected at designated time points via the femoral vein volume from each animal pool. An aliquot of pooled urine was centrifuged catheter into lithium heparin blood collection tubes and placed on wet ice. The at 20,817g for 10 minutes. The supernatants were submitted to liquid collected blood was temporarily stored on wet ice and then centrifuged at 4500g chromatography-14C-high resolution mass spectrometry (LC-14C-HRMS) anal- for 10 minutes to separate the plasma. The plasma was acidified with aqueous FA ysis. To inform the structure of the biotransformation products in urine, all AMG (2 ml of 1:1 v/v stock for 100 ml of plasma). 416-derived products were reduced with TCEP to liberate total M11. An aliquot Downloaded from Group 8. Animals in this group were male BN rats. Each rat underwent a bilateral of pooled urine was added TCEP (20 mM final concentration), vortex-mixed, nephrectomy procedure after a 48-hour diet consisting of sugar cubes. Animals were and allowed to stand at room temperature overnight. The resultant reduced urine 14 anesthetized with isoflurane (1%–4% in O2 at a rate of 1.5 liters/min), and the was then subjected to LC- C-HRMS analysis. kidneys were located and aseptically removed using the procedure described by Plasma: Equal volumes of plasma from each rat were first pooled at each time Waynforth and Flecknell (1992). Within 2 hours after surgery, each rat received point (0–48 hours for group 8 or 0–24 hours for other groups), and then a time- an i.v. dose of [14C]Ac-AMG 416 (Table 1) and was housed in a metabolic cage. proportional pool was prepared according to the Hamilton method (Hamilton dmd.aspetjournals.org Blood and feces were collected at designated time points. Immediately after et al., 1981) to obtain a single sample for rats in the respective group. Pooled euthanasia, select tissues were excised and stored for 14C-analysis. plasma samples were diluted 8-fold with 0.1% FA and then directly injected for 14 14 Groups 9 and 10. The study was conducted to determine the CO2 in expired LC- C-HRMS analysis. Additionally, an aliquot of the diluted pooled plasma air (Table 1). The setup consisted of an airflow gauge placed in line before an air was added TCEP aqueous solution (10 mM final concentration) and incubated at 14 metabolism cage (Metabowl MKIII system; Jencons Scientific, Bridgeville, PA) 37C for 1.5 hour before LC- C-HRMS analysis. This same procedure was used followed by two sequential Nilox columns (Jencons) each containing Carbosorb to prepare the TCEP-reduced plasma from in vitro whole blood incubations. The (Carbosorb E; PerkinElmer, Waltham, MA). A unidirectional airflow of 900 ml/min workflow to track the protein modifications is shown in Supplemental Fig. 1 and is described in Supplemental Material. was maintained through the metabolism cages, and the exhaust air was bubbled at ASPET Journals on September 30, 2021 LC-14C-HRMS Analysis. through the two Nilox chambers arranged in series to trap expired CO2.After [14C]AMG 416 dose administration, the rats were housed in a metabolic cage. Plasma and urine. Plasma samples were diluted 8-fold with 0.1% aqueous FA (v/v) before LC-14C-HRMS analysis. Analyte separations were achieved Before each collection interval, each CO2 trap chamber was filled with Carbosorb (approximately 300 ml each). After each collection interval, Carbosorb in the bubbling on an Agilent 1200 system (Agilent Technologies Inc., Wilmington, DE), chambers was removed for 14C-analysis, and its volume was measured and recorded. including a binary pump (G1312B), an autosampler (G1367C), and a Groups 11–13. These groups were used to characterize the tissue distribu- temperature controlled column compartment (G1316B) with a C18 column  m tion of [14C]AMG 416 by QWBA in LE and SD rats (Table 1). Animals were (XSelect HSS T3 C18, 250 4.6 mm, 3.5 m; Waters Corp., Bedford, MA) sacrificed via exsanguination (cardiac puncture) under isoflurane anesthesia, and maintained at 45 C. Mobile phases consisted of 0.1% TFA in water (solvent A) and 0.1% TFA in 50:50 water:acetonitrile (solvent B). For the in vitro plasma blood (2–10 ml) was collected into a tube containing K2EDTA immediately analysis, the following solvent gradient conditions were used at a flow rate of before collection of carcasses for QWBA. An aliquot of collected blood from 1 ml/min: 0–1 minute, 10% B; 1–31 minutes, 10%–16.4% B; 31–36.5 minutes, each time point was analyzed for 14C content, and the remainder was centrifuged 16.4%–100% B; 36.5–50.5 minutes, 100% B; 50.5–51 minutes, 100%–10% B; to separate the plasma. The carcasses were immediately frozen in a hexane/dry 51–65 minutes, 10% B. Postcolumn fraction collection for 14C-profiling and ice bath for approximately 8 minutes. Each carcass was drained, blotted dry, MS analyses were performed with a CollectPal HTC fraction collector (Leap placed into an appropriately labeled bag, and placed on dry ice or stored at Technologies, Carrboro, NC) and a LTQ Orbitrap Velos high-resolution mass approximately 270C for at least 2 hours. The frozen carcasses were embedded spectrometer (Thermo Fisher Scientific Inc.), respectively. The postcolumn in chilled carboxymethylcellulose (;6C; 2% w/v) and frozen at approxi- flow was split with 75% of the flow directed to the fraction collector and the mately 220C into blocks. Frozen blocks were imbedded with [14C]glucose remaining 25% to the MS. A total of 384 fractions were collected onto a ; ( 140,000 dpm/g) quality control reference standards to verify intersection and 384-well Deepwell LumaPlate (PerkinElmer) at 8 seconds/well. The column intrasection thickness variability. recovery of the radioactivity was 93.5% for the direct analysis of human 14 C analysis. Blood, RBCs, plasma, bile, urine, feces, and expired air were plasma. After the LumaPlate was dried in a vacuum centrifuge, the processed and counted in a liquid scintillation counter according to system radioactivity of each well was counted by a TopCount reader (PerkinElmer) manufacturer instructions and are described in Supplemental Material. using a normalized 14C protocol. Radiometric data were then imported and QWBA sample preparation and analysis. Sagittal whole-body sections analyzed using Laura software (version 4.1.13.91; IN/US Systems, Tampa (40-mm thickness) of rat carcasses were collected on adhesive tape using a Bay, FL). All MS data were acquired using a heated electrospray ionization cryomicrotome (Leica CM 3600; Buffalo Grove, IL) maintained at 220C. source in the positive-ion mode. The MS instrument conditions were as Sections were collected to show major tissues, organs, and biologic fluids. follows: capillary temperature, 350C; source temperature, 300C; source 2 Collected sections were dried in the microtome at approximately 20 C. Sections voltage, 5 kV; sheath gas (N2) flow rate, 35 (arbitrary units); auxiliary gas (N2) were attached to matting board, wrapped with Mylar film, and exposed for 4 days flow rate, 10 (arbitrary units); S-Lens RF level, 47%. MS2 data were collected to phosphor imaging screens. Screens were coexposed to [14C]glucose blood using a collision energy of 35%. The in vivo plasma and urine and in vitro S9 standards (0.980 to 10300 nCi/g) for radioactivity quantification. Exposed screens and hepatocyte supernatants were analyzed using a similar method and are were scanned using a PhosporImager (Storm; GE Life Sciences, Pittsburgh, PA), and described in Supplemental Material. tissue concentrations of radioactivity were determined using MCID Analysis Serum albumin peptide digest. The LC-14C-HRMS analysis was the same as software (InterFocus Imaging Ltd, Cambridge, UK). Tissue concentrations of plasma analysis with the following exceptions. Analyte separations were Nonclinical Pharmacokinetics and Disposition of Etelcalcetide 1323 achieved with a C18 column (Vydac 218MS52, 250  2.1 mm, 5 mm; Grace, P450 and Transporter Interactions Deerfield, IL) maintained at 40 C. Mobile phases consisted of 0.1% FA in water AMG 416 stability in HLM and HLS9 was not significantly affected by (solvent A) and 0.1% FA in acetonitrile (solvent B). The following solvent the presence or absence of NADPH, and was not significantly different gradient conditions were used: 0–2 minutes, 3% B, 0.2 ml/min; 2–45 minutes, 3%–40% B, 0.2 ml/min; 45–46 minutes, 40%–80% B, 0.2–0.3 ml/min; 46– between incubations that were preincubated with NADPH in the presence 58 minutes, 80% B, 0.3 ml/min; 58–60 minutes, 80%–3% B, 0.3–0.2 ml/min; or absence of 1-aminobenzotriazole. In the absence of any role of P450 in its 60–65 minutes, 3% B, 0.2 ml/min. The fractions were collected at 10 s/well. The biotransformation, AMG 416 concentrations decreased with time in HLM MS instrument conditions were as follows: capillary temperature, 275C; source and HLS9 incubations at pH 7.4. AMG 416 disappearance was completely temperature, 275C; source voltage, 4 kV; sheath gas (N2) flow rate, 25 (arbitrary inhibited in presence of 1% FA in HLM and HLS9 solutions at pH 3. units); auxiliary gas (N2) flow rate, 10 (arbitrary units); S-Lens RF level, 61%. AMG 416 was not a reversible or time-dependent inhibitor of the tested The column recovery of the radioactivity was 99.0%. P450 isoforms in HLM, the enzymatic activity in all AMG 416 treated Plasma protein conjugate. The intact protein MS analysis was conducted on groups was within 88% to 117% of the corresponding negative control an ultra-high-performance liquid chromatography-Q-TOF system, which con- group. AMG 416 was not an inducer of P450 isoforms 1A2, 2B6, and 3A4 sisted of an autosampler (CTC PAL, Leap Technologies) and an Agilent 1290 in human hepatocytes; the activities of all three P450 isoforms were ,2% system (Agilent Technologies Inc.), including a binary pump (G4220A), a of the corresponding positive controls in all three donors. temperature-controlled column compartment (G1316C), and a DAD detector  26 (G4212A). Analyte separations were achieved with a C18 column (POROS The average apparent permeability for AMG 416 was 0.7 10 cm/s R2/10, 100  2.1 mm, 10 mm; Applied Biosystems, Foster City, CA) maintained in LLC-PK1 cells. AMG 416 was not a substrate or inhibitor of Pgp or

at 45C. Mobile phases consisted of 0.1% FA in water (solvent A) and 0.1% FA BCRP transporters. The average Pgp and BCRP efflux ratio was close Downloaded from in acetonitrile (solvent B). The following solvent gradient conditions were used: to 1.0 and independent of the AMG 416 concentration tested. AMG 416 0–1 minute, 20% B, 1.0 ml/min; 1–2 minutes, 20%–35% B, 1.0–0.4 ml/min; 2– was neither a substrate nor an inhibitor of the uptake transporters tested. 6minutes,35%–45% B, 0.4 ml/min; 6–6.5 minutes, 45%–95% B, 0.4–1.0 ml/min; Probe substrate uptake into OAT1, OAT3, OATP1B1, OATP1B3, or 6.5–8.5 minutes, 95% B, 1.0 ml/min; 8.5–9 minutes, 95%–20% B, 1.0 ml/min; 9– OCT2 transfected cells was 0.7–1.04-fold of the respective controls 10 minutes, 20% B, 1.0 ml/min. Mass spectrometric analysis was performed with a + values and independent of the AMG 416 concentration tested. Also, TripleTOF 5600 system (AB Sciex, Foster City, CA) operating in the positive AMG 416 had no effect on human BSEP activity. ion and intact protein mode using a DuoSpray Ion Source. The instrument was dmd.aspetjournals.org calibrated using atmospheric pressure chemical ionization positive calibration Excretion Balance solution (AB Sciex) before analysis. The MS parameters were as follows: curtain gas, 10 (arbitrary units); ion source gas 1, 50 (arbitrary units); ion source gas 2, The excretion data after a single i.v. administration of [14C]AMG 416 50 (arbitrary units); temperature, 0C; ion spray voltage floating, 5.5 kV; to bile duct cannulated rats are presented in Table 2. In the 72 hours after a declustering potential, 250 V. single dose of [14C]Ac-AMG 416 (groups 1 and 2) or [14C]Ala-AMG PK analysis. Concentrations of radioactivity were converted into ng equivalent 416 (group 5), a total of 83%–88% of the radioactive dose was recovered. units before PK analysis. PK parameters were calculated by noncompartmental The radioactivity was predominantly recovered in urine (77%–84%; analysis using Phoenix Winnonlin (version 6; Pharsight, Cary, NC) for the following .90% of recovered dose) with minor amounts in feces (,4%) and in bile at ASPET Journals on September 30, 2021 concentration data sets: total radioactivity in blood and plasma, LC-MS/MS (,1%). 14C excretion was similar for both sexes and the two radiolabels. determined AMG 416 in plasma, and LC-MS/MS determined Total M11 in plasma. In BN rats (group 8; Supplemental Table 2), a small amount of the dose Identification of biotransformation products. Structure assignments of AMG was excreted in feces (1.2%), and approximately 11% of the dose was 416 biotransformation products were based on comparisons to authentic standards, calculated elemental composition of the products from high- recovered from the gastrointestinal tract by 48 hours. In rats with intact , resolution MS data, the 12C and 14C isotopic peak pattern, and interpretation kidney function (groups 9 and 10), minimal amount of the dose ( 0.2%) of the LC-MS/MS data. Authentic standards were available for M10, M11, and was eliminated in expired air within 24 hours. M12 and the identity of the biotransformation products was verified by 14 comparison of the retention time, MS, and MS2 spectra. Plasma PK ( C, AMG 416, and Total M11) A representative concentration-time profile of 14C in blood and Results plasma, AMG 416 in plasma, and total M11 in plasma from groups 4, 7, and 8 are shown in Fig. 2. Noncompartmental analysis-derived PK Blood-to-Plasma Ratio and Uptake into Red Blood Cells In vitro, the 14C blood to plasma ratio in rat, healthy human, and TABLE 2 CKD patient was 0.51, 0.50, and 0.69, respectively, and independent of 14 AMG 416 concentration. In vivo, the 14C area under the curve (AUC) in Excretion of radioactivity after a single i.v. dose administration of [ C]AMG 416 to bile-duct cannulated Sprague-Dawley rats blood was smaller than 14C AUC in plasma in normal and BN rats 14 (Supplemental Table 1). These results indicate that [ C]AMG 416- Recovery of Radioactivity (% of Administered Dose) derived radioactivity was preferentially retained in plasma. The uptake Group → 12 5 experiment of [14C]AMG 416 into RBC indicated approximately 10% 14 14 14 14 of [14C]AMG 416 was recovered in the RBC lysate. There was no [ C] label → [ C]Ac-AMG 416 [ C]Ac-AMG 416 [ C]Ala-AMG 416 14 change in C concentration in RBCs or in the buffer over the 2-hour Sex → Male Female Male incubation time indicating AMG 416-derived radioactivity was poorly Matrix Time (h) Mean S.D. Mean S.D. Mean S.D. permeable into RBCs. 0–8 67.9 7.54 58.5 7.30 63.3 7.34 8–24 11.0 3.13 13.6 4.71 9.30 1.74 Noncovalent Protein Binding in Plasma Urine 24–48 3.52 0.94 3.41 1.81 3.01 0.87 48–72 1.35 0.72 1.16 0.24 1.69 0.32 The mean noncovalent unbound fraction of AMG 416 in plasma was Subtotal 83.8 4.13 76.6 5.94 77.3 7.91 0.77 (rat), 0.67 (dog), 0.53 (healthy volunteers), and 0.59 (CKD Bile 0–72 0.52 0.34 0.28 0.09 0.64 0.62 patients). Plasma protein binding was independent of AMG 416 Feces 0–72 1.34 0.69 3.90 2.77 3.79 1.98 – concentration (50–10,000 ng/ml; approximates 0.05–10 mM) in all Cage wash 0 72 2.13 0.11 2.30 0.81 2.30 1.09 All Total 87.7 4.47 83.1 4.79 84.1 9.32 species. 1324 Subramanian et al.

Fig. 2. Mean concentration-time profiles of (A) 14C in blood and Downloaded from plasma and (B) 14C, total M11 and AMG 416 in plasma following a single i.v. administration of [14C]Ac-AMG416tonormal (group 4) or bilaterally nephrectomized (group 8) male rats and [14C]Ala-AMG 416 to normal male rats (group 7). dmd.aspetjournals.org at ASPET Journals on September 30, 2021

parameters are summarized in Supplemental Table 1. PK profiles in blood volume of distribution (Vss) in LE rats was high (7230 liters/kg; and plasma were similar for male rats dosed with [14C]Ac-AMG 416 group 11) and greater than total body water. For the LE rats (group and [14C]Ala-AMG 416 (Fig. 2A) and the female rats dosed with 11), maximal levels of radioactivity occurred in most tissues by 0.5 [14C]Ac-AMG 416 (data not shown). 14C concentrations in blood hour postdose followed by a decline in 14C concentration over time. and plasma declined through 24 hours postdose followed by a slower Maximal 14C concentration in kidney, kidney cortex, and liver was elimination phase; the latter was better captured in 14C PK profile observed at 12 hours postdose. Matrices with the highest 14C obtained in the QWBA analysis (groups 12 and 13; Supplemental concentrations were urine, epiphyseal line, kidney cortex, interver- Fig. 2). The rate of decline in 14C and AMG 416 plasma concentrations tebral cartilage, hyaline cartilage, kidney, kidney medulla, articular was significantly lower in BN rats compared with the normal rats (Fig. 2B). cartilage, and bile. Tissues with the lowest Cmax values (,500 ng-eq/g) The plasma PK profiles for the total M11 closely matched the profile of were the eye lens, brain (medulla, olfactory lobe, cerebrum, and 14 total radioactivity (Fig. 2B). The plasma C clearance in male rats dosed cerebellum), spinal cord, and abdominal fat. At 2016 hours post 14 14 with [ C]Ac-AMG 416 or [ C]Ala-AMG 416 was similar (approxi- dose, 18 tissues had quantifiable concentrations of radioactivity, mately 0.30 liter/h per kilogram). Removal of both kidneys resulted in an including hyaline cartilage, red pulp of spleen, spleen, kidney cortex, 14 18-fold increase in plasma C AUC and a substantially lower clearance kidney, and kidney medulla. The highest tissue-to-plasma concen- (approximately 0.013 l/h per kg), which was approximately 25 times lower tration ratios were observed in the kidney, liver, spleen, bone than that observed in rats with intact kidneys. The plasma AMG 416 marrow, lymph nodes, and cartilages. Tissues with the highest 14 clearance was significantly higher than the C clearance at approximately radioactivity exposures included kidney cortex, kidney, kidney 3-fold and 5.5-fold in normal and BN rats, respectively. medulla, hyaline cartilage, liver, spleen, bone marrow, intervertebral cartilage, lymph nodes, and epiphyseal line. Exposures of radioac- Tissue Distribution tivity were low (,500 ng-eq × h/g) in eye lens, brain (all regions), A plot of 14C concentration versus time for select tissues in male LE and spinal cord. 14 14 rats is shown in Fig. 3. A listing of the select C concentration-time The plasma C and AMG 416 Vss in SD rats was high (Supplemental data and the corresponding Cmax, AUC, and half-life for select tissues Table 1) and greater than the total body water. Tissue distribution and are presented in Supplemental Table 3 for male LE rats. The plasma 14C kinetics of radioactivity in male and female SD rats (groups 12 and 13; Nonclinical Pharmacokinetics and Disposition of Etelcalcetide 1325

Fig. 3. Concentrations of radioactivity in high and low exposure tissues as determined by quantitative whole-body autoradiography after a single i.v. administration of [14C]AMG 416 to male LE rats (group 11). Downloaded from data not shown) were similar and consistent with that observed in male binding (Wong et al., 1981; Iyer et al., 2001; Wait et al., 2006). Thus, LE rats. No substantive sex-dependent differences were observed in a method was developed wherein the diluted plasma was directly tissue distribution of radioactivity in SD rats. subjected to LC separation. Using this method, the qualitative and quantitative information of all biotransformation products, including Biotransformation Product Profiles the protein conjugates in the plasma, were simultaneously determined 14 14 The relative quantities of [ C]AMG 416 and its biotransformation from the resultant MS- and C-chromatograms. dmd.aspetjournals.org products in vitro and in vivo matrices are summarized in Table 3 and The chromatographic analyses of plasma obtained after incubation of 14 Supplemental Table 4, respectively. Excretion into feces and bile was [ C]AMG 416 in rat and human whole blood are shown in Fig. 4, and minor; therefore, these matrices were not chromatographically analyzed. the components detected in plasma are listed in Table 3. For both Plasma. The conventional extraction methods using protein precip- species, late eluting peaks (Fig. 4, A and B) postulated to be protein itation with an organic solvent or solid-phase extraction typically work conjugate(s) were the most abundant components, accounting for 59% well if the drug-related materials are noncovalently bound to plasma and 71% of the total radioactivity in rat and human plasma, respectively 14

proteins. The C plasma recovery in the supernatant after protein (Table 3). The abundance of the other biotransformation products was at ASPET Journals on September 30, 2021 precipitation was low (,50%), which is consistent with previous minor. In rat plasma, the most abundant nonprotein product M10 (14%) reports for thiol-containing drugs owing to high covalent protein was a glutathione (GSH) conjugate formed upon disulfide exchange

TABLE 3 Summary of biotransformation products in plasma from whole blood incubation, liver (L) fraction incubation, and time- proportionally pooled plasma obtained after a single i.v. administration of [14C]AMG 416 to normal and bilaterally nephrectomized (BN) male Sprague-Dawley rats

% 14C-Chromatograma

In Vitro Component Plasma Liver S9 Kidney S9 Hepatocytes In Vivo Rat Plasma

Rat Human Rat Human Rat Human Rat Human Normal Kidney BN AMG 416 11.7 14.1 16.3 72.7 75.4 45.3 12.9 35.1 34.5 24.3 M1 5.6 1.4 ND ND L ND L L 4.3 17.8 M5 ND ND ND 3.5 2.2 1.7 ND 1.4 ND ND M9 ND ND ND 9.4 ND 3.8 ND ND ND ND M10 14.2 1.1 74.0 ND 3.8 ND 68.9 30.3 10.8 19.9 M11 2.3 1.2 ND 3.6 ND 20.7 ND ND 2.0 5.8 M12 1.6 1.7 1.2 ND 3.8 3.8 15.8 26.1 2.3 4.7 M13 1.4 3.9 ND ND ND ND ND 2.6 ND 2.0b M14 1.9c ND 3.2 6.6 ND 3.1 ND 0.5 0.5 0.6 M15 1.5 1.5 ND ND ND ND ND 2.9 2.5b 2.9b M16 NDNDNDNDNDND2.41.1NDND M22 ND ND ND ND ND 1.4 ND ND ND ND M28 1.6 1.8 L L L L L L ND 1.1b Protein conjugate(s) 58.9 71.4 ND ND ND ND ND ND 42.3 18.7 U2 ND ND ND ND ND ND ND ND ND 2.2 Otherd 0.8 1.9 5.3 4.1 14.8 20.3 ND ND ND ND

BN, bilaterally nephrectomized; ND, not detected; U2, unknown component. %14C-chromatogram = 100*cpm counts in the area under the region of interest (ROI)/sum of cpm counts in all ROI. bNo mass spectral data were available because the peak intensity was below the detection limit. Identity was based on retention time matches with corresponding peaks observed in the in vitro plasma profile. cM14 and M15 were coeluted. dThe sum of all other peaks. In liver and kidney S9 fractions, the highest component was the late eluting putative protein conjugates (not characterized further). Up to nine additional metabolites were detected with each at ,1% administered radioactivity. 1326 Subramanian et al. Downloaded from

Fig. 5. Representative high-performance liquid chromatography radiochromato- grams of pooled urine without (A) or with (B) TCEP reduction after i.v. administration of [14C]Ac-AMG 416 to bile duct cannulated rats (group 1). TM11, total M11; M5-DesCys, sulfhydryl (reduced) form of M5; U1, unknown peak. dmd.aspetjournals.org

(Fig. 2B) and the TCEP-reduced plasma biotransformation profile affirmed that the covalent binding to serum albumin was via a disulfide bond and that the D-amino acid backbone remained intact in each AMG 416–related component detected in plasma. Urine. The radiochromatogram of the pooled urine from [14C]Ac-AMG 416 dosed male rats (group 1) is shown in Fig. 5A. The biotransfor- mation profiles were similar for both sexes and both radiolabels at ASPET Journals on September 30, 2021 (Supplemental Table 4). Intact AMG 416 was the most abundant component (approximately 35%–47% of the administered dose) in urine. M1was the most abundant biotransformation product. M3 (acetylated glutathione disulfide) and M2b (a disulfide; exact identity Fig. 4. Radiochromatograms of direct analysis for plasma from (A) rat and (B) unknown) coeluted under the chromatographic conditions used, and healthy human whole blood incubated with [14C]Ac-AMG 416 (5 mM)and time- therefore their individual quantities could not be determined. Treatment proportionally pooled plasma after i.v. administration of [14C]Ac-AMG 416 to (C) of urine (Fig. 5B) obtained from [14C]Ac-AMG 416-dosed male rats normal male rats (group 4), (D) bilaterally nephrectomized male rats (group 8), and (group 1) with TCEP resulted in the formation of a single predominant (E) TCEP-reduced plasma, bilaterally nephrectomized male rats (group 8). M5-DesCys, sulfhydryl (reduced) form of M5; TM11, total M11; U2, unknown peak. peak, total M11, and a minor peak, M5-DesCys, the reduced product from M5. An unassigned minor peak U1 was observed in both nonreduced and TCEP-reduced urine. This demonstrated that the with the L-cysteine of AMG 416. The next most abundant product was D-amino acid backbone remained intact in almost all biotransformation M1 (6%), a product of thiol sulfation. In comparison, M13 (4%), an products that were present in urine. L-cysteinylglycine conjugate, was the most abundant nonprotein bio- transformation product in the human plasma. M11 (2%), the thiol or Liver and Kidney S9 Fractions. The observed biotransformation sulfhydryl (reduced) form of the D-amino acid backbone in AMG 416 products in both rat and human S9 fractions are listed in Table 3. The most was a minor component. Reduction of the plasma samples resulted predominant products formed were GSH conjugate (M10) or the disulfide predominantly in the formation of a single total M11 peak (92% in both bond reduced product (M11) formed in rat liver and human kidney S9, species, data not shown) in the 14C-chromatograms. The biotransforma- respectively. Peaks that eluted late in the chromatographic analyses of all tion products present in plasma prepared from whole blood of both incubations were putatively protein conjugate(s) and were not further healthy humans and CKD patients were also present in plasma from rat. characterized. Reduction with TCEP treatment resulted in the formation of a In plasma obtained after AMG 416 dosing in normal and BN rats, 42% single predominant product, total M11. The biotransformation products and 19% of 14C, respectively, were covalently bound to plasma proteins formed from both acetyl and alanine labeled AMG 416 were very similar in (Fig. 4, C and D; Table 3). The predominant protein conjugate in plasma humanandratliverandkidneyS9fractions with or without TCEP treatment. was presumed to be serum albumin peptide conjugate (SAPC) based on Hepatocytes. The observed biotransformation products from both the in vitro characterization. The most abundant nonprotein conjugated rat and human hepatocytes are summarized in Table 3. The GSH drug-related components in in vivo plasma were the same as observed conjugate M10 was the most abundant product formed in hepatocytes in vitro. Total M11 was the predominant biotransformation product from both species, and after TCEP reduction, a single predominant observed in the TCEP-reduced plasma (Fig. 4E). Additional analyses product (total M11) was formed (data not shown). The products formed showed that 14C profiles were similar for both sexes and the two in hepatocytes from AMG 416 radiolabeled at both sites were very radiolabels (data not shown). Collectively, the total M11 and 14C PK data similar with or without TCEP treatment. Nonclinical Pharmacokinetics and Disposition of Etelcalcetide 1327

which was used to aid in structural assignment. These ions included m/z 448.276 (2+; D-cysteine S-C bond cleavage), m/z 464.262 (2+; disulfide bond cleavage), and m/z 481.256 (2+; L-cysteine S-C bond cleavage).

SAPC SAPC was characterized in plasma from in vitro whole-blood incubations. Figure 7A displays the deconvoluted mass spectrum of the human SA peak in the LC-HRMS chromatogram of the plasma from incubation of [14C]Ala-AMG 416 (200 mM) in human whole blood. Four major peaks were observed. The peak at m/z 66,438 Da was the unmodified human SA. Peaks at m/z 66,557 and 66,601 had mass shifts of 119 and 163 Da compared with the SA and represented cysteine and glycosylated conjugates of albumin, respectively. The peak at m/z 67,367 had a mass increase of 929 Da, which was equivalent to the 14 addition of the D-amino acid backbone of [ C]AMG 416 to SA. In the analysis of whole-blood incubations from CKD patients and rats, the same conjugates were observed (data not shown). Thus, in both rats Downloaded from Fig. 6. Proposed biotransformation of AMG 416 in in vitro whole-blood incubation and humans, the molecular weight of the conjugate corresponds to the and rat. addition of one molecule of the D-amino acid backbone from AMG 416 to one molecule of serum albumin. No other protein conjugates were detected in the analyses. 14 Structure Assignment of Biotransformation Products The Coomassie blue–stained and C images of the SDS-PAGE of affinity gel column fractions P1-P3 obtained from plasma isolated from dmd.aspetjournals.org A total of 19 nonprotein-conjugated biotransformation products were [14C]Ala-AMG 416 incubated in human whole blood are shown in detected in the analyzed in vitro and in vivo matrices. The proposed Fig. 8. In the Coomassie blue–stained gel (Fig. 8A), multiple protein biotransformation scheme is shown in Fig. 6. The MS and MS2 bands were observed for all three fractions. The most abundant band fragmentation data of all identified biotransformation products are in P2 and P3 (lanes 2 and 3 in Fig. 8A) had a molecular weight of summarized in Table 4. Characterization of the parent AMG 416 approximately 66 kDa and matched the band from human SA control molecule and the predominant biotransformation product in plasma, (lane 4 in Fig. 8A). Therefore, this band was identified as the human SAPC, are described below. 14

SA. The C image (Fig. 8B) of the same gel shows that a single at ASPET Journals on September 30, 2021 radioactive band was observed in the position of human SA in both P2 AMG 416 and P3 lanes. Thus, SA was the predominant plasma protein that was A doubly charged molecular ion at m/z 524.770 was observed in the covalently adducted with [14C]Ala-AMG 416. In the SDS-PAGE full-scan HRMS of AMG 416. Collision-induced dissociation (CID) of analysis, unmodified and modified SA were not separated and appeared this molecular ion resulted in multiple-fragment ions (Table 4), each of as a single band. In the Coomassie blue–stained gel (Fig. 8A), the

TABLE 4 Summary of precursor and product ions observed in high-resolution mass spectra for AMG 416 and its in vivo nonprotein biotransformation products

Observed Precursor Ion Component Mass Shifta Observed Product Ionsb (m/z; +2 Charge State) Da m/z AMG 416 524.770 20.003 448.276, 464.262, 481.257 M1 505.248 239.047 448.276, 464.263 M2a 617.288 185.033 448.277, 464.264, 481.256 M2b 617.288 185.033 448.277, 464.262, 481.255 M3 638.808 228.073 448.276, 464.261, 481.257 M4 574.780 100.017 448.276, 464.262, 481.256, 524.771 M5 525.263 0.983 448.768, 464.753, 481.748 M6 671.309 293.075 448.278, 464.272 M7 698.830 348.117 448.277, 465.264, 481.265, 617.806 M8 568.780 88.017 448.277, 464.264, 481.257 M9 560.767 71.991 448.279, 464.265, 481.263 M10 617.804 186.065 448.275, 465.262 M11 465.272 2118.999 448.284 M12 464.764c 807.498 448.271, 481.255 M13 553.285 57.027 448.278, 465.268 M14 531.777 14.011 448.274, 465.271 M15 589.296 129.049 448.279, 465.269, 481.254, 524.773 M16 643.313 237.083 448.279, 488.262, 634.805 M22 575.774 102.005 448.279, 464.263, 553.778 M28 489.264 271.015 481.751

aMass shift is the difference of observed singly charged m/z 2 calculated AMG 416 singly charged m/z (1,048.536). bProducts of the 12C precursor ion except for M28, which was obtained from fragmentation of [14C]M28. c+4 charge state. 1328 Subramanian et al.

Fig. 7. Mass spectra of serum albumin (SA) and serum albumin peptide conjugate (SAPC) from plasma obtained Downloaded from after incubation of [14C]Ala-AMG 416 in human whole blood. (A) Deconvoluted mass spectrum of whole proteins; 14 (B) MS of the [ C]AMG 416 D-amino acid backbone modified peptide 21ALVLIAFAQYLQQCPFEDHV41K; (C) MS2 of m/z 673.56 (5+) in (B). dmd.aspetjournals.org at ASPET Journals on September 30, 2021

abundance of the serum albumin bands in lanes P2 and P3 were similar; expected 2-Da mass difference between a labeled and nonlabeled AMG however, in the 14C image (Fig. 8B), the SA band in the P3 lane was 416 molecule in the 5+ charge state. These findings show that the much more intense than the corresponding band in P2 lane and, peptide was modified by a single molecule of the D-amino acid therefore, indicated an increased proportion of SAPC in P3 fraction. backbone of AMG 416. The identities of the modified serum albumin P1 was the wash solution expected to contain non-albumin proteins; peptides were confirmed by their MS2 spectra (Fig. 7C), in which accordingly, no band was observed corresponding to the SA molecular product ions from [14C]Ala-AMG 416 moiety and the albumin peptide weight in the Coomassie blue. were observed. Two additional Cys34 conjugated peptides (peaks C34- The Lys-C digests of fractions P2 and P3 from the [14C]Ala-AMG 2 and C34-3 in Fig. 8, C and D) of minor abundance were also detected: 416 incubations in human whole blood from both healthy volunteers C34-2 was not yet fully characterized; C34-3 was a nonspecifically and CKD patients were profiled by LC-14C-HRMS (Fig. 8, C and D). cleaved shorter peptide from C34-1 (Supplemental Table 5). The The composition of the radiolabeled peaks in the chromatogram and modified Cys34 containing peptides contribute to approximately 76% their abundance are summarized in Supplemental Table 5. Similar 14C- of the total 14C count in the digested fraction. The same MS analyses profiles of the Lys-C digest were observed for the sample from a healthy were also performed on tryptic digests obtained from human whole- volunteer and a CKD patient receiving hemodialysis. Peptide C34-1 was blood incubation with [14C]Ac-AMG 416 at 1 and 10 mM (data not 14 the predominant peak in the chromatogram (Fig. 8, C and D). The shown); the conjugation of [ C]Ac-AMG 416 D-amino acid backbone molecular ion and mass spectrum were consistent with conjugation of one to Cys34 in human SA was confirmed from the HRMS and MS2 data of D-amino acid backbone from AMG 416 via a disulfide bond to cysteine at the detected peptide C34-1. position 34 (Cys34) in the 21ALVLIAFAQYLQQCPFEDHV41Kpep- tide within serum albumin. Similar retention times were observed for nonlabeled AMG 416 and [14C]Ala-AMG 416–modified serum Discussion albumin peptide in their extracted ion chromatograms (data AMG 416 is a novel synthetic peptide composed almost entirely of not shown). The MS spectra show that the precursor ion (z = 5+) of D-amino acids. The only natural L-amino acid in the molecule is not in [14C]Ala-AMG 416 (Fig. 7B)–modified peptide has a mass shift of the peptide backbone but instead is linked via a disulfide bond. Thus, all 0.4 Da compared with the nonlabeled counterpart, indicative of the peptide bonds in AMG 416 are between D-amino acids, which are Nonclinical Pharmacokinetics and Disposition of Etelcalcetide 1329

Fig. 8. SDS-PAGE of affinity gel column fractions P1 (lane 1), P2 (lane 2), P3 (lane 3), control human serum albumin (lane 4) and radiolabeled protein marker standards (lane 5). Plasma obtained after incubation with [14C]Ala-AMG 416 in healthy volunteer human whole blood was subjected to affinity gel chromatography.

The same PAGE gel was stained with Coomassie Blue and also Downloaded from imaged on a 14C-phosphor system. (A) Coomassie Blue–stained image; (B) 14C image. 14C chromatogram of Lys-C digest from plasma obtained after incubation of human whole blood from (C) healthy volunteer or (D) CKD patient with [14C]Ala-AMG 416 (200 mM). The plasma proteins were subjected to affinity gel column separation; fractions P2 and P3 obtained thereafter were combined and subjected to Lys-C digestion. C34-1, 2, and 3 refer to

peptides shown to contain cysteine 34 in albumin; composition of dmd.aspetjournals.org the modified peptides is shown in Supplemental Table 5. CKD, chronic kidney disease; SA, serum albumin; U3 and U4 are unknown peaks. at ASPET Journals on September 30, 2021

sometimes incorporated into therapeutic peptide drugs to prevent of the D-amino acid backbone, which was likely to be metabolically peptide bond hydrolysis by peptidases (Chan et al., 1991; Hennan stable but required a far more complicated synthesis procedure to et al., 2006; Bloedon et al., 2008; Charignon et al., 2012). This is an prepare. The PK and disposition profiles after a single i.v. dose of the effective strategy to improve the metabolic stability of peptides; two 14C-labeled AMG 416 molecules to rats were similar, and no sex 14 14 however, because the biologic activity of most peptides is likely differences were observed. Excretion of C, presumably as CO2,in dependent on interactions between their target and L-amino acid expired air was low (,0.2%) with both labels. Des-acetylated bio- sequences in the peptide, the number of D-amino acids that can typically transformation products were specifically searched for in the in vitro be incorporated is limited. Accordingly, disposition of few, if any, and in vivo LC-MS data sets, and no des-acetyl products were detected peptide drugs composed primarily of D-amino acids has been de- in any of the samples. These findings demonstrated that the entire termined. In vitro investigations showed that the D-amino acid D-amino acid backbone—including the N-acetyl group—in AMG 416 backbone of AMG 416 was essentially inert to peptidase-mediated was metabolically stable. Accordingly, no distinction is made in hydrolysis. Additional studies showed that other metabolizing en- descriptions between the two labeled compounds, and the test material zymes, such as the P450s, catalyzed little if any biotransformation of is described solely as [14C]AMG 416. AMG 416. Finally, in vitro studies also showed that AMG 416 was not The intended clinical route of AMG 416 is by i.v. administration; a substrate of the typical transporters that are involved in the disposition therefore, the kinetics of absorption upon delivery by a non-IV route of some drugs. Collectively, these studies showed that the D-amino acid was not studied. The total recovery of radioactivity was similar in the backbone of AMG 416 is metabolically quite inert and unlikely to be male and female rats with normal kidney function. AMG 416 has a low subject to clearance routes dependent on hydrolysis, oxidation, or active molecular weight and positive polarity, and in vitro findings showed transport into excreta. that its cellular permeability was limited and its plasma protein binding Two AMG 416 radiolabels were prepared and tested—the first on relatively low; therefore, glomerular filtration was expected to be a the conveniently prepared and potentially metabolically labile acetyl predominant mechanism of plasma clearance. Indeed, when AMG 416 position, the second on the D-alanine (next to D-cysteine) in the interior was administered to rats with intact kidney function, its observed 1330 Subramanian et al. plasma clearance was 0.97 L/h per killogram (Supplemental Table 1), 0.19 liters/kg (Hennan et al., 2006). The Vss for AMG 416 and total and the estimated renal clearance (fraction unbound in plasma (0.77) Â 14C, however, was significantly higher (.8-fold) than the extracellular observed plasma clearance = 0.75 liter/h per kilogram) was close to the volume. This result could be attributed in part to reversible disulfide glomerular filtration rate in rat of 0.65 liter/h per kilogram (Sadick et al., exchange of the AMG 416 D-amino acid backbone between all 2011). However, because the drug is intended for treatment of patients available endogenous thiols and the L-cysteine readily available in with little or no kidney function, this route of elimination would be plasma. This reversible exchange was investigated and will be reported highly restricted, and assessment of its disposition in the absence of in a separate publication. The D-amino acid backbone reversibly bound kidney clearance was of critical importance. In BN rats, where the as a disulfide to macromolecules such as albumin would constitute kidney function was totally absent, the total recovery of radioactivity distribution into a peripheral compartment with a volume that exceeds was low and the elimination by nonrenal pathways was extremely slow. extracellular water. Indeed, the D-amino acid backbone of AMG 416 Another important feature of AMG 416 is the presence of the probably exists in equilibrium among many disulfide forms, one of disulfide bond between a D-amino acid in the peptide backbone, and which is the parent molecule, and which in plasma is dominated by L-cysteine. The relative facility with which disulfide exchange between SAPC because of the high concentration of albumin in serum. High AMG 416 and other thiol-containing molecules can occur is illustrated levels of radioactivity in some tissues may be a consequence of by the fact that the biologic activity of AMG 416 is mediated by phagocytosis (spleen, liver), binding of the labeled D-amino acid formation of a disulfide bond between the D-amino acid backbone of backbone to calcium-sensing receptor via disulfide bond (kidney, AMG 416 and a cysteine in the calcium-sensing receptor (Alexander cartilage, bone medullary space) and reabsorption of SAPC (kidney) Downloaded from et al., 2015). Disulfide bond formation between a drug and endogenous (Christensen and Birn, 2002; Dvorak et al., 2004; Chang et al., 2008). thiols has been studied and described with other drugs, such as captopril Distribution of radioactivity to the brain was low, which reflects the low (Migdalof et al., 1984). The dispositional fate of captopril and other passive permeability of AMG 416 and lack of active uptake transport similar thiol-containing drugs suggests that the disposition of AMG 416 resulting in a poor distribution across the blood-brain barrier. would not be determined solely by its peptide structure but also by thiol AMG 416 presents a low risk for P450 or transporter mediated drug- exchange mechanisms. Indeed, in vitro studies demonstrated that drug interactions. AMG 416 was not a substrate, a reversible or time- disulfide exchange readily occurred in whole blood between low- dependent inhibitor, or an inducer of the P450 isozymes tested in this dmd.aspetjournals.org molecular-weight endogenous thiols, such as glutathione, and high- study. This finding should provide an advantage over the other molecular-weight thiols, such as serum albumin. These findings are calcimimetic, cinacalcet, the only drug of this class for the treatment similar to findings with other thiol drugs (Migdalof et al., 1984; Iyer of secondary HPT (Torres, 2006). AMG 416 was not a substrate or et al., 2001; Wait et al., 2006). These in vivo studies demonstrated that an inhibitor of the common efflux and uptake human transporters the biotransformation products formed after in vitro incubation in whole examined in the current study. blood were also formed in vivo. In vitro studies also showed that the In summary, the nonclinical PK, disposition, and drug-drug in- same biotransformation products formed in blood could be formed in teraction potential of AMG 416 were assessed. Disposition of AMG at ASPET Journals on September 30, 2021 S9 fractions from liver and kidney; however, because of the low 416 was predominantly by renal elimination. AMG 416 was biotrans- permeability of AMG 416, the intracellular contribution to biotrans- formed via disulfide exchange of its L-cysteine moiety with thiols formation is likely to be small. present in whole blood, predominantly to albumin. The risk of P450 or In both rat and human whole blood, SAPC was the predominant transporter mediated drug-drug interactions with AMG 416 is antici- covalently bound-protein biotransformation product. The covalent pated to be very low. These studies demonstrated a 14C label on either interaction of AMG 416 with plasma proteins is likely, if not the acetyl or the D-alanine (next to D-cysteine) in AMG 416 peptide exclusively, via disulfide bond formation with cysteine residues present backbone would be appropriate for other nonclinical and clinical in the plasma proteins based on the following: AMG 416 activity absorption, distribution, metabolism, and excretion studies. requires disulfide exchange with a cysteine residue on the calcium- sensing receptor, as stated already herein; other thiol drugs are known to undergo disulfide exchange (Wong et al., 1981; Wait et al., 2006); Acknowledgments lastly, liberation of total M11 by TCEP indicates that the covalent bonds The authors thank Michael Hayashi for performing hepatocyte incubations; are likely to be disulfides. SAPC was formed via disulfide conjugation Dean Hickman for helping design the protein binding experiment; Robert Ortiz for assistance with SDS-PAGE experiments and 14C-phosphoimager analysis; of the AMG 416 D-amino acid backbone to serum albumin. In addition Andrew Hui and Jun Lammawin for performing the transporter experiments; to the SDS-PAGE evidence, binding to a specific amino acid was Ryan Morgan for human BSEP transport experiment; Yihong Zhou for determined using HRMS. Human SA contains a total of 35 cysteines, conducting the time-dependent P450 experiments; Erin Ballard and Phil with Cys34 the only one with a free thiol group, and therefore it should Manteufel for the QWBA study; Mark Fielden, James Tomlinson, and Charles be more readily than other cysteines to undergo disulfide exchange with Dean, Jr., for QWBA data interpretation; and Holly Tomlin (employee and the AMG 416 D-amino acid backbone to form SAPC. No other cysteine stockholder of Amgen Inc) for medical writing and journal formatting assistance. residue in serum albumin was found to be modified, confirming this hypothesis. Disulfide exchange reactions proceed through a nucleophilic substi- Authorship Contributions Participated in research design: Zhu, Fitzsimmons, Esmay, Louie, Edson, tution (SN2) reaction via the thiolate anion intermediate (Fernandes and Ramos, 2004). AMG 416 was stable in in vitro incubations at pH 3, and Walter, Soto, Wagner, Wilson, Skiles, Subramanian. this was attributed to a decrease in thiolate anion formation from the Conducted experiments: Zhu, Esmay, Fitzsimmons, Louie, Edson, Kerr, Pham, Soto, Wagner. endogenous thiols and consequently a decrease in the disulfide ex- Contributed new reagents or analytic tools: Zhu, Esmay, Pham, Wilson, change (Nagy, 2013). 14 Subramanian. After a single i.v. dose of [ C]AMG 416, radioactivity appeared Performed data analysis: Zhu, Esmay, Louie, Edson, Kerr, Pham, Wagner, rapidly in most of the tissues evaluated. A peptide not subject to rapid Subramanian. metabolic clearance would be expected to have a Vss limited to Wrote or contributed to the writing of the manuscript: Zhu, Esmay, extracellular volume. For example, the Vss for rotigaptide was Fitzsimmons, Louie, Edson, Kerr, Wilson, Skiles, Subramanian. Nonclinical Pharmacokinetics and Disposition of Etelcalcetide 1331

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(2006) Development and characterization of LLC-PK1 cells pathogenesis of secondary hyperparathyroidism. Am J Physiol Renal Physiol 288:F253–F264. containing Sprague-Dawley rat Abcb1a (Mdr1a): comparison of rat P-glycoprotein transport to Sadick M, Attenberger U, Kraenzlin B, Kayed H, Schoenberg SO, Gretz N, and Schock-Kusch D human and mouse. J Pharmacol Toxicol Methods 54:78–89. (2011) Two non-invasive GFR-estimation methods in rat models of polycystic kidney disease: Chan RL, Hsieh SC, Haroldsen PE, Ho W, and Nestor JJ, Jr (1991) Disposition of RS-26306, a 3.0 Tesla dynamic contrast-enhanced MRI and optical imaging. Nephrol Dial Transplant 26: potent luteinizing hormone-releasing hormone antagonist, in monkeys and rats after single 3101–3108. intravenous and subcutaneous administration. Drug Metab Dispos 19:858–864. Schinkel AH, Wagenaar E, van Deemter L, Mol CA, and Borst P (1995) Absence of the mdr1a Downloaded from Chang W, Tu C, Chen TH, Bikle D, and Shoback D (2008) The extracellular calcium-sensing P-Glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, receptor (CaSR) is a critical modulator of skeletal development. Sci Signal 1:ra1–13. digoxin, and cyclosporin A. J Clin Invest 96:1698–1705. Charignon D, Späth P, Martin L, and Drouet C (2012) Icatibant, the bradykinin B2 receptor antagonist Tfelt-Hansen J and Brown EM (2005) The calcium-sensing receptor in normal physiology and with target to the interconnected kinin systems. Expert Opin Pharmacother 13:2233–2247. pathophysiology: a review. Crit Rev Clin Lab Sci 42:35–70. Christensen EI and Birn H (2002) Megalin and cubilin: multifunctional endocytic receptors. Nat Torres PU (2006) Cinacalcet HCl: a novel treatment for secondary hyperparathyroidism caused Rev Mol Cell Biol 3:256–266. by chronic kidney disease. J Ren Nutr 16:253–258. Chu XY, Bleasby K, Yabut J, Cai X, Chan GH, Hafey MJ, Xu S, Bergman AJ, Braun MP, van Staden CJ, Morgan RE, Ramachandran B, Chen Y, Lee PH, and Hamadeh HK(2012) and Dean DC, et al. (2007) Transport of the dipeptidyl peptidase-4 inhibitor sitagliptin by Membrane Vesicle ABC Transporter Assays for Drug Safety Assessment. Current Protocols in human organic anion transporter 3, organic anion transporting polypeptide 4C1, and multidrug Toxicology series, Unit 23.5.1–23.5.24, Wiley, New York. dmd.aspetjournals.org resistance P-glycoprotein. J Pharmacol Exp Ther 321:673–683. Wait JC, Vaccharajani N, Mitroka J, Jemal M, Khan S, Bonacorsi SJ, Rinehart JK, and Iyer RA Cozzolino M, Tomlinson J, Walsh L, and Bellasi A (2015) Emerging drugs for secondary (2006) Metabolism of [14C]gemopatrilat after oral administration to rats, dogs, and humans. hyperparathyroidism. Expert Opin Emerg Drugs 20:197–208. Drug Metab Dispos 34:961–970. Cunningham J, Locatelli F, and Rodriguez M (2011) Secondary hyperparathyroidism: patho- Walsky RL and Obach RS (2004) Validated assays for human cytochrome P450 activities. Drug genesis, disease progression, and therapeutic options. Clin J Am Soc Nephrol 6:913–921. Metab Dispos 32:647–660. Dvorak MM, Siddiqua A, Ward DT, Carter DH, Dallas SL, Nemeth EF, and Riccardi D (2004) Walter S, Baruch A, Alexander ST, Janes J, Sho E, Dong J, Yin Q, Maclean D, Mendel DB, Physiological changes in extracellular calcium concentration directly control osteoblast func- and Karim F, et al. (2014) Comparison of AMG 416 and cinacalcet in rodent models of uremia. tion in the absence of calciotropic hormones. Proc Natl Acad Sci USA 101:5140–5145. BMC Nephrol 15:81. Fernandes PA and Ramos MJ (2004) Theoretical insights into the mechanism for thiol/disulfide Walter S, Baruch A, Dong J, Tomlinson JE, Alexander ST, Janes J, Hunter T, Yin Q, Maclean D, exchange. Chemistry 10:257–266. and Bell G, et al. (2013) Pharmacology of AMG 416 (Velcalcetide), a novel peptide agonist of

Goodman WG and Quarles LD (2008) Development and progression of secondary hyperpara- the calcium-sensing receptor, for the treatment of secondary hyperparathyroidism in hemodi- at ASPET Journals on September 30, 2021 thyroidism in chronic kidney disease: lessons from molecular genetics. Kidney Int 74:276–288. alysis patients. J Pharmacol Exp Ther 346:229–240. Hamilton RA, Garnett WR, and Kline BJ (1981) Determination of mean valproic acid serum level Waynforth HB and Flecknell PA (1992) Specific Surgical Procedures, in Experimental and by assay of a single pooled sample. Clin Pharmacol Ther 29:408–413. Surgical Technique in the Rat (Waynforth HB ed), Academic Press, London. Hennan JK, Swillo RE, Morgan GA, Keith JC, Jr, Schaub RG, Smith RP, Feldman HS, Haugan Wong KK, Lan S, and Migdalof BH (1981) In vitro biotransformations of [14C]captopril in the K, Kantrowitz J, and Wang PJ, et al. (2006) Rotigaptide (ZP123) prevents spontaneous ven- blood of rats, dogs and humans. Biochem Pharmacol 30:2643–2650. tricular arrhythmias and reduces infarct size during myocardial ischemia/reperfusion injury in Yamazaki M, Li B, Louie SW, Pudvah NT, Stocco R, Wong W, Abramovitz M, Demartis A, open-chest dogs. J Pharmacol Exp Ther 317:236–243. Laufer R, and Hochman JH, et al. (2005) Effects of fibrates on human organic anion- Hewitt NJ, Lechón MJ, Houston JB, Hallifax D, Brown HS, Maurel P, Kenna JG, Gustavsson L, transporting polypeptide 1B1-, multidrug resistance protein 2- and P-glycoprotein-mediated Lohmann C, and Skonberg C, et al. (2007) Primary hepatocytes: current understanding of the transport. Xenobiotica 35:737–753. regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev 39:159–234. Iyer RA, Mitroka J, Malhotra B, Bonacorsi S, Jr, Waller SC, Rinehart JK, Roongta VA, Address correspondence to: Raju Subramanian, Amgen Inc. One Amgen Center and Kripalani K (2001) Metabolism of [(14)C]omapatrilat, a sulfhydryl-containing Drive, Thousand Oaks, CA 91320. E-mail: [email protected] vasopeptidase inhibitor in humans. Drug Metab Dispos 29:60–69. DMD #68007

Drug Metabolism and Disposition Supplemental Information

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

Pham, Sarah F. Wilson, and Gary L. Skiles

Pharmacokinetics and Drug Metabolism, Amgen Inc., Thousand Oaks, California (R.S., X.Z, 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 Laboratories, Madison, Wisconsin (M.F.)

Corresponding Author:

Raju Subramanian Amgen Inc. One Amgen Center Drive Thousand Oaks, CA 91320 Phone: 805-447-6301 Email: [email protected]

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Materials

Solvents and reagents were of the highest analytical grade available. Fresh healthy volunteer

human and rat whole blood (n = 4) were obtained from Amgen’s departments of Occupational

Health and Comparative Animal Research, respectively (Thousand Oaks, CA). Whole blood

from beagle dogs and late-stage CKD patients were obtained from BioReclamation (East

Meadow, NY). All whole blood samples had K2-EDTA as the anti-coagulant. Fresh rat hepatocytes were prepared at Amgen. Pooled male Sprague-Dawley rat liver and kidney S9

fractions, male human liver and kidney S9 fractions, rat cryopreserved hepatocytes, and two

separate 20-donor pooled human cryopreserved hepatocyte preparations were purchased from

Celsis In Vitro Technologies (Baltimore, MD). Human liver microsomes (HLM) were acquired

from Gibco (Carlsbad, CA) or CellzDirect (Durham, NC). The parental LLC-PK1 (porcine renal epithelial cells) and HEK-293 cell line (human embryonic kidney epithelial cells) were

purchased from American Type Culture Collection (Manassas, VA) and Invitrogen (Grand

Island, NY), respectively. The parental cell line MDCKII (Madin Darby canine kidney epithelial

cells) was licensed from the National Institute of Health (Bethesda, MD). All human transporter

gene transfected cells were generated at Amgen (Thousand Oaks, CA). Red blood cell (RBC)

buffer was prepared by Amgen and contained 10 mM HEPES, 130 mM NaCl, 4.2 mM KCl, 0.5

mM MgCl2, 5 mM glucose, 50 mM LiCl, 1.2 mM CaCl2, pH 7.75. ACK buffer was obtained

from Amgen Media Services and contained 0.155 M NH4Cl, 0.01 M KHCO3, and 0.1 mM

K2EDTA.

Formic acid (FA) and trifluoroacetic acid (TFA) were purchased from Thermo Fisher Scientific

Inc. Lysyl Endopeptidase (Lys-C) was purchased from Wako Chemicals USA, Inc (Richmond,

VA). Econo-Pac 10 DG and affinity gel column were acquired from Bio-Rad (Hercules, CA).

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Vivaspin 20 mL tube (molecular weight cutoff 10,000 Da) was purchased from Sartorius Stedim

Biotech GmbH (Goettingen, Germany). Cryopreserved hepatocyte recovery medium, NuPAGE®

Novex® 4-12% Bis-Tris Gel, and NuPAGE® MOPS SDS Buffer Kit were purchased from Life

Technologies (Carlsbad, CA). TFA, formic acid (FA), Tris(2-carboxyethyl)phosphine

hydrochloride (TCEP), and human serum albumin were purchased from Sigma Aldrich (St.

Louis, MO). 14C-labeled protein molecular weight marker mixture (P/N: NEC811) containing

phosphorylase B, bovine serum albumin, gammaglobulin, ovalbumin, and cytochrome C was

purchased from PerkinElmer (Waltham, MA).

Coomassie Plus™ protein assay solution, bovine serum albumin (2 mg/mL) standard, and M-

Per® mammalian protein extraction reagent were purchased from Thermo Scientific, Pierce

(Rockford, IL). 1-aminobenzotriazole (ABT), antifoam A emulsion atenolol, dextromethorphan, dextrorphan, diclofenac, digoxin, estradiol-17β-glucuronide, estrone-3-sulfate, furafylline, paclitaxel, paroxetine, phenacetin, prazosin, probenecid, reduced β-nicotinamide adenine dinucleotide phosphate (NADPH), ticlopidine, and tienilic acid were purchased from Sigma

Aldrich (St. Louis, MO). (S)-mephenytoin and troleandomycin were purchased from Enzo life

Science (Farmingdale, NY). 1-OH midazolam, 6-OH paclitaxel, midazolam, 4-OH S-

13 15 mephenytoin, 4-OH diclofenac, acetaminophen-[ C2, N), 6α-OH taxol-[D5], 4-OH diclofenac-

13 13 [ C6], 4-OH S-mephenytoin-[D3], dextrorphan-[D3], 1-OH midazolam-[ C3] were purchased

from BD Biosciences (Bedford, MA). Gemfibrozil 1-O-glucuronide was purchased from Toronto

Research Chemicals (North York, Ontario, Canada). 3H-digoxin, 14C-aminohippuric acid, 3H-

prazosin, 3H-estradiol-17β-glucuronide (E-17βD-Gluc), and 3H-estrone sulfate (ES), and 3H-

cholecystokinin (CCK8) were purchased from PerkinElmer (Waltham, MA). 3H-Atenolol (ATN)

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and 14C-Glycylsarcosine (GlySarc) were purchased from Moravek (Brea, CA). 14C-metformin

(MET) was purchased from American Radiolabeled Chemicals (St. Louis, MO).

AMG Non-Covalent Plasma Protein Binding Determination AMG 416 and internal standard

13 15 (IS) ( C3D3 N-AMG 416) were isolated from 50 μL of acidified plasma and ultra-filtrate by

protein precipitation with trichloroacetic acid and LC/MS. The LC system (LC-20ADXR;

Shimadzu Co., Columbia, MD) consisted of two pumps, a controller, vacuum degasser, autosampler, and column heater. The chromatographic separation was performed on a C18 column (Acquity HSS T3, 50 × 2.1 mm, 1.8 µm; Waters Corp.). Mobile phase A was 0.07%

TFA and 0.1% FA in water and mobile phase B was acetonitrile. The LC method was a gradient method going from 0% mobile phase B to 60% mobile phase B over 3 min, then stepped up to

95% mobile phase B in 0.2 min and held for 0.8 min at 0.5 mL/min. The gradient was then stepped down to 0% mobile phase B and re-equilibrated for 2 min. The MS analysis was performed on a API 4000 Q-Trap mass spectrometer (AB Sciex) equipped with a Turbo Ion

Spray interface operated in the positive ion mode with the following MRM transitions: AMG

13 15 416, m/z 525.0 → m/z 448.4; C3D3 N-AMG 416 (IS), m/z 528.5 → m/z 451.9. The linear

calibration range was 2.9 to 1500 ng/mL.

AMG 416 concentrations in plasma control, filtered plasma and ultra-filtrate were determined by

interpolation from a standard curve using linear regression of standards with 1/x weighting.

Fraction unbound was calculated as shown below:

[ 416] = [ 416] 퐴퐴퐴 푢푢푢푢푢−푓푓𝑓𝑓�푓 퐹퐹퐹퐹퐹퐹퐹퐹 푈�푈�푈�푈 퐴퐴퐴 푝𝑝푝푝� 푐푐푐𝑐푐� Recovery of AMG 416 in plasma was calculated as shown below:

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([ 416] × ) + ([ 416] × ) = ([ 416] × ) 퐴퐴퐴 푉�푉�푉� 푓푓𝑓푓�푓푓 푝𝑝푝푝� 퐴퐴퐴 푉�푉�푉� 푢푢푢푢푢−푓푓𝑓𝑓�푓 푅푅𝑅푅푅�푅 퐴퐴퐴 푉�푉�푉� 푝𝑝푝푝� 푐푐푐𝑐푐� Preliminary experiment with blank plasma indicated that 12 minute centrifugation at 2000 × g

produced > 120 µL of ultra-filtrate from 500 µL starting plasma. Average volumes of 170 µL

ultra-filtrate and 330 µL filtered plasma were used for calculation of recovery.

14C Analysis in Blood, Plasma, Bile, Urine and Feces 100 µL each of the whole blood incubate

and the harvested plasma was pipetted into a pre-weighed sample oxidation cone (Combusto-

Cones, PerkinElmer, Waltham, MA) and the sample weights were recorded. Sample cones were dried at ambient temperature and combusted in a Tri-Carb Model 307 Sample Oxidizer

14 (PerkinElmer, City, ST). The resultant CO2 was trapped in Carbsorb (PerkinElmer), then liquid

scintillant (Permafluor, PerkinElmer) was added and vortex mixed, and then 14C levels were determined by liquid scintillation counting (LSC; Model 2900TR Tri-Carb; PerkinElmer).

Plasma (20-100 µL), bile (100 µL), and urine (100 µL) were added directly to 6 mL of scintillation cocktail (UltimaGold XR, PerkinElmer). The mixture was vortex-mixed and equilibrated for at least 24 hr prior to 14C counting. The samples were counted by LSC and corrected for quenching with automatic external standardization. Feces were homogenized in

70% ethanol in water at an approximate ratio of 4 to 1 (v/v) using a high frequency mechanical

homogenizer. Tissues (from Group 8 only) were homogenized in PBS. Triplicate aliquots of homogenates were transferred to Combusto-Cones (PerkinElmer), weighed, air-dried and

14 combusted using a sample oxidizer (model 307, PerkinElmer). The resultant CO2 was trapped

with Carbosorb (9 mL) and then combined with 9 mL of scintillation cocktail (Permafluor,

PerkinElmer). Triplicate aliquots of blood samples (20-50 µL) from Groups 3-8 were added on

to Combusto-Cones, weighed, and air-dried. Oxidation of dried blood samples was performed as

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described above. The combustion efficiency was separately determined using commercial radiolabeled standards. Duplicate aliquots of blood samples (0.1 g) from Groups 11-13 were

combined with solubilizing agent (Solvable; PerkinElmer) and incubated for 1 hr at 60 °C to

digest the proteins, then mixed with Na2-EDTA followed by 30% hydrogen peroxide to remove color. The bleached blood samples were combined with scintillation cocktail (UltimaGold XR),

vortex-mixed and counted on the LSC. An aliquot of Carbosorb (200 µL) in each CO2 trap from

Groups 9 and 10 was combined with scintillation cocktail (6mL; UltimaGold XR) and counted

on the LSC. 14C administered dose and dose recovery calculations were performed using

Debra software. 14C recoveries were expressed as percent of administered radioactive dose.

AMG 416 and Total M11 Plasma Bioanalysis

AMG 416 AMG 416 and IS were isolated from 50 μL of acidified rat plasma using a 96-well protein precipitation extraction procedure. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) system consisted of two pumps (LC-10AD; Shimadzu Co.,

Columbia, MD), a controller (SCL-10A; Shimadzu Co.), an autosampler (LEAP Technologies,

Carrboro, NC), and a triple quadrupole MS (API 5000; AB Sciex, Concord, Ontario, Canada) equipped with a Turbo Ion Spray interface. The chromatographic separation was performed on a

C18 column (BetaBasic, 2.1 × 50 mm, 5 μm; Thermo Fisher Scientific Inc.). Mobile phases A and B were 0.07% TFA with 0.1% FA in water and acetonitrile, respectively. The liquid chromatography (LC) method was a gradient method as following: 0-1 min, 5-70% B, 0.5

mL/min; 1-1.2 min, 70-95% B, 0.5 mL/min; 1.2-2.5 min, 95% B, 0.5-1 mL/min; 2.5-2.6 min,

95% B, 1-0.5 mL/min; 2.6-3.0 min, 95-5% B, 0.5 mL/min.

The MS analysis was performed in the positive ion mode and the analytes were followed via

their selected reaction monitoring (SRM) transitions: AMG 416, m/z 524.9 → m/z 448.4;

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14 13 15 [ C]AMG 416, m/z 525.9 → m/z 449.4; C3D3 N-AMG 416 (IS), m/z 528.4 → m/z 451.8.

The linear calibration range was from 1.00 to 500 ng/mL.

Total M11 Aliquots (50 μL) of acidified rat plasma standards, QC’s, samples, and blanks were reduced to M11 upon a 30 min incubation at 37°C with TCEP followed by protein precipitation, dry-down, and reconstitution in a 96-well format. The LC-MS/MS system consisted of two pumps (LC-20AD; Shimadzu Co.), a controller (SCL-20A; Shimadzu Co.), an autosampler (SIL-

20AC; Shimadzu Co.), and a triple quadrupole MS (API 4000; AB Sciex) equipped with a Turbo

Ion Spray interface. The chromatographic separation was performed on a C18 column (Luna, 2.1

× 50 mm, 5 μm; Phenomenex Inc., Torrance, CA) at a flow rate of 0.5 mL/min. Mobile phases A and B were 0.1% TFA in water and acetonitrile-water (0.1% TFA; 50:50, v/v), respectively. The

LC method was a gradient method as following: 0-4 min, 0-90% B; 4-4.1 min, 90-100% B; 4.1-

5.1 min, 100-0% B.

The MS analysis was performed in the positive ion mode and monitored the following SRM transitions: M11, m/z 465.5 → m/z 448.5; [14C]M11, m/z 466.5 → m/z 449.5; reduced

13 15 C3D3 N-AMG 416 (IS) m/z 468.9 → m/z 451.9. The linear calibration range was from 50 to

2000 ng/mL.

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In Vivo Plasma and Urine LC-14C-HRMS Analysis For the urine analysis, a XSelect HSS T3

C18 column (250 x 4.6 mm, 3.5 µm; Waters Corp., Bedford, MA) maintained at 45 °C was used

for chromatographic separation at a flow rate of 1 mL/min using the following gradient: 0-1 min,

5% B; 1-34 min, 5-30% B; 34-36.5 min, 30-100% B; 36.5-45.5 min, 100% B; 45.5-46 min, 100-

5% B; 46-60 min, 5% B. For the plasma analysis, the same chromatographic condition as the in vitro plasma analysis was used. The post column eluate was diverted with 3/4 of flow directed to a fraction collector and the remainder to a mass spectrometer. Fractions were collected via a

CollectPAL (Leap Technologies) onto a 384-deepwell LumaPlate (PerkinElmer) at 7 seconds/well for analysis of urine. For the plasma analysis, fractions were collected onto 4 × 96- deepwell plates at 8 seconds/well with multiple injections and then transferred to 4 × 96- deepwell LumaPlates multiple times due to low radioactivity of the diluted plasma. Each

LumaPlate was dried in a vacuum centrifuge once it was replenished with the eluate. The dried

LumaPlates were counted at 5 min/well on a TopCount reader (PerkinElmer) using a normalized

14C protocol. Radiometric data were then imported and analyzed using Laura software (version

4.1; LabLogic Systems, Brandon, FL).

Plasma Protein and Peptide Conjugates Analysis The work flow to track the protein modifications is shown in Figure S1. For whole protein analysis, human (healthy volunteer and

CKD patient) and rat plasma obtained following whole blood incubations was diluted 320-fold with 0.1% aqueous FA (v/v) before analysis by LC-HRMS. For peptide analysis, the plasma was processed in five steps before analysis by liquid chromatography-14C-high resolution mass spectrometry (LC-14C-HRMS). In step 1, human or rat plasma (~1 mL) was diluted to 1.5 mL

with 20 mM pH 7.1 sodium phosphate buffer (buffer A) and loaded on to an Econo-Pac 10 DG

column. The column was eluted with 10 mL of buffer A and ten 1-mL fractions (labeled

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fractions 1 to 10) were collected and the radioactivity in a 20-µL aliquot from each fraction was

determined by LSC. In step 2, the fractions previously determined to contain protein were then

loaded onto an albumin affinity gel column. The affinity gel column contents were then washed

with 25 mL of buffer A to remove non-albumin proteins and residual small molecules and 5

fractions (labeled 2-1 to 2-5; 5 mL each) were collected. Then, 30 mL of saline phosphate buffer

(1.4 M NaCl, 20 mM sodium phosphate in water, pH 7.1) was used to elute the serum albumin

protein and 6 fractions (identified as 3-1 to 3-6; 5 mL each) were collected. Finally, the column was washed with 15 mL of NaSCN phosphate buffer (1.5 M NaSCN, 20 mM sodium phosphate in water, pH 7.1) to elute the tightly bound albumin and to regenerate the affinity gel column and three fractions (labeled 4-1 to 4-3, 5 mL each) were collected. Step 2 resulted in pooled fractions labeled P1 (fractions 2-1 to 2-4), P2 (fractions 3-1 to 3-4), and P3 (fractions 4-1 to 4-3). In step

3, a desalting procedure was applied to pooled fractions P1, P2, and P3 by loading each onto

individual inside (concentrator) chambers in Vivaspin tubes (20 mL volume) and concentrated to

approximately 1 mL by centrifugation at 2500 × g at 20 ºC. Water was added to the residual

volume in the concentrator chamber (20 mL each time) and this wash process was repeated three

times. In step 4, the desalted solutions (~1 mL/each) of P1, P2 and P3 were transferred to 4 mL

vials and lyophilized (ModulyoD Freeze Dryer; Thermo Fisher Scientific Inc, Waltham, MA).

Aliquots of P1, P2, and P3 from healthy volunteer plasma were subjected to SDS-PAGE

analysis.

The remaining P2 and P3 solids were dissolved in water then combined and lyophilized. In step

5, an aliquot of the combined P2 and P3 solid (0.25 mg) was added to buffer (375 µL; pH 5.5)

containing urea (4 M), ammonium acetate (50 mM), and hydroxylamine (20 mM), and digested

with Lys-C (12.5 µL; 1 µg/µL) with a substrate to enzyme ratio of 20/1 (w/w). The digestion

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mixture was gently mixed and incubated at 37 oC in a water bath for 24 hr. The digestion

reaction was stopped by addition of FA (3.9 µL; 1% v/v final solution) and analyzed by LC-14C-

HRMS.

Gel electrophoresis analysis was performed on a NuPAGE® SDS-PAGE Gel Electrophoresis

System (Life Technologies, Carlsbad, CA). Aliquot of P1 (5 µL; 1 µg/µL), P2 (5 µL; 1 µg/µL),

P3 (7.5 µL; 1 µg/µL), control human serum albumin (SA) (5 µL; 1 µg/µL), and 14C-labeled

protein molecular weight marker mixture (5 µL; 0.17 µg/µL, 0.005 µCi/µL) were each loaded

into a well of a NuPAGE® Novex® 4-12% Bis-Tris Gel. The loaded gel was run using

NuPAGE® MOPS SDS Buffer Kit under non-reducing condition at a constant voltage (200 V)

for 50 min and stained using SimplyBlue™ SafeStain. For 14C-analysis, the same gel was dried using the DryEase® Mini-Gel Drying System and then 14C-labeled proteins were detected and quantified using a Cyclone Plus Storage Phosphor System (C431200; PerkinElmer).

Hepatocyte and S9 Fractions The LC-14C-HRMS analysis was the same as plasma analysis

except for the following. The sample was separated with a C18 column (Acquity HSS T3, 150 ×

3.0 mm, 1.8 µm; Waters Corp.), maintained at 50 °C. The following solvent gradient conditions

were employed at a flow rate of 0.5 mL/min: 0-1 min, 5% B; 1-34 min, 5-30% B; 34-39.5, 30-

100% B; 39.5-44.5 min, 100% B; 44.5-45 min, 100-5% B; 45-60 min, 5% B. The fractions were

collected at 7 seconds/well.

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Supplemental Table 1. Plasma pharmacokinetic parameters calculated using non compartmental analysis. 14C label → [14C]Ac-AMG 416 [14C]Ala-AMG 416 [14C]Ac-AMG 416 Kidney Function Normal Normal None Parameter* ↓ Group 4 Group 7 Group 8 Analyte → 14C 14C TM11 AMG 416 14C 14C 14C 14C TM11 AMG 416 Matrix → B P P P B P B P P P

λz 0.11 0.12 0.32 0.17 0.13 0.14 0.02 0.03 0.03 0.05

Cmax 4863 7563 8930 3050 3673 5050 3859 11303 10900 4090

AUCall 4961 7419 6971 2557 7337 9283 52308 134451 118150 24415

AUCinf 5101 7574 7278 2571 7472 9384 75148 184963 161134 26971

CL 0.49 0.33 NR 0.97 0.33 0.27 0.033 0.013 NR 0.09

VSS 1.63 0.95 NR 1.92 0.94 0.63 1.30 0.48 NR 1.66 Legend: B, blood; P, plasma; TM11, Total M11; NR, not reported due to limited data * Phoenix winnonlin reported NCA parameters: λz (1/hr), first order rate constant associated with the terminal (log-linear) portion of the curve. Estimated by linear regression of time vs. log concentration; Cmax (ng/mL), Maximum observed concentration; AUCall (ng*hr/mL), Area under the curve from the time of dosing to the time of the last observation; AUCinf (ng*hr/mL), AUC from dosing time extrapolated to infinity, based on the last observed concentration; CL (L/hr/kg), Total body clearance; Vss (L/kg), Volume of distribution at steady state.

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Supplemental Table 2. Excretion of radioactivity after a single IV dose administration of [14C]AMG 416 to bilaterally nephrectomized male rats (Group 8).

Recovery of Radioactivity (% of Administered [14C]Ac- Time a Matrix point AMG 416 Dose) (hr) Mean SD

G.I. Tract 0-72 10.9 2.76

Feces 0-48 1.20 0.57 Cage wash 0-72 0.03 0.02 All Total 12.1 2.45 a % Radioactivity in the HPLC profile = 100 × CPM counts in the area under the region of interest (ROI)/ sum of CPM counts in all ROI. CPM = 14C-counts per min. Legend: SD, standard deviation

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Supplemental Table 3. Concentrations of radioactivity, tissue to plasma ratio, Cmax, AUC, and t1/2 for select tissues after a single intravenous administration of [14C]AMG 416 to male Long Evans rats (Group 11). Tissue Distribution Time (hr) 0.083 1 48 168 1008a 2016a Pharmacokinetic Parameters

T T/P T T/P T T/P T T/P T T Cmax AUC t1/2 Bone 618 0.058 634 0.292 25.2 2.68 ND NA ND ND 863 6710 145b Bone marrow 1850 0.175 856 0.394 901 95.6 1110 232 242 69.3 1850 757969 559b Brain cerebellum 104 0.010 47.0 0.022 ND NA ND NA ND ND 152 104 0.295b Brain choroid plexus 619 0.058 159 0.073 ND NA ND NA ND ND 619 365 0.384b Cartilage (articular) 11700 1.10 9170 4.23 536 56.9 400 83.7 80.9 ND 18200 317614 369 Cartilage (hyaline) 16400 1.55 30700 14.1 2910 309 539 113 1190 532 36000 2178111 868b Cartilage (intervertebral) 21600 2.04 36200 16.7 1020 108 592 124 229 85.1 45400 711452 706b Epididymis 2340 0.221 1400 0.645 129 13.7 73.2 15.3 BLQ ND 2340 34584 107 Epiphyseal line 34900 3.29 44300 20.4 1010 107 524 110 124 ND 64100 652412 403 Eye lens 27.6 0.003 30.2 0.014 ND NA ND NA ND ND 30.2 12.1 NC Eye uveal tract 6250 0.590 2380 1.10 342 36.3 289 60.5 220 BLQ 6250 366379 NC Eye 684 0.065 312 0.144 53.7 5.70 36.3 7.59 22.3 18.4 684 60141 521b Kidney cortex 24700 2.33 27100 12.5 34900 3700 20600 4310 1560 163 50500 9801320 250 Kidney medulla 25600 2.42 10300 4.75 6580 699 5330 1120 640 151 25600 2342365 563 Kidney 25000 2.36 18700 8.62 24600 2610 17700 3700 1400 155 32300 7791905 283 Large intestine 5530 0.522 1960 0.903 196 20.8 125 26.2 BLQ ND 5530 54642 372b Liver 1930 0.182 2260 1.04 2000 212 1170 245 70.9 ND 3850 840783 231 Lung 6370 0.601 1720 0.793 184 19.5 85.6 17.9 17.5 ND 6370 77749 359 Lymph node 3050 0.288 1350 0.622 901 95.6 579 121 402 43.7 3050 664771 468 Myocardium 3690 0.348 820 0.378 66.6 7.07 37.7 7.89 BLQ ND 3690 19158 248b Pancreas 2050 0.193 534 0.246 82.0 8.70 66.3 13.9 BLQ BLQ 2050 46028 NC Plasma 10600 1.00 2170 1.00 9.42 1.00 4.78 1.00 BLQ BLQ 6170 12373 142b Skin (pigmented) 3860 0.364 1330 0.613 106 11.3 99.8 20.9 BLQ ND 3860 41434 196b Small intestine 2890 0.273 905 0.417 141 15.0 137 28.7 34.9 32.1 2890 109218 702 Spleen 2440 0.230 1250 0.576 1010 107 774 162 202 176 2440 763669 NC Stomach 4690 0.442 1930 0.889 107 11.4 91.5 19.1 23.4 BLQ 4690 73764 649 Testis 828 0.078 487 0.224 57.5 6.10 40.8 8.54 18.8 ND 828 36315 726 Thymus 1460 0.138 372 0.171 110 11.7 99.1 20.7 40.1 49.8 1460 111182 NC Thyroid 3490 0.329 983 0.453 168 17.8 134 28.0 59.5 53.7 3490 149490 8584b

Legend: AUC = area under the concentration-time curve from time 0 to the time of last observation (ng-eq/g); BLQ = below limit of quantitation; Cmax = maximum observed concentration (ng-eq/g); NA = not applicable; NC = Not calculated; ND = not detected; T = tissue concentration (ng-eq/g) ; P = plasma 14C concentration counted by liquid scintillation counting (ng-eq/g); t1/2 (hr), calculated terminal half-life; T/P = tissue to plasma concentration ratio. a T/P not determined because plasma concentrations were BLQ at these time-points b Number of sampling time points does not support the t1/2; t1/2 should be interpreted with caution

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Supplemental Table 4. Excretion profiles of biotransformation products from pooled urine after single IV dose administration of [14C]AMG 416 to bile duct cannulated rats.

Component ↓ Mean 14C Recovery (% of Administered Dose) in Urinea

Group → 1 2 5

[14C] label → [14C]Ac-AMG 416 [14C]Ac-AMG 416 [14C]Ala-AMG 416

Sex → Male Female Male

AMG416 36.8 34.9 46.9 M1 8.13 8.72 5.98 M2a 5.23 4.31 2.66 b M3,M2b 11.4 10.6 5.14 M4 2.36 2.08 1.51 M5 1.36 1.32 1.84 M6 0.78 1.10 0.58 M8 2.54 1.75 1.34 M9 1.73 1.29 1.21 M10 1.63 0.89 1.36 M11 < 0.1 <0.1 0.85 M12 3.11 2.05 1.67 b M14,M7 2.63 2.01 2.35 U1 1.62 2.22 1.33 a % Radioactivity in the HPLC profile = 100 × CPM counts in the area under the region of interest (ROI)/ sum of CPM counts in all ROI. CPM = 14C-counts per min. b Co-eluting metabolites. U1, unknown component

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Supplemental Table 5. Composition of AMG 416 conjugated peptides in the 14C-chromatogram of the Lys-C digest. Plasma obtained following incubation of [14C]Ala-AMG 416 (200 µM) with

human whole blood from healthy volunteer (HV) or chronic kidney disease patient (CKD) was

first subjected to affinity gel column chromatography. Peaks P2 and P3 from affinity gel column

separation were then digested with Lys-C.

Sequence Modified %14C-peak areab Name Peptide Sequence or Comments RT (min) Position Residue HV CKD C34-1 ALVLIAFAQYLQQCPFEDHVK 21-41 34 34.17 69.7 68.2 C34-2 ALVLIAFAQYLQQCPFEDHVKa 21-41 34 35.67 5.9 6.4 C34-3 IAFAQYLQQCPFEDHVK 25-41 34 24.33 * 1.5 U1 Unknown NA NA 4.00 2.1 3.4 U2 Unknown NA NA 32.00 8.5 4.1 Others Each component was <2% NA NA NA 13.8 16.4 NA, not applicable aThe peptide sequence within serum albumin was confirmed from the MS2 data, but the nature of modification was not fully characterized. b%14C-peak area, determined by (100*Selected Peak CPM)/Total CPM of identified peaks. *The peptide was detected in the MS, but the 14C peak area was below the limit of quantification.

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Supplemental Figure 1. Sample analysis workflow for identification of protein and peptide conjugates in plasma obtained following whole blood incubation of AMG 416. Details of incubation, sample preparation steps, and analysis are provided in methods.

Incubation Sample Preparation Analysis

14 [ C]AMG 416 + Plasma LC-HRMS Whole Blood

(1) Buffer Exchange

(2) Affinity-Gel Chromatography

(3) Desalt

(4) Lyophilize SDS-PAGE

14 (5) Lys-C Digestion LC- C-HRMS

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Supplemental Figure 2. Concentrations of radioactivity in blood and plasma after a single intravenous dose of 14C-AMG 416 to male (Group 12) and female (Group 13) Sprague Dawley rats

100000

Blood - Males 10000 Plasma - Males Blood - Females

C-AMG416/g) Plasma - Females 14 1000

100

10 ConcentrationEquivalents (ng 1 0 24 48 72 96 120 144 168 Time (Hours)

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