Special Section on Emerging Novel Enzyme Pathways in ...each hemodialysis session in CKD patients,...

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

Special Section on Emerging Novel Enzyme Pathways inDrug Metabolism

Nonclinical Pharmacokinetics, Disposition, and Drug-DrugInteraction 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

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

AMG 416 (etelcalcetide) is a novel synthetic peptide agonist of thecalcium-sensing receptor composed of a linear chain of sevenD-amino acids (referred to as the D-amino acid backbone) with aD-cysteine linked to an L-cysteine via a disulfide bond. AMG 416contains four basic D-arginine residues and is a +4 charged peptide atphysiologic pH with a mol. wt. of 1048.3 Da. The pharmacokinetics(PK), disposition, and potential of AMG 416 to cause drug-druginteraction were investigated in nonclinical studies with two single14C-labels placed either at a potentially metabolically labile acetylposition or on the D-alanine next to D-cysteine in the interior of theD-amino acid backbone. After i.v. dosing, the PK and disposition ofAMG 416 were similar in male and female rats. Radioactivity rapidlydistributed to most tissues in rats with intact kidneys, and renal

elimination was the predominant clearance pathway. No strain-dependent differences were observed. In bilaterally nephrectomizedrats, minimal radioactivity (1.2%) was excreted via nonrenal path-ways. Biotransformation occurred primarily via disulfide exchangewith endogenous thiol-containing molecules in whole blood ratherthan metabolism by enzymes, such as proteases or cytochromeP450s; the D-amino acid backbone remained unaltered. A substantialproportion of the plasma radioactivity was covalently conjugated toalbumin. AMG 416 presents a low risk for P450 or transporter-mediated drug-drug interactions because it showed no interactionsin vitro. These studies demonstrated a 14C label on either the acetyl orthe D-alanine in the D-amino acid backbone would be appropriate forclinical studies.

Introduction

Secondary hyperparathyroidism (HPT), a consequence of chronickidney disease (CKD) in humans, is a chronic and progressive dis-ease characterized by elevated levels of parathyroid hormone (PTH),disturbances in the homeostasis of calcium, phosphorus, vitamin D,

and fibroblast growth factor 23 (FGF-23) levels and parathyroid glandhyperplasia (Rodriguez et al., 2005; Tfelt-Hansen and Brown, 2005;Goodman and Quarles, 2008; Cunningham et al., 2011). Left untreated,secondary HPT leads to disturbances in mineral metabolism, bone disease,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, andphosphate binders (Nemeth et al., 1998; Block et al., 2004).AMG 416 (etelcalcetide, Amgen Inc, Thousand Oaks, CA) (Fig. 1) is a

novel calcimimetic currently in clinical development for the treatment ofsecondaryHPT. Studies have shown that AMG416 lowers PTH levels andnormalizes mineral metabolism in uremic animal models (Walter et al.,2013, 2014). AMG 416, administered i.v. three times weekly at the end of

This study was funded by Amgen Inc., Thousand Oaks, California.1Current affiliation: Department of Pharmaceutics, University of Washington,

Seattle, Washington.2Current affiliation: Antiva Biosciences, South San Francisco, California.dx.doi.org/10.1124/dmd.115.068007.s This article has supplemental material available at dmd.aspetjournals.org.

ABBREVIATIONS: AUC, area under the curve; BCRP, breast cancer resistance protein; BSEP, bile salt export pump; BN, bilaterallynephrectomized; CKD, chronic kidney disease; FA, formic acid; HPT, hyperparathyroidism; IS, internal standard (13C3D3

15N-AMG 416); LC, liquidchromatography; LC-14C-HRMS, liquid chromatography-14C-high resolution mass spectrometry; LC-MS/MS, liquid chromatography-tandem massspectrometry; 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

http://dmd.aspetjournals.org/content/suppl/2016/02/19/dmd.115.068007.DC1Supplemental material to this article can be found at:

at ASPE

T Journals on July 2, 2021

dmd.aspetjournals.org

Dow

nloaded from

http://dx.doi.org/10.1124/dmd.115.068007http://dx.doi.org/10.1124/dmd.115.068007http://dmd.aspetjournals.orghttp://dmd.aspetjournals.org/content/suppl/2016/02/19/dmd.115.068007.DC1http://dmd.aspetjournals.org/

each hemodialysis session in CKD patients, has demonstrated PTH-lowering effect and improved treatment adherence with reduced adverseevents (Martin et al., 2014; Bell et al., 2015; Cozzolino et al., 2015).AMG 416 is a novel synthetic peptide and is composed of a linear

chain of seven D-amino acids, referred herein as the “D-amino acidbackbone” of the molecule, and an L-cysteine linked to an N-terminalD-cysteine through a disulfide bond. The N-terminal D-cysteine and theC-terminal arginine in AMG 416 are capped with acetyl and amidegroups, respectively. AMG 416 contains four basic D-arginine residuesand is a positively charged peptide (+4) at physiologic pH (7.4) witha mol. wt. of 1048.3 Da. Given the structure of AMG 416, it led us tohypothesize that the molecule may be expected to be: 1) poorlypermeable unless assisted by active transport; 2) unlikely to interactwith cytochrome P450s (P450s) because it bears little resemblance totheir known substrates; 3) resistant to protease-mediated hydrolysisowing to the presence of a D-amino acid backbone; 4) biotransformedby disulfide exchange, specifically by endogenous thiols in blood, andthis pathway will likely be a significant metabolic pathway for thismolecule; and 5) mainly cleared by renal elimination. However, AMG416 is administered clinically to CKD patients with minimal kidneyfunction and on maintenance hemodialysis.There is no approved drug that consists primarily of D-amino acids and

minimal literature is available on the fate of predominantly D-amino acid–containing peptides. The objective of the present study was to test thehypotheses stated above and overall, to characterize the nonclinicalpharmacokinetics and disposition of AMG416 using in vitromethods andin vivo in rats with normal and no kidney function. Also, the potential ofAMG 416 to cause P450 or transported mediated drug-drug interactionswas determined in vitro. The findings from these studies helped in theselection of a radiolabel site in AMG 416 for clinical studies.

Materials and Methods

Test Articles and Reagents

The structures and nomenclature for AMG 416 are shown in Fig. 1. AMG416 was synthesized by Bachem (Torrance, CA) and stored in 0.1%trifluoroacetic acid (TFA) in water at pH 4.0. Three lots of [14C]AMG 416,each incorporating a single 14C label, were used in the in vivo studies. Ofthose, two lots designated [14C]Ac-AMG 416, incorporated 14C into thecarbonyl carbon of the acetyl moiety. The first, [14C]Ac-AMG 416 lot(specific activity 28.2 mCi/mmol; 95.1% radioactive purity), was synthesizedby PolyPeptide Laboratories (San Diego, CA). The second, [14C]Ac-AMG 416lot (specific activity 60.3 mCi/mmol, 98.8% radioactive purity), wassynthesized at Amgen (Thousand Oaks, CA). The third lot, designated[14C]Ala-AMG416 (specific activity 56.3 mCi/mmol; 97.0% radioactive purity),contained the 14C-label on the carbonyl carbon of the D-alanine adjacent toD-cysteine moiety and was synthesized by PolyPeptide Laboratories. The firstand third lots of radiolabeled AMG 416 contained a single degradant (M5, adeamidation product of AMG 416) present at approximately 2% of the total 14Ccontent. AMG 416 internal standard (IS; 13C3D3

15N-AMG 416), M10,M11, andM12were supplied by Amgen. (Waltham,MA). Tris(2-carboxyethyl phosphine)(TCEP) hydrochloride was obtained from Sigma-Aldrich (St. Louis, MO).

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

In Vitro Methods

Blood-to-Plasma Ratio and Red Blood Cell Uptake. Pooled whole bloodfrom rats, healthy human volunteers, and CKD patients was incubated with[14C]Ac-AMG 416 at three concentrations (0.1, 1.0, and 10 mM) at 37�C for4 hours. An aliquot of blood (100 ml) was removed, and the remainder of themixture was centrifuged at 14,000g for 5 minutes at 4�C 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 redblood cell (RBC) experiment buffer (see Supplemental Material) and gentlymixed. Samples were centrifuged at 150g for 5 minutes at 5�C. The supernatantswere then removed and replaced with one volume of fresh cold RBC buffer. Theprevious step was repeated four times to obtain washed RBSs. After the finalwash, the RBC pellet was suspended in an equal volume of the cold RBCexperiment buffer and used for direct uptake experiments. [14C]Ac-AMG 416(10 mM) was incubated in the washed RBC-buffer mixture at 37�C for 2 hours.After centrifugation, the supernatant was separated, and the pelleted RBCs werelysed with cold ammonium-chloride-potassium (ACK) lysing buffer. Theradioactivity of both supernatant and the lysate was determined by 14C analysis.

Noncovalent Plasma Protein Binding. An ultrafiltration method was usedto determine the noncovalent protein binding of AMG 416 while minimizing itscovalent binding to plasma proteins. An ultrafiltration method was used for thispurpose because the unbound fraction could be separated from the plasma within12 minutes, whereas other methods, including equilibrium dialysis andultracentrifugation, require hours to obtain the unbound fraction.

Whole blood from rats, dogs, healthy human volunteers, and CKD patientswas incubated with AMG 416 (50, 1000 or 10,000 ng/ml) on an orbital shaker at100 rpm for 4 hours at 37�C in 5% CO2 and 90% relative humidity and thenimmediately centrifuged at 5250g for 10 minutes to obtain plasma. The plasmawas divided into two aliquots. One aliquot of the plasma was acidified with theaddition of formic acid (FA; 1% final concentration, v/v) and subsequentlyprepared and analyzed as plasma control representing the total startingconcentration of AMG 416 in plasma. The other aliquot of plasma from wholeblood incubations was placed in 30,000 Da cutoff ultrafiltration devices (500 mleach in quadruplicate) and centrifuged at 2000g in a fixed angle rotor for12 minutes. At the end of centrifugation, the residual filtered plasma and theultrafiltrate were added to FA to achieve 1% final acid (v/v). The AMG 416 inplasma and filtrate was quantified by liquid chromatography-tandem massspectrometry (LC-MS/MS) method described in Supplemental Material.

P450 and Transporter Interactions. The P450 metabolic stability of AMG416 (5 mM) was determined in HLM and human liver S9 fractions (HLS9) at1 mg/ml protein concentration in pH 7.4 phosphate buffer (100 mM) with orwithout NADPH and with or without 1% FA at 37�C. In addition, HLM andHLS9 were preincubated with 1-aminobenzotriazole (5 mM) and NADPH toinactivate P450 enzymes before the addition of AMG 416.

The reversible inhibition potential of AMG 416 (0.2, 1, and 5 mg/ml) towardhuman P450 isozymes 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, 2B6, and 3A4 wasevaluated following a published method (Walsky and Obach, 2004). The time-dependent inhibition potential of AMG 416 (5 mM) toward human P450 isozymes1A2, 2C8, 2C9, 2C19, 2D6, and 3A was evaluated according to published methods(Atkinson et al., 2005; Obach et al., 2007). Induction of human P450 isozymes1A2, 2B6, and 3A4 by AMG 416 was evaluated in cryopreserved primary humanhepatocytes following a previously published method (Hewitt et al., 2007). Thehepatocytes were incubated with AMG 416 at 0.2, 1, and 5 mg/ml for the first day;higher concentrations of 0.4, 2, and 10mg/mlwere used on the second and third days.

The permeability and transporter assays measured vectorial transport or ac-cumulation of [14C]AMG 416 or the radiolabeled probe substrates in cell-linemonolayers using published methods with slight modifications. Appropriatepositive and negative controls were used in each assessment. The transcellularpermeability with LLC-PK1 and efflux transport by P-glycoprotein (Pgp) andbreast cancer resistance protein (BCRP) was assessed in MDR1-LLC-PK1 andBCRP-MDCKII cell monolayers (Schinkel et al., 1995; Booth-Genthe et al.,2006). The [14C] AMG 416 direct uptake assay was conducted in HEK293 cellstransfected with organic anion transporter (OAT) 1, OAT3, OAT polypeptide(OATP) 1B1, OATP1B3, peptide transporter 1, 2, or organic cation transporter

Fig. 1. Structure of AMG 416 (etelcalcetide). Ac, the acetyl group on theN terminus of the D-cysteine; NH2, denotes amidation on the C terminus of theD-arginine. 14C-label was placed on the carbonyl carbon on either the acetyl moiety.([14C]Ac-AMG 416,denoted by *) or the D-alanine moiety ([14C]Ala-AMG 416,denoted by #).

1320 Subramanian et al.

at ASPE



Dow

nloaded from

http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/

(OCT) 2 transporter (Yamazaki et al., 2005; Chu et al., 2007). The effect of AMG416 on human bile salt export pump (BSEP) transporter was evaluated accordingto a published method (van Staden et al., 2012).

Whole-Blood Incubation for Plasma Analysis. Whole blood from rats,healthy human volunteers, and CKD patients was incubated with AMG 416(200 mM), [14C]Ac-AMG 416 (1, 5,10, or 200 mM), or [14C]Ala-AMG 416(200 mM) at 37�C. Mixtures were shaken at 300 rpm for 4 hours and thenimmediately centrifuged at 14,000g for 5 minutes at 4�C. Plasma wastransferred to a clean vial containing an appropriate amount of aqueous FA(final concentration, 1% FA v/v), and then stored at 280�C until furtheranalysis.

S9 Fraction and Hepatocyte Incubations. Rat and human liver and kidneyS9 fraction (2 mg/ml final concentration) incubations were conducted in a finalvolume of 1 ml of buffer (100 mM phosphate buffer with 3 mMMgCl2, pH 7.4)with either [14C]Ac-AMG 416 or [14C]Ala-AMG 416 (7.5 mM) and AMG 416(2.5 mM) at 37�C. Reaction mixtures were terminated after 6 hours by theaddition of FA (final concentration, 10% v/v). For the 0-hour incubations, 10mMof the corresponding 14C-labeled AMG416was added to 0.5 ml of S9 suspendedbuffer and immediately quenched. The quenched reaction mixture was vortex-mixed, centrifuged at 20,000g for 20 minutes at 4�C, and then stored immediatelyat 280�C until further analysis.

Pooled cryopreserved human hepatocytes (two separate lots) were suspendedin 1 ml of Krebs-Henseleit buffer at a final concentration of two million cells permilliliter. To these cell suspensions, either [14C]Ac-AMG 416 or [14C]Ala-AMG416 (10 mM) was added. Cell mixtures were incubated at 37�C in a water bathshaken at 75 rpm in a humidified (95% relative humidity) atmosphere of 95%oxygen and 5% CO2 and terminated after 2 hours by the addition of FA (finalconcentration, 10% v/v). The quenched reaction mixture was vortex-mixed,centrifuged at 14000g for 6 minutes at 4�C, and then stored at 280�C untilLC-14C-HRMS analysis. For the 0-hour incubations, 10 mM of [14C]AMG 416was added to 1 ml of hepatocyte suspension, quenched immediately, andprocessed as described herein.

In Vivo Methods

Study Design. The study overview is outlined in Table 1, and the individualexperiment details are provided as follows under the respective group identifier.All animals were acclimated to study conditions for 4 to 5 days before dosing.During acclimation and the test periods, animals were housed in individual,suspended, stainless steel wire-mesh cages. All studies were performed in 7- to12-week old male and female Sprague-Dawley (SD) rats except group 11 withmale Long-Evans (LE) rats, weighing 190–350 g, which were obtained fromCharles River Laboratories (Hollister, CA) and were cared for in accordance tothe Guide for the Care and Use of Laboratory Animals, 8th edition (NationalResearch Council, 2011). Animals were single-housed at an Association forAssessment and Accreditation of Laboratory Animal Care, internationalaccredited facility, and all research protocols were approved by the InstitutionalAnimal Care and Use Committee. All animals were maintained on a 12-hourlight/dark cycle in rooms at 18–26�C and 10%–70% humidity. Animals in allgroups had access to water ad libitum for the duration of each study. Bilaterallynephrectomized (BN) rats received sugar cubes as food, and animals in all theremaining groups received the Harlan Laboratory diet (no. 2020X or no. 2016C).All radioanalysis data were generated with Debra (versions 5.7 or 6.0; LabLogicSystems Ltd., Sheffield, UK). All studies were conducted at Amgen except thequantitative whole-body autoradiography (QWBA; groups 11–13), which wereperformed at Covance Laboratories Inc (Madison, WI). All animals weredetermined free of specific pathogens (http://www.criver.com/files/pdfs/app/direct-animal-sampling-rat-chart.aspx). All collected samples were storedimmediately after collection at 280�C until further analysis.

Dose Solution Preparation. For all studies, AMG 416 was formulated inphosphate-buffered saline to achieve the target dose volume of 0.5 ml/kg. Analiquot of each dose-solution was analyzed for both total 14C and the radio-puritybefore and after dose administration. All rats received a single i.v. bolus dose,which was administered via a surgically implanted jugular vein catheter (groups1–10) or tail vein (groups 11–13).

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

2F (4) Bile: 0–4, 4–8, 8 24, 24–48, 48–72 h

Urine: 0–8, 8–24, 24–48, 48–72 hFeces: 0–24, 24–48, 48–72 h

3F (4) B/P, plasma met ID

Blood: 0.25, 1, 4, 8, 24 h

4M (10) 2.5 (50) B/P, plasma AMG 416 and total M11 bioanalysis, and plasma met ID

Blood: 0.083, 0.25, 0.5, 1, 2, 4, 8, 24 h

5

Ala M (4) 2.5 (38) Mass balance, excreta met IDBile: 0–4, 4–8, 8–24, 24–48, 48–72 hUrine: 0–8, 8–24, 24–48, 48–72 hFeces: 0–24, 24–48, 48–72 h

6M (4) plasma met ID

Blood: 0.25, 1, 4, 8 h

7M (2) B/P

Blood: 0.25, 1, 4, 8 h

8

None Ac M (6) 2.5 (50) Mass balance, B/P, plasma AMG 416 and total M11 bioanalysis, andplasma met ID

Feces: 0–24, 24–48Blood: 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48 h

9 Normal Ac M (1) 2.5 (50) 14C in expired air10 Ala M (1) 2.5 (38) Expired air: 0–8, 8–24

11b Ac M (16) 2.5 (50) B/P, QWBA12 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.

Nonclinical Pharmacokinetics and Disposition of Etelcalcetide 1321

at ASPE



Dow

nloaded from

http://www.criver.com/files/pdfs/app/direct-animal-sampling-rat-chart.aspxhttp://www.criver.com/files/pdfs/app/direct-animal-sampling-rat-chart.aspxhttp://dmd.aspetjournals.org/

Groups 1, 2, and 5. Both groups consisted of the jugular vein cannulated andbile duct cannulated from male and female rats, respectively. Each rat underwentsurgery 7–10 days before study initiation to implant two catheters: one into thejugular vein for i.v. dose administration, and the other into the bile duct with aduodenum return. Before drug administration, flow from the bile duct catheterwas diverted to collect bile and a solution of taurocholic acid (0.0134 g/ml) insaline (9 mg/ml NaCl, 0.5 mg/ml KCl) was continuously infused into theduodenum at a rate of 1 ml/hour for the duration of the study. Each rat wasadministered [14C]AMG 416 (see details in Table 1) and placed in a metaboliccage. Following dosing, bile and urine were collected into prefrozen (280�C)BASi I-Cup devices (Bioanalytical Systems, Inc., West Lafayette, IN), and feceswere collected at room temperature.

Group 3, 4, 6, and 7. Rats were implanted with jugular and femoral veincannulae 7–10 days before study initiation. After dose administration, blood(200–1000 ml) was collected at designated time points via the femoral veincatheter into lithium heparin blood collection tubes and placed on wet ice. Thecollected blood was temporarily stored on wet ice and then centrifuged at 4500gfor 10 minutes to separate the plasma. The plasmawas acidified with aqueous FA(2 ml of 1:1 v/v stock for 100 ml of plasma).

Group 8. Animals in this groupweremaleBN rats. Each rat underwent a bilateralnephrectomy procedure after a 48-hour diet consisting of sugar cubes. Animals wereanesthetized with isoflurane (1%–4% in O2 at a rate of 1.5 liters/min), and thekidneys were located and aseptically removed using the procedure described byWaynforth and Flecknell (1992). Within 2 hours after surgery, each rat receivedan i.v. dose of [14C]Ac-AMG 416 (Table 1) and was housed in a metabolic cage.Blood and feces were collected at designated time points. Immediately aftereuthanasia, select tissues were excised and stored for 14C-analysis.

Groups 9 and 10. The study was conducted to determine the 14CO2 in expiredair (Table 1). The setup consisted of an airflow gauge placed in line before an airmetabolism cage (Metabowl MKIII system; Jencons Scientific, Bridgeville, PA)followed by two sequential Nilox columns (Jencons) each containing Carbosorb(Carbosorb E; PerkinElmer, Waltham, MA). A unidirectional airflow of 900 ml/minwas maintained through the metabolism cages, and the exhaust air was bubbledthrough 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.Before each collection interval, each CO2 trap chamber was filled with Carbosorb(approximately 300 ml each). After each collection interval, Carbosorb in the bubblingchambers was removed for 14C-analysis, and its volume was measured and recorded.

Groups 11–13. These groups were used to characterize the tissue distribu-tion of [14C]AMG 416 by QWBA in LE and SD rats (Table 1). Animals weresacrificed via exsanguination (cardiac puncture) under isoflurane anesthesia, andblood (2–10 ml) was collected into a tube containing K2EDTA immediatelybefore collection of carcasses for QWBA. An aliquot of collected blood fromeach time point was analyzed for 14C content, and the remainder was centrifugedto separate the plasma. The carcasses were immediately frozen in a hexane/dryice bath for approximately 8 minutes. Each carcass was drained, blotted dry,placed into an appropriately labeled bag, and placed on dry ice or stored atapproximately270�C for at least 2 hours. The frozen carcasses were embeddedin chilled carboxymethylcellulose (;6�C; 2% w/v) and frozen at approxi-mately 220�C into blocks. Frozen blocks were imbedded with [14C]glucose(;140,000 dpm/g) quality control reference standards to verify intersection andintrasection thickness variability.

14C analysis. Blood, RBCs, plasma, bile, urine, feces, and expired air wereprocessed and counted in a liquid scintillation counter according to systemmanufacturer instructions and are described in Supplemental Material.

QWBA sample preparation and analysis. Sagittal whole-body sections(40-mm thickness) of rat carcasses were collected on adhesive tape using acryomicrotome (Leica CM 3600; Buffalo Grove, IL) maintained at 220�C.Sections were collected to show major tissues, organs, and biologic fluids.Collected sections were dried in the microtome at approximately220�C. Sectionswere attached to matting board, wrapped with Mylar film, and exposed for 4 daysto phosphor imaging screens. Screens were coexposed to [14C]glucose bloodstandards (0.980 to 10300 nCi/g) for radioactivity quantification. Exposed screenswere scanned using a PhosporImager (Storm; GE Life Sciences, Pittsburgh, PA), andtissue concentrations of radioactivity were determined using MCID Analysissoftware (InterFocus Imaging Ltd, Cambridge, UK). Tissue concentrations of

radioactivity were interpolated from each standard curve and then converted tong-equivalents/g (ng-eq/g) using the [14C]AMG 416 specific activity.

AMG 416 and total M11 bioanalysis. AMG 416 and total M11 weremeasured in plasma by LC-MS/MS as described in Supplemental Material. Todetermine the proportion of biotransformation products in plasma that retainedthe intact D-amino acid backbone, all AMG 416-related homo- and hetero-disulfides were reduced with TCEP, a disulfide bond reducing agent. The M11formed upon TCEP reduction is referred to as “Total M11” because it representsall AMG 416–related material present in incubations. Total M11 (also calledTM11 in biotransformation profiles) is distinct from M11 present in the in vitroincubates due to biotransformation (described in Results).

Sample preparation for metabolite analysis. Urine: Urine was first pooled inproportion to the volume collected at each time period (0–72 hour) from eachanimal in the same group. A grand pool was then prepared by combining equalvolume from each animal pool. An aliquot of pooled urine was centrifugedat 20,817g for 10 minutes. The supernatants were submitted to liquidchromatography-14C-high resolution mass spectrometry (LC-14C-HRMS) anal-ysis. To inform the structure of the biotransformation products in urine, all AMG416-derived products were reduced with TCEP to liberate total M11. An aliquotof pooled urine was added TCEP (20 mM final concentration), vortex-mixed,and allowed to stand at room temperature overnight. The resultant reduced urinewas then subjected to LC-14C-HRMS analysis.

Plasma: Equal volumes of plasma from each rat were first pooled at each timepoint (0–48 hours for group 8 or 0–24 hours for other groups), and then a time-proportional pool was prepared according to the Hamilton method (Hamiltonet al., 1981) to obtain a single sample for rats in the respective group. Pooledplasma samples were diluted 8-fold with 0.1% FA and then directly injected forLC-14C-HRMS analysis. Additionally, an aliquot of the diluted pooled plasmawas added TCEP aqueous solution (10 mM final concentration) and incubated at37�C for 1.5 hour before LC-14C-HRMS analysis. This same procedure was usedto prepare the TCEP-reduced plasma from in vitro whole blood incubations. Theworkflow to track the protein modifications is shown in Supplemental Fig. 1 andis described in Supplemental Material.

LC-14C-HRMS Analysis.Plasma and urine. Plasma samples were diluted 8-fold with 0.1% aqueous

FA (v/v) before LC-14C-HRMS analysis. Analyte separations were achievedon an Agilent 1200 system (Agilent Technologies Inc., Wilmington, DE),including a binary pump (G1312B), an autosampler (G1367C), and atemperature controlled column compartment (G1316B) with a C18 column(XSelect HSS T3 C18, 250 � 4.6 mm, 3.5 mm; Waters Corp., Bedford, MA)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 plasmaanalysis, the following solvent gradient conditions were used at a flow rate of1 ml/min: 0–1 minute, 10% B; 1–31 minutes, 10%–16.4% B; 31–36.5 minutes,16.4%–100%B; 36.5–50.5 minutes, 100% B; 50.5–51 minutes, 100%–10%B;51–65 minutes, 10% B. Postcolumn fraction collection for 14C-profiling andMS analyses were performed with a CollectPal HTC fraction collector (LeapTechnologies, Carrboro, NC) and a LTQ Orbitrap Velos high-resolution massspectrometer (Thermo Fisher Scientific Inc.), respectively. The postcolumnflow was split with 75% of the flow directed to the fraction collector and theremaining 25% to the MS. A total of 384 fractions were collected onto a384-well Deepwell LumaPlate (PerkinElmer) at 8 seconds/well. The columnrecovery of the radioactivity was 93.5% for the direct analysis of humanplasma. After the LumaPlate was dried in a vacuum centrifuge, theradioactivity of each well was counted by a TopCount reader (PerkinElmer)using a normalized 14C protocol. Radiometric data were then imported andanalyzed using Laura software (version 4.1.13.91; IN/US Systems, TampaBay, FL). All MS data were acquired using a heated electrospray ionizationsource in the positive-ion mode. The MS instrument conditions were asfollows: capillary temperature, 350�C; source temperature, 300�C; sourcevoltage, 5 kV; sheath gas (N2) flow rate, 35 (arbitrary units); auxiliary gas (N2)flow rate, 10 (arbitrary units); S-Lens RF level, 47%. MS2 data were collectedusing a collision energy of 35%. The in vivo plasma and urine and in vitro S9and hepatocyte supernatants were analyzed using a similar method and aredescribed in Supplemental Material.

Serum albumin peptide digest. The LC-14C-HRMS analysis was the same asplasma analysis with the following exceptions. Analyte separations were


at ASPE



Dow

nloaded from

http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/

achieved with a C18 column (Vydac 218MS52, 250 � 2.1 mm, 5 mm; Grace,Deerfield, IL) maintained at 40�C. Mobile phases consisted of 0.1% FA in water(solvent A) and 0.1% FA in acetonitrile (solvent B). The following solventgradient 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; 46–58 minutes, 80% B, 0.3 ml/min; 58–60 minutes, 80%–3% B, 0.3–0.2 ml/min;60–65 minutes, 3% B, 0.2 ml/min. The fractions were collected at 10 s/well. TheMS instrument conditions were as follows: capillary temperature, 275�C; sourcetemperature, 275�C; source voltage, 4 kV; sheath gas (N2) flow rate, 25 (arbitraryunits); auxiliary gas (N2) flow rate, 10 (arbitrary units); S-Lens RF level, 61%.The column recovery of the radioactivity was 99.0%.

Plasma protein conjugate. The intact protein MS analysis was conducted onan ultra-high-performance liquid chromatography-Q-TOF system, which con-sisted of an autosampler (CTC PAL, Leap Technologies) and an Agilent 1290system (Agilent Technologies Inc.), including a binary pump (G4220A), atemperature-controlled column compartment (G1316C), and a DAD detector(G4212A). Analyte separations were achieved with a C18 column (POROSR2/10, 100� 2.1 mm, 10mm; Applied Biosystems, Foster City, CA) maintainedat 45�C. Mobile phases consisted of 0.1% FA in water (solvent A) and 0.1% FAin acetonitrile (solvent B). The following solvent gradient conditions were used:0–1 minute, 20% B, 1.0 ml/min; 1–2 minutes, 20%–35% B, 1.0–0.4 ml/min; 2–6minutes, 35%–45%B, 0.4 ml/min; 6–6.5 minutes, 45%–95%B, 0.4–1.0 ml/min;6.5–8.5 minutes, 95% B, 1.0 ml/min; 8.5–9 minutes, 95%–20% B, 1.0 ml/min; 9–10minutes, 20%B, 1.0ml/min. Mass spectrometric analysis was performedwith aTripleTOF 5600+ system (AB Sciex, Foster City, CA) operating in the positiveion and intact protein mode using a DuoSpray Ion Source. The instrument wascalibrated using atmospheric pressure chemical ionization positive calibrationsolution (AB Sciex) before analysis. TheMS parameters were as follows: curtaingas, 10 (arbitrary units); ion source gas 1, 50 (arbitrary units); ion source gas 2,50 (arbitrary units); temperature, 0�C; ion spray voltage floating, 5.5 kV;declustering potential, 250 V.

PK analysis. Concentrations of radioactivity were converted into ng equivalentunits before PK analysis. PK parameters were calculated by noncompartmentalanalysis using PhoenixWinnonlin (version 6; Pharsight, Cary, NC) for the followingconcentration data sets: total radioactivity in blood and plasma, LC-MS/MSdetermined AMG 416 in plasma, and LC-MS/MS determined Total M11 in plasma.

Identification of biotransformation products. Structure assignments of AMG416 biotransformation products were based on comparisons to authenticstandards, calculated elemental composition of the products from high-resolution MS data, the 12C and 14C isotopic peak pattern, and interpretationof the LC-MS/MS data. Authentic standards were available for M10, M11, andM12 and the identity of the biotransformation products was verified bycomparison of the retention time, MS, and MS2 spectra.

Results

Blood-to-Plasma Ratio and Uptake into Red Blood Cells

In vitro, the 14C blood to plasma ratio in rat, healthy human, andCKD patient was 0.51, 0.50, and 0.69, respectively, and independent ofAMG 416 concentration. In vivo, the 14C area under the curve (AUC) inblood was smaller than 14C AUC in plasma in normal and BN rats(Supplemental Table 1). These results indicate that [14C]AMG 416-derived radioactivity was preferentially retained in plasma. The uptakeexperiment of [14C]AMG 416 into RBC indicated approximately 10%of [14C]AMG 416 was recovered in the RBC lysate. There was nochange in 14C concentration in RBCs or in the buffer over the 2-hourincubation time indicating AMG 416-derived radioactivity was poorlypermeable into RBCs.

Noncovalent Protein Binding in Plasma

The mean noncovalent unbound fraction of AMG 416 in plasma was0.77 (rat), 0.67 (dog), 0.53 (healthy volunteers), and 0.59 (CKDpatients). Plasma protein binding was independent of AMG 416concentration (50–10,000 ng/ml; approximates 0.05–10 mM) in allspecies.

P450 and Transporter Interactions

AMG 416 stability in HLM and HLS9 was not significantly affected bythe presence or absence of NADPH, and was not significantly differentbetween incubations that were preincubated with NADPH in the presenceor absence of 1-aminobenzotriazole. In the absence of any role of P450 in itsbiotransformation, AMG 416 concentrations decreased with time in HLMand HLS9 incubations at pH 7.4. AMG 416 disappearance was completelyinhibited in presence of 1% FA in HLM and HLS9 solutions at pH 3.AMG 416 was not a reversible or time-dependent inhibitor of the tested

P450 isoforms in HLM, the enzymatic activity in all AMG 416 treatedgroups was within 88% to 117% of the corresponding negative controlgroup. AMG416was not an inducer of P450 isoforms 1A2, 2B6, and 3A4in human hepatocytes; the activities of all three P450 isoforms were,2%of the corresponding positive controls in all three donors.The average apparent permeability for AMG 416was 0.7� 1026 cm/s

in LLC-PK1 cells. AMG 416 was not a substrate or inhibitor of Pgp orBCRP transporters. The average Pgp and BCRP efflux ratio was closeto 1.0 and independent of the AMG 416 concentration tested. AMG 416was neither a substrate nor an inhibitor of the uptake transporters tested.Probe substrate uptake into OAT1, OAT3, OATP1B1, OATP1B3, orOCT2 transfected cells was 0.7–1.04-fold of the respective controlsvalues and independent of the AMG 416 concentration tested. Also,AMG 416 had no effect on human BSEP activity.

Excretion Balance

The excretion data after a single i.v. administration of [14C]AMG 416to bile duct cannulated rats are presented in Table 2. In the 72 hours after asingle dose of [14C]Ac-AMG 416 (groups 1 and 2) or [14C]Ala-AMG416 (group 5), a total of 83%–88% of the radioactive dose was recovered.The radioactivity was predominantly recovered in urine (77%–84%;.90% of recovered dose) withminor amounts in feces (,4%) and in bile(,1%). 14C excretion was similar for both sexes and the two radiolabels.In BN rats (group 8; Supplemental Table 2), a small amount of the dosewas excreted in feces (1.2%), and approximately 11% of the dose wasrecovered from the gastrointestinal tract by 48 hours. In rats with intactkidney function (groups 9 and 10), minimal amount of the dose (,0.2%)was eliminated in expired air within 24 hours.

Plasma PK (14C, AMG 416, and Total M11)

A representative concentration-time profile of 14C in blood andplasma, AMG 416 in plasma, and total M11 in plasma from groups 4, 7,and 8 are shown in Fig. 2. Noncompartmental analysis-derived PK

TABLE 2

Excretion of radioactivity after a single i.v. dose administration of [14C]AMG 416 tobile-duct cannulated Sprague-Dawley rats

Recovery of Radioactivity (% of Administered Dose)

Group → 1 2 5

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

Sex → Male Female Male

Matrix Time (h) Mean S.D. Mean S.D. Mean S.D.

Urine

0–8 67.9 7.54 58.5 7.30 63.3 7.348–24 11.0 3.13 13.6 4.71 9.30 1.74

24–48 3.52 0.94 3.41 1.81 3.01 0.8748–72 1.35 0.72 1.16 0.24 1.69 0.32Subtotal 83.8 4.13 76.6 5.94 77.3 7.91

Bile 0–72 0.52 0.34 0.28 0.09 0.64 0.62Feces 0–72 1.34 0.69 3.90 2.77 3.79 1.98Cage wash 0–72 2.13 0.11 2.30 0.81 2.30 1.09All Total 87.7 4.47 83.1 4.79 84.1 9.32


at ASPE



Dow

nloaded from

http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/

parameters are summarized in Supplemental Table 1. PK profiles in bloodand plasma were similar for male rats dosed with [14C]Ac-AMG 416and [14C]Ala-AMG 416 (Fig. 2A) and the female rats dosed with[14C]Ac-AMG 416 (data not shown). 14C concentrations in bloodand plasma declined through 24 hours postdose followed by a slowerelimination phase; the latter was better captured in 14C PK profileobtained in the QWBA analysis (groups 12 and 13; SupplementalFig. 2). The rate of decline in 14C and AMG 416 plasma concentrationswas significantly lower in BN rats comparedwith the normal rats (Fig. 2B).The plasma PK profiles for the total M11 closely matched the profile oftotal radioactivity (Fig. 2B). The plasma 14C clearance in male rats dosedwith [14C]Ac-AMG 416 or [14C]Ala-AMG 416 was similar (approxi-mately 0.30 liter/h per kilogram). Removal of both kidneys resulted in an18-fold increase in plasma 14C AUC and a substantially lower clearance(approximately 0.013 l/h per kg), which was approximately 25 times lowerthan that observed in rats with intact kidneys. The plasma AMG 416clearance was significantly higher than the 14C clearance at approximately3-fold and 5.5-fold in normal and BN rats, respectively.

Tissue Distribution

A plot of 14C concentration versus time for select tissues in male LErats is shown in Fig. 3. A listing of the select 14C concentration-timedata and the corresponding Cmax, AUC, and half-life for select tissuesare presented in Supplemental Table 3 for male LE rats. The plasma 14C

volume of distribution (Vss) in LE rats was high (7230 liters/kg;group 11) and greater than total body water. For the LE rats (group11), maximal levels of radioactivity occurred in most tissues by 0.5hour postdose followed by a decline in 14C concentration over time.Maximal 14C concentration in kidney, kidney cortex, and liver wasobserved at 12 hours postdose. Matrices with the highest 14Cconcentrations were urine, epiphyseal line, kidney cortex, interver-tebral cartilage, hyaline cartilage, kidney, kidney medulla, articularcartilage, and bile. Tissues with the lowest Cmax values (,500 ng-eq/g)were the eye lens, brain (medulla, olfactory lobe, cerebrum, andcerebellum), spinal cord, and abdominal fat. At 2016 hours postdose, 18 tissues had quantifiable concentrations of radioactivity,including hyaline cartilage, red pulp of spleen, spleen, kidney cortex,kidney, and kidney medulla. The highest tissue-to-plasma concen-tration ratios were observed in the kidney, liver, spleen, bonemarrow, lymph nodes, and cartilages. Tissues with the highestradioactivity exposures included kidney cortex, kidney, kidneymedulla, hyaline cartilage, liver, spleen, bone marrow, intervertebralcartilage, lymph nodes, and epiphyseal line. Exposures of radioac-tivity were low (,500 ng-eq × h/g) in eye lens, brain (all regions),and spinal cord.The plasma 14C and AMG 416 Vss in SD rats was high (Supplemental

Table 1) and greater than the total body water. Tissue distribution andkinetics of radioactivity in male and female SD rats (groups 12 and 13;

Fig. 2. Mean concentration-time profiles of (A) 14C in blood andplasma and (B) 14C, total M11 and AMG 416 in plasma followinga single i.v. administration of [14C]Ac-AMG 416 to normal(group 4) or bilaterally nephrectomized (group 8) male rats and[14C]Ala-AMG 416 to normal male rats (group 7).


at ASPE



Dow

nloaded from

http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/

data not shown) were similar and consistent with that observed in maleLE rats. No substantive sex-dependent differences were observed intissue distribution of radioactivity in SD rats.

Biotransformation Product Profiles

The relative quantities of [14C]AMG 416 and its biotransformationproducts in vitro and in vivo matrices are summarized in Table 3 andSupplemental Table 4, respectively. Excretion into feces and bile wasminor; therefore, these matrices were not chromatographically analyzed.Plasma. The conventional extraction methods using protein precip-

itation with an organic solvent or solid-phase extraction typically workwell if the drug-related materials are noncovalently bound to plasmaproteins. The 14C plasma recovery in the supernatant after proteinprecipitation was low (,50%), which is consistent with previousreports for thiol-containing drugs owing to high covalent protein

binding (Wong et al., 1981; Iyer et al., 2001; Wait et al., 2006). Thus,a method was developed wherein the diluted plasma was directlysubjected to LC separation. Using this method, the qualitative andquantitative information of all biotransformation products, includingthe protein conjugates in the plasma, were simultaneously determinedfrom the resultant MS- and 14C-chromatograms.The chromatographic analyses of plasma obtained after incubation of

[14C]AMG 416 in rat and human whole blood are shown in Fig. 4, andthe components detected in plasma are listed in Table 3. For bothspecies, late eluting peaks (Fig. 4, A and B) postulated to be proteinconjugate(s) were the most abundant components, accounting for 59%and 71% of the total radioactivity in rat and human plasma, respectively(Table 3). The abundance of the other biotransformation products wasminor. In rat plasma, the most abundant nonprotein product M10 (14%)was a glutathione (GSH) conjugate formed upon disulfide exchange

Fig. 3. Concentrations of radioactivity in highand low exposure tissues as determined byquantitative whole-body autoradiography after asingle i.v. administration of [14C]AMG 416 tomale LE rats (group 11).

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

Component

% 14C-Chromatograma

In Vitro

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 24.3M1 5.6 1.4 ND ND L ND L L 4.3 17.8M5 ND ND ND 3.5 2.2 1.7 ND 1.4 ND NDM9 ND ND ND 9.4 ND 3.8 ND ND ND NDM10 14.2 1.1 74.0 ND 3.8 ND 68.9 30.3 10.8 19.9M11 2.3 1.2 ND 3.6 ND 20.7 ND ND 2.0 5.8M12 1.6 1.7 1.2 ND 3.8 3.8 15.8 26.1 2.3 4.7M13 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 0.6M15 1.5 1.5 ND ND ND ND ND 2.9 2.5b 2.9b

M16 ND ND ND ND ND ND 2.4 1.1 ND NDM22 ND ND ND ND ND 1.4 ND ND ND NDM28 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 18.7U2 ND ND ND ND ND ND ND ND ND 2.2Otherd 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.


at ASPE



Dow

nloaded from

http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/

with the L-cysteine of AMG 416. The next most abundant product wasM1 (6%), a product of thiol sulfation. In comparison, M13 (4%), anL-cysteinylglycine conjugate, was the most abundant nonprotein bio-transformation product in the human plasma. M11 (2%), the thiol orsulfhydryl (reduced) form of the D-amino acid backbone in AMG 416was a minor component. Reduction of the plasma samples resultedpredominantly in the formation of a single total M11 peak (92% in bothspecies, data not shown) in the 14C-chromatograms. The biotransforma-tion products present in plasma prepared from whole blood of bothhealthy humans and CKD patients were also present in plasma from rat.In plasma obtained after AMG 416 dosing in normal and BN rats, 42%

and 19% of 14C, respectively, were covalently bound to plasma proteins(Fig. 4, C and D; Table 3). The predominant protein conjugate in plasmawas presumed to be serum albumin peptide conjugate (SAPC) based onthe in vitro characterization. The most abundant nonprotein conjugateddrug-related components in in vivo plasma were the same as observedin vitro. Total M11 was the predominant biotransformation productobserved in the TCEP-reduced plasma (Fig. 4E). Additional analysesshowed that 14C profiles were similar for both sexes and the tworadiolabels (data not shown). Collectively, the total M11 and 14C PK data

(Fig. 2B) and the TCEP-reduced plasma biotransformation profileaffirmed that the covalent binding to serum albumin was via a disulfidebond and that the D-amino acid backbone remained intact in each AMG416–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(Supplemental Table 4). Intact AMG 416 was the most abundantcomponent (approximately 35%–47% of the administered dose) inurine. M1was the most abundant biotransformation product. M3(acetylated glutathione disulfide) and M2b (a disulfide; exact identityunknown) coeluted under the chromatographic conditions used, andtherefore their individual quantities could not be determined. Treatmentof urine (Fig. 5B) obtained from [14C]Ac-AMG 416-dosed male rats(group 1) with TCEP resulted in the formation of a single predominantpeak, total M11, and a minor peak, M5-DesCys, the reduced productfrom M5. An unassigned minor peak U1 was observed in bothnonreduced and TCEP-reduced urine. This demonstrated that theD-amino acid backbone remained intact in almost all biotransformationproducts that were present in urine.

Liver and Kidney S9 Fractions. The observed biotransformationproducts in both rat and human S9 fractions are listed in Table 3. The mostpredominant products formed were GSH conjugate (M10) or the disulfidebond reduced product (M11) formed in rat liver and human kidney S9,respectively. Peaks that eluted late in the chromatographic analyses of allincubations were putatively protein conjugate(s) and were not furthercharacterized. Reduction with TCEP treatment resulted in the formation of asingle predominant product, total M11. The biotransformation productsformed from both acetyl and alanine labeled AMG 416 were very similar inhuman and rat liver and kidney S9 fractionswith orwithout TCEP treatment.

Hepatocytes. The observed biotransformation products from bothrat and human hepatocytes are summarized in Table 3. The GSHconjugate M10 was the most abundant product formed in hepatocytesfrom both species, and after TCEP reduction, a single predominantproduct (total M11) was formed (data not shown). The products formedin hepatocytes from AMG 416 radiolabeled at both sites were verysimilar with or without TCEP treatment.

Fig. 4. Radiochromatograms of direct analysis for plasma from (A) rat and (B)healthy human whole blood incubated with [14C]Ac-AMG 416 (5 mM)and time-proportionally pooled plasma after i.v. administration of [14C]Ac-AMG 416 to (C)normal male rats (group 4), (D) bilaterally nephrectomized male rats (group 8), and(E) TCEP-reduced plasma, bilaterally nephrectomized male rats (group 8).M5-DesCys, sulfhydryl (reduced) form of M5; TM11, total M11; U2, unknown peak.

Fig. 5. Representative high-performance liquid chromatography radiochromato-grams of pooled urine without (A) or with (B) TCEP reduction afteri.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, unknownpeak.


at ASPE



Dow

nloaded from


Structure Assignment of Biotransformation Products

A total of 19 nonprotein-conjugated biotransformation products weredetected in the analyzed in vitro and in vivo matrices. The proposedbiotransformation scheme is shown in Fig. 6. The MS and MS2

fragmentation data of all identified biotransformation products aresummarized in Table 4. Characterization of the parent AMG 416molecule and the predominant biotransformation product in plasma,SAPC, are described below.

AMG 416

A doubly charged molecular ion at m/z 524.770 was observed in thefull-scan HRMS of AMG 416. Collision-induced dissociation (CID) ofthis molecular ion resulted in multiple-fragment ions (Table 4), each of

which was used to aid in structural assignment. These ions includedm/z448.276 (2+; D-cysteine S-C bond cleavage),m/z 464.262 (2+; disulfidebond cleavage), and m/z 481.256 (2+; L-cysteine S-C bond cleavage).

SAPC

SAPC was characterized in plasma from in vitro whole-bloodincubations. Figure 7A displays the deconvoluted mass spectrum ofthe human SA peak in the LC-HRMS chromatogram of the plasma fromincubation 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 theunmodified human SA. Peaks atm/z 66,557 and 66,601 had mass shiftsof 119 and 163 Da compared with the SA and represented cysteineand glycosylated conjugates of albumin, respectively. The peak atm/z 67,367 had a mass increase of 929 Da, which was equivalent to theaddition of the D-amino acid backbone of [14C]AMG 416 to SA. Inthe analysis of whole-blood incubations from CKD patients and rats,the same conjugates were observed (data not shown). Thus, in both ratsand humans, the molecular weight of the conjugate corresponds to theaddition of one molecule of the D-amino acid backbone from AMG 416to one molecule of serum albumin. No other protein conjugates weredetected in the analyses.The Coomassie blue–stained and 14C images of the SDS-PAGE of

affinity gel column fractions P1-P3 obtained from plasma isolated from[14C]Ala-AMG 416 incubated in human whole blood are shown inFig. 8. In the Coomassie blue–stained gel (Fig. 8A), multiple proteinbands were observed for all three fractions. The most abundant bandin P2 and P3 (lanes 2 and 3 in Fig. 8A) had a molecular weight ofapproximately 66 kDa and matched the band from human SA control(lane 4 in Fig. 8A). Therefore, this band was identified as the humanSA. The 14C image (Fig. 8B) of the same gel shows that a singleradioactive band was observed in the position of human SA in both P2and P3 lanes. Thus, SA was the predominant plasma protein that wascovalently adducted with [14C]Ala-AMG 416. In the SDS-PAGEanalysis, unmodified and modified SA were not separated and appearedas a single band. In the Coomassie blue–stained gel (Fig. 8A), the

Fig. 6. Proposed biotransformation of AMG 416 in in vitro whole-blood incubationand rat.

TABLE 4

Summary of precursor and product ions observed in high-resolution mass spectra for AMG 416 and its in vivononprotein biotransformation products

ComponentObserved Precursor Ion(m/z; +2 Charge State)

Mass Shifta Observed Product Ionsb

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


at ASPE



Dow

nloaded from

http://dmd.aspetjournals.org/

abundance of the serum albumin bands in lanes P2 and P3 were similar;however, in the 14C image (Fig. 8B), the SA band in the P3 lane wasmuch more intense than the corresponding band in P2 lane and,therefore, indicated an increased proportion of SAPC in P3 fraction.P1 was the wash solution expected to contain non-albumin proteins;accordingly, no band was observed corresponding to the SA molecularweight in the Coomassie blue.The Lys-C digests of fractions P2 and P3 from the [14C]Ala-AMG

416 incubations in human whole blood from both healthy volunteersand CKD patients were profiled by LC-14C-HRMS (Fig. 8, C and D).The composition of the radiolabeled peaks in the chromatogram andtheir abundance are summarized in Supplemental Table 5. Similar 14C-profiles of the Lys-C digest were observed for the sample from a healthyvolunteer and a CKD patient receiving hemodialysis. Peptide C34-1 wasthe predominant peak in the chromatogram (Fig. 8, C and D). Themolecular ion andmass spectrumwere consistent with conjugation of oneD-amino acid backbone fromAMG416 via a disulfide bond to cysteine atposition 34 (Cys34) in the 21ALVLIAFAQYLQQCPFEDHV41K pep-tide within serum albumin. Similar retention times were observedfor nonlabeled AMG 416 and [14C]Ala-AMG 416–modified serumalbumin peptide in their extracted ion chromatograms (datanot shown). The MS spectra show that the precursor ion (z = 5+) of[14C]Ala-AMG 416 (Fig. 7B)–modified peptide has a mass shift of0.4 Da compared with the nonlabeled counterpart, indicative of the

expected 2-Da mass difference between a labeled and nonlabeled AMG416 molecule in the 5+ charge state. These findings show that thepeptide was modified by a single molecule of the D-amino acidbackbone of AMG 416. The identities of the modified serum albuminpeptides were confirmed by their MS2 spectra (Fig. 7C), in whichproduct ions from [14C]Ala-AMG 416 moiety and the albumin peptidewere observed. Two additional Cys34 conjugated peptides (peaks C34-2 and C34-3 in Fig. 8, C and D) of minor abundance were also detected:C34-2 was not yet fully characterized; C34-3 was a nonspecificallycleaved shorter peptide from C34-1 (Supplemental Table 5). Themodified Cys34 containing peptides contribute to approximately 76%of the total 14C count in the digested fraction. The same MS analyseswere also performed on tryptic digests obtained from human whole-blood incubation with [14C]Ac-AMG 416 at 1 and 10 mM (data notshown); the conjugation of [14C]Ac-AMG 416 D-amino acid backboneto Cys34 in human SA was confirmed from the HRMS andMS2 data ofthe detected peptide C34-1.

Discussion

AMG 416 is a novel synthetic peptide composed almost entirely ofD-amino acids. The only natural L-amino acid in the molecule is not inthe peptide backbone but instead is linked via a disulfide bond. Thus, allpeptide bonds in AMG 416 are between D-amino acids, which are

Fig. 7. Mass spectra of serum albumin (SA) and serumalbumin peptide conjugate (SAPC) from plasma obtainedafter incubation of [14C]Ala-AMG 416 in human wholeblood. (A) Deconvoluted mass spectrum of whole proteins;(B) MS of the [14C]AMG 416 D-amino acid backbonemodified peptide 21ALVLIAFAQYLQQCPFEDHV41K;(C) MS2 of m/z 673.56 (5+) in (B).


at ASPE



Dow

nloaded from

http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/lookup/suppl/doi:10.1124/dmd.115.068007/-/DC1http://dmd.aspetjournals.org/

sometimes incorporated into therapeutic peptide drugs to preventpeptide bond hydrolysis by peptidases (Chan et al., 1991; Hennanet al., 2006; Bloedon et al., 2008; Charignon et al., 2012). This is aneffective strategy to improve the metabolic stability of peptides;however, because the biologic activity of most peptides is likelydependent on interactions between their target and L-amino acidsequences in the peptide, the number of D-amino acids that can typicallybe incorporated is limited. Accordingly, disposition of few, if any,peptide drugs composed primarily of D-amino acids has been de-termined. In vitro investigations showed that the D-amino acidbackbone of AMG 416 was essentially inert to peptidase-mediatedhydrolysis. Additional studies showed that other metabolizing en-zymes, such as the P450s, catalyzed little if any biotransformation ofAMG 416. Finally, in vitro studies also showed that AMG 416 was nota substrate of the typical transporters that are involved in the dispositionof some drugs. Collectively, these studies showed that the D-amino acidbackbone of AMG 416 is metabolically quite inert and unlikely to besubject to clearance routes dependent on hydrolysis, oxidation, or activetransport into excreta.Two AMG 416 radiolabels were prepared and tested—the first on

the conveniently prepared and potentially metabolically labile acetylposition, the second on the D-alanine (next to D-cysteine) in the interior

of the D-amino acid backbone, which was likely to be metabolicallystable but required a far more complicated synthesis procedure toprepare. The PK and disposition profiles after a single i.v. dose of thetwo 14C-labeled AMG 416 molecules to rats were similar, and no sexdifferences were observed. Excretion of 14C, presumably as 14CO2, inexpired air was low (,0.2%) with both labels. Des-acetylated bio-transformation products were specifically searched for in the in vitroand in vivo LC-MS data sets, and no des-acetyl products were detectedin any of the samples. These findings demonstrated that the entireD-amino acid backbone—including the N-acetyl group—in AMG 416was metabolically stable. Accordingly, no distinction is made indescriptions between the two labeled compounds, and the test materialis described solely as [14C]AMG 416.The intended clinical route of AMG 416 is by i.v. administration;

therefore, the kinetics of absorption upon delivery by a non-IV routewas not studied. The total recovery of radioactivity was similar in themale and female rats with normal kidney function. AMG 416 has a lowmolecular weight and positive polarity, and in vitro findings showedthat its cellular permeability was limited and its plasma protein bindingrelatively low; therefore, glomerular filtration was expected to be apredominant mechanism of plasma clearance. Indeed, when AMG 416was administered to rats with intact kidney function, its observed

Fig. 8. SDS-PAGE of affinity gel column fractions P1 (lane 1), P2(lane 2), P3 (lane 3), control human serum albumin (lane 4) andradiolabeled protein marker standards (lane 5). Plasma obtainedafter incubation with [14C]Ala-AMG 416 in healthy volunteerhuman whole blood was subjected to affinity gel chromatography.The same PAGE gel was stained with Coomassie Blue and alsoimaged on a 14C-phosphor system. (A) Coomassie Blue–stainedimage; (B) 14C image. 14C chromatogram of Lys-C digest fromplasma 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 gelcolumn separation; fractions P2 and P3 obtained thereafter werecombined and subjected to Lys-C digestion. C34-1, 2, and 3 refer topeptides shown to contain cysteine 34 in albumin; composition ofthe modified peptides is shown in Supplemental Table 5. CKD,chronic kidney disease; SA, serum albumin; U3 and U4 areunknown peaks.


at ASPE



Dow

nloaded from


plasma clearance was 0.97 L/h per killogram (Supplemental Table 1),and the estimated renal clearance (fraction unbound in plasma (0.77)�observed plasma clearance = 0.75 liter/h per kilogram) was close to theglomerular filtration rate in rat of 0.65 liter/h per kilogram (Sadick et al.,2011). However, because the drug is intended for treatment of patientswith little or no kidney function, this route of elimination would behighly restricted, and assessment of its disposition in the absence ofkidney clearance was of critical importance. In BN rats, where thekidney function was totally absent, the total recovery of radioactivitywas low and the elimination by nonrenal pathways was extremely slow.Another important feature of AMG 416 is the presence of the

disulfide bond between a D-amino acid in the peptide backbone, andL-cysteine. The relative facility with which disulfide exchange betweenAMG 416 and other thiol-containing molecules can occur is illustratedby the fact that the biologic activity of AMG 416 is mediated byformation of a disulfide bond between the D-amino acid backbone ofAMG 416 and a cysteine in the calcium-sensing receptor (Alexanderet al., 2015). Disulfide bond formation between a drug and endogenousthiols has been studied and described with other drugs, such as captopril(Migdalof et al., 1984). The dispositional fate of captopril and othersimilar thiol-containing drugs suggests that the disposition of AMG 416would not be determined solely by its peptide structure but also by thiolexchange mechanisms. Indeed, in vitro studies demonstrated thatdisulfide exchange readily occurred in whole blood between low-molecular-weight endogenous thiols, such as glutathione, and high-molecular-weight thiols, such as serum albumin. These findings aresimilar to findings with other thiol drugs (Migdalof et al., 1984; Iyeret al., 2001; Wait et al., 2006). These in vivo studies demonstrated thatthe biotransformation products formed after in vitro incubation in wholeblood were also formed in vivo. In vitro studies also showed that thesame biotransformation products formed in blood could be formed inS9 fractions from liver and kidney; however, because of the lowpermeability of AMG 416, the intracellular contribution to biotrans-formation is likely to be small.In both rat and human whole blood, SAPC was the predominant

covalently bound-protein biotransformation product. The covalentinteraction of AMG 416 with plasma proteins is likely, if notexclusively, via disulfide bond formation with cysteine residues presentin the plasma proteins based on the following: AMG 416 activityrequires disulfide exchange with a cysteine residue on the calcium-sensing receptor, as stated already herein; other thiol drugs are known toundergo disulfide exchange (Wong et al., 1981; Wait et al., 2006);lastly, liberation of totalM11 by TCEP indicates that the covalent bondsare likely to be disulfides. SAPC was formed via disulfide conjugationof the AMG 416 D-amino acid backbone to serum albumin. In additionto the SDS-PAGE evidence, binding to a specific amino acid wasdetermined using HRMS. Human SA contains a total of 35 cysteines,with Cys34 the only one with a free thiol group, and therefore it shouldbe more readily than other cysteines to undergo disulfide exchange withthe AMG 416 D-amino acid backbone to form SAPC. No other cysteineresidue in serum albumin was found to be modified, confirming thishypothesis.Disulfide exchange reactions proceed through a nucleophilic substi-

tution (SN2) reaction via the thiolate anion intermediate (Fernandes andRamos, 2004). AMG 416 was stable in in vitro incubations at pH 3, andthis was attributed to a decrease in thiolate anion formation from theendogenous thiols and consequently a decrease in the disulfide ex-change (Nagy, 2013).After a single i.v. dose of [14C]AMG 416, radioactivity appeared

rapidly in most of the tissues evaluated. A peptide not subject to rapidmetabolic clearance would be expected to have a Vss limited toextracellular volume. For example, the Vss for rotigaptide was

0.19 liters/kg (Hennan et al., 2006). The Vss for AMG 416 and total14C, however, was significantly higher (.8-fold) than the extracellularvolume. This result could be attributed in part to reversible disulfideexchange of the AMG 416 D-amino acid backbone between allavailable endogenous thiols and the L-cysteine readily available inplasma. This reversible exchange was investigated and will be reportedin a separate publication. The D-amino acid backbone reversibly boundas a disulfide to macromolecules such as albumin would constitutedistribution into a peripheral compartment with a volume that exceedsextracellular water. Indeed, the D-amino acid backbone of AMG 416probably exists in equilibrium among many disulfide forms, one ofwhich is the parent molecule, and which in plasma is dominated bySAPC because of the high concentration of albumin in serum. Highlevels of radioactivity in some tissues may be a consequence ofphagocytosis (spleen, liver), binding of the labeled D-amino acidbackbone to calcium-sensing receptor via disulfide bond (kidney,cartilage, bone medullary space) and reabsorption of SAPC (kidney)(Christensen and Birn, 2002; Dvorak et al., 2004; Chang et al., 2008).Distribution of radioactivity to the brain was low, which reflects the lowpassive permeability of AMG 416 and lack of active uptake transportresulting in a poor distribution across the blood-brain barrier.AMG 416 presents a low risk for P450 or transporter mediated drug-

drug interactions. AMG 416 was not a substrate, a reversible or time-dependent inhibitor, or an inducer of the P450 isozymes tested in thisstudy. This finding should provide an advantage over the othercalcimimetic, cinacalcet, the only drug of this class for the treatmentof secondary HPT (Torres, 2006). AMG 416 was not a substrate oran inhibitor of the common efflux and uptake human transportersexamined in the current study.In summary, the nonclinical PK, disposition, and drug-drug in-

teraction potential of AMG 416 were assessed. Disposition of AMG416 was predominantly by renal elimination. AMG 416 was biotrans-formed via disulfide exchange of its L-cysteine moiety with thiolspresent in whole blood, predominantly to albumin. The risk of P450 ortransporter mediated drug-drug interactions with AMG 416 is antici-pated to be very low. These studies demonstrated a 14C label on eitherthe acetyl or the D-alanine (next to D-cysteine) in AMG 416 peptidebackbone would be appropriate for other nonclinical and clinicalabsorption, distribution, metabolism, and excretion studies.

Acknowledgments

The authors thank Michael Hayashi for performing hepatocyte incubations;Dean Hickman for helping design the protein binding experiment; Robert Ortizfor assistance with SDS-PAGE experiments and 14C-phosphoimager analysis;Andrew Hui and Jun Lammawin for performing the transporter experiments;Ryan Morgan for human BSEP transport experiment; Yihong Zhou forconducting the time-dependent P450 experiments; Erin Ballard and PhilManteufel for the QWBA study; Mark Fielden, James Tomlinson, and CharlesDean, Jr., for QWBA data interpretation; and Holly Tomlin (employee andstockholder of Amgen Inc) for medical writing and journal formatting assistance.

Authorship ContributionsParticipated in research design: Zhu, Fitzsimmons, Esmay, Louie, Edson,

Walter, Soto, Wagner, Wilson, Skiles, Subramanian.Conducted experiments: Zhu, Esmay, Fitzsimmons, Louie, Edson, Kerr,

Pham, Soto, Wagner.Contributed new reagents or analytic tools: Zhu, Esmay, Pham, Wilson,

Subramanian.Performed data analysis: Zhu, Esmay, Louie, Edson, Kerr, Pham, Wagner,

Subramanian.Wrote or contributed to the writing of the manuscript: Zhu, Esmay,

Fitzsimmons, Louie, Edson, Kerr, Wilson, Skiles, Subramanian.


at ASPE



Dow

nloaded from


References

Alexander ST, Hunter T, Walter S, Dong J, Maclean D, Baruch A, Subramanian R,and Tomlinson JE (2015) Critical cysteine residues in both the calcium-sensing receptor andthe allosteric activator AMG 416 underlie the mechanism of action. Mol Pharmacol 88:853–865.

Atkinson A, Kenny JR, and Grime K (2005) Automated assessment of time-dependent inhibitionof human cytochrome P450 enzymes using liquid chromatography-tandem mass spectrometryanalysis. Drug Metab Dispos 33:1637–1647.

Bell G, Huang S, Martin KJ, and Block GA (2015) A randomized, double-blind, phase 2 studyevaluating the safety and efficacy of AMG 416 for the treatment of secondary hyperparathy-roidism in hemodialysis patients. Curr Med Res Opin 31:943–952.

Block GA, Martin KJ, de Francisco AL, Turner SA, Avram MM, Suranyi MG, Hercz G,Cunningham J, Abu-Alfa AK, and Messa P, et al. (2004) Cinacalcet for secondary hyper-parathyroidism in patients receiving hemodialysis. N Engl J Med 350:1516–1525.

Bloedon LT, Dunbar R, Duffy D, Pinell-Salles P, Norris R, DeGroot BJ, Movva R, Navab M,Fogelman AM, and Rader DJ (2008) Safety, pharmacokinetics, and pharmacodynamics of oralapoA-I mimetic peptide D-4F in high-risk cardiovascular patients. J Lipid Res 49:1344–1352.

Booth-Genthe CL, Louie SW, Carlini EJ, Li B, Leake BF, Eisenhandler R, Hochman JH, Mei Q,Kim RB, and Rushmore TH, et al. (2006) Development and characterization of LLC-PK1 cellscontaining Sprague-Dawley rat Abcb1a (Mdr1a): comparison of rat P-glycoprotein transport tohuman and mouse. J Pharmacol Toxicol Methods 54:78–89.

Chan RL, Hsieh SC, Haroldsen PE, Ho W, and Nestor JJ, Jr (1991) Disposition of RS-26306, apotent luteinizing hormone-releasing hormone antagonist, in monkeys and rats after singleintravenous and subcutaneous administration. Drug Metab Dispos 19:858–864.

Chang W, Tu C, Chen TH, Bikle D, and Shoback D (2008) The extracellular calcium-sensingreceptor (CaSR) is a critical modulator of skeletal development. Sci Signal 1:ra1–13.

Charignon D, Späth P, Martin L, and Drouet C (2012) Icatibant, the bradykinin B2 receptor antagonistwith target to the interconnected kinin systems. Expert Opin Pharmacother 13:2233–2247.

Christensen EI and Birn H (2002) Megalin and cubilin: multifunctional endocytic receptors. NatRev Mol Cell Biol 3:256–266.

Chu XY, Bleasby K, Yabut J, Cai X, Chan GH, Hafey MJ, Xu S, Bergman AJ, Braun MP,and Dean DC, et al. (2007) Transport of the dipeptidyl peptidase-4 inhibitor sitagliptin byhuman organic anion transporter 3, organic anion transporting polypeptide 4C1, and multidrugresistance P-glycoprotein. J Pharmacol Exp Ther 321:673–683.

Cozzolino M, Tomlinson J, Walsh L, and Bellasi A (2015) Emerging drugs for secondaryhyperparathyroidism. Expert Opin Emerg Drugs 20:197–208.

Cunningham J, Locatelli F, and Rodriguez M (2011) Secondary hyperparathyroidism: patho-genesis, disease progression, and therapeutic options. Clin J Am Soc Nephrol 6:913–921.

Dvorak MM, Siddiqua A, Ward DT, Carter DH, Dallas SL, Nemeth EF, and Riccardi D (2004)Physiological changes in extracellular calcium concentration directly control osteoblast func-tion in the absence of calciotropic hormones. Proc Natl Acad Sci USA 101:5140–5145.

Fernandes PA and Ramos MJ (2004) Theoretical insights into the mechanism for thiol/disulfideexchange. Chemistry 10:257–266.

Goodman WG and Quarles LD (2008) Development and progression of secondary hyperpara-thyroidism in chronic kidney disease: lessons from molecular genetics. Kidney Int 74:276–288.

Hamilton RA, Garnett WR, and Kline BJ (1981) Determination of mean valproic acid serum levelby assay of a single pooled sample. Clin Pharmacol Ther 29:408–413.

Hennan JK, Swillo RE, Morgan GA, Keith JC, Jr, Schaub RG, Smith RP, Feldman HS, HauganK, Kantrowitz J, and Wang PJ, et al. (2006) Rotigaptide (ZP123) prevents spontaneous ven-tricular arrhythmias and reduces infarct size during myocardial ischemia/reperfusion injury inopen-chest dogs. J Pharmacol Exp Ther 317:236–243.

Hewitt NJ, Lechón MJ, Houston JB, Hallifax D, Brown HS, Maurel P, Kenna JG, Gustavsson L,Lohmann C, and Skonberg C, et al. (2007) Primary hepatocytes: current understanding of theregulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for theuse of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicitystudies. Drug Metab Rev 39:159–234.

Iyer RA, Mitroka J, Malhotra B, Bonacorsi S, Jr, Waller SC, Rinehart JK, Roongta VA,and Kripalani K (2001) Metabolism of [(14)C]omapatrilat, a sulfhydryl-containingvasopeptidase inhibitor in humans. Drug Metab Dispos 29:60–69.

Martin KJ, Bell G, Pickthorn K, Huang S, Vick A, Hodsman P, and Peacock M (2014) Velcalcetide(AMG 416), a novel peptide agonist of the calcium-sensing receptor, reduces serum parathyroidhormone and FGF23 levels in healthy male subjects. Nephrol Dial Transplant 29:385–392.

Migdalof BH, Antonaccio MJ, McKinstry DN, Singhvi SM, Lan SJ, Egli P, and Kripalani KJ(1984) Captopril: pharmacology, metabolism and disposition. Drug Metab Rev 15:841–869.

Moe SM, Chertow GM, Coburn JW, Quarles LD, Goodman WG, Block GA, Drüeke TB,Cunningham J, Sherrard DJ, and McCary LC, et al. (2005) Achieving NKF-K/DOQI bonemetabolism and disease treatment goals with cinacalcet HCl. Kidney Int 67:760–771.

Nagy P (2013) Kinetics and mechanisms of thiol-disulfide exchange covering direct substitutionand thiol oxidation-mediated pathways. Antioxid Redox Signal 18:1623–1641.

National Research Council. (2011) Guide for the Care and Use of Laboratory Animals: 8thEdition. Washington, DC: National Academies Press.

Nemeth EF, Steffey ME, Hammerland LG, Hung BC, Van Wagenen BC, DelMar EG,and Balandrin MF (1998) Calcimimetics with potent and selective activity on the parathyroidcalcium receptor. Proc Natl Acad Sci USA 95:4040–4045.

Obach RS, Walsky RL, and Venkatakrishnan K (2007) Mechanism-based inactivation of humancytochrome p450 enzymes and the prediction of drug-drug interactions. Drug Metab Dispos35:246–255.

Rodriguez M, Nemeth E, and Martin D (2005) The calcium-sensing receptor: a key factor in thepathogenesis of secondary hyperparathyroidism. Am J Physiol Renal Physiol 288:F253–F264.

Sadick M, Attenberger U, Kraenzlin B, Kayed H, Schoenberg SO, Gretz N, and Schock-Kusch D(2011) Two non-invasive GFR-estimation methods in rat models of polycystic kidney disease:3.0 Tesla dynamic contrast-enhanced MRI and optical imaging. Nephrol Dial Transplant 26:3101–3108.

Schinkel AH, Wagenaar E, van Deemter L, Mol CA, and Borst P (1995) Absence of the mdr1aP-Glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone,digoxin, and cyclosporin A. J Clin Invest 96:1698–1705.

Tfelt-Hansen J and Brown EM (2005) The calcium-sensing receptor in normal physiology andpathophysiology: a review. Crit Rev Clin Lab Sci 42:35–70.

Torres PU (2006) Cinacalcet HCl: a novel treatment for secondary hyperparathyroidism causedby chronic kidney disease. J Ren Nutr 16:253–258.

van Staden CJ, Morgan RE, Ramachandran B, Chen Y, Lee PH, and Hamadeh HK(2012)Membrane Vesicle ABC Transporter Assays for Drug Safety Assessment. Current Protocols inToxicology series, Unit 23.5.1–23.5.24, Wiley, New York.

Wait JC, Vaccharajani N, Mitroka J, Jemal M, Khan S, Bonacorsi SJ, Rinehart JK, and Iyer RA(2006) Metabolism of [14C]gemopatrilat after oral administration to rats, dogs, and humans.Drug Metab Dispos 34:961–970.

Walsky RL and Obach RS (2004) Validated assays for human cytochrome P450 activities. DrugMetab Dispos 32:647–660.

Walter S, Baruch A, Alexander ST, Janes J, Sho E, Dong J, Yin Q, Maclean D, Mendel DB,and Karim F, et al. (2014) Comparison of AMG 416 and cinacalcet in rodent models of uremia.BMC Nephrol 15:81.

Walter S, Baruch A, Dong J, Tomlinson JE, Alexander ST, Janes J, Hunter T, Yin Q, Maclean D,and Bell G, et al. (2013) Pharmacology of AMG 416 (Velcalcetide), a novel peptide agonist ofthe calcium-sensing receptor, for the treatment of secondary hyperparathyroidism in hemodi-alysis patients. J Pharmacol Exp Ther 346:229–240.

Waynforth HB and Flecknell PA (1992) Specific Surgical Procedures, in Experimental andSurgical Technique in the Rat (Waynforth HB ed), Academic Press, London.

Wong KK, Lan S, and Migdalof BH (1981) In vitro biotransformations of [14C]captopril in theblood of rats, dogs and humans. Biochem Pharmacol 30:2643–2650.

Yamazaki M, Li B, Louie SW, Pudvah NT, Stocco R, Wong W, Abramovitz M, Demartis A,Laufer R, and Hochman JH, et al. (2005) Effects of fibrates on human organic anion-transporting polypeptide 1B1-, multidrug resistance protein 2- and P-glycoprotein-mediatedtransport. Xenobiotica 35:737–753.

Address correspondence to: Raju Subramanian, Amgen Inc. One Amgen CenterDrive, Thousand Oaks, CA 91320. E-mail: [email protected]


at ASPE



Dow

nloaded from
mailto:[email protected]://dmd.aspetjournals.org/

Special Section on Emerging Novel Enzyme Pathways in ...each hemodialysis session in CKD patients,...

Documents

Transcript of Special Section on Emerging Novel Enzyme Pathways in ...each hemodialysis session in CKD patients,...