Drug Metabolism and Disposition Fast Forward
First published on August 24, 2004; DOI: 10.1124/dmd.104.000604
0090-9556/04/3212-1491-1500$20.00
DMD 32:1491-1500, 2004
METABOLISM AND DISPOSITION OF CALCIMIMETIC AGENT CINACALCET HCL IN HUMANS AND ANIMAL MODELS
Gondi N. Kumar,
Christopher Sproul,
Leszek Poppe,
Sharon Turner,
Mark Gohdes,
Hesham Ghoborah,
Desmond Padhi, and
Lorin Roskos
Amgen Inc., Thousand Oaks, California (G.N.K., C.S., L.P., S.T., H.G., D.P., L.R.); and Covance Laboratories Inc., Madison, Wisconsin (M.G.)
(Received May 14, 2004;
accepted August 20, 2004)
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Abstract
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The metabolism and disposition of calcimimetic agent cinacalcet HCl was examined after a single oral administration to mice, rats, monkeys, and human volunteers. In all species examined, cinacalcet was well absorbed, with greater than 74% oral bioavailability of cinacalcet-derived radioactivity in monkeys and humans. In rats, cinacalcet-derived radioactivity was widely distributed into most tissues, with no marked gender-related differences. In all animal models examined, radioactivity was excreted rapidly via both hepatobiliary and urinary routes. In humans, radioactivity was cleared primarily via the urinary route (80%), with 17% excreted in the feces. Cinacalcet was not detected in the urine in humans. The primary routes of metabolism of cinacalcet were N-dealkylation leading to carboxylic acid derivatives (excreted in urine as glycine conjugates) and oxidation of naphthalene ring to form dihydrodiols (excreted in urine and bile as glucuronide conjugates). The plasma radioactivity in both animals and humans was primarily composed of carboxylic acid metabolites and dihydrodiol glucuronides, with <1% circulating radioactivity accounting for the unchanged cinacalcet. Overall, the circulating and excreted metabolite profile of cinacalcet in humans was qualitatively similar to that observed in preclinical animal models.
Calcimimetic agents act as positive allosteric modulators of the calcium receptor located on the surface of the parathyroid cells (Nemeth et al., 1998
; Cohen and Silverberg, 2002). Cinacalcet HCl (AMG 073; hereinafter referred to as cinacalcet), a calcimimetic agent, acts on the calcium-sensing receptor of the parathyroid, the principal regulator of parathyroid hormone release, to increase its sensitivity to activation by extracellular calcium, thus decreasing parathyroid hormone. The clinical efficacy of cinacalcet has been demonstrated in several studies in subjects with secondary hyperparathyroidism (Drueke et al., 2001; Goodman et al., 2002; Quarles et al., 2003).
Nonclinical biodisposition studies (ADME) of a new chemical entity play a critical role in its initial selection as well as in its subsequent clinical development. Biodisposition studies provide important information regarding the absorption of the drug from the site of administration, distribution of the drug-related material into the target tissues, metabolic pathways to which the drug is subject, and the eventual excretion of the drug-related material from the body (Campbell, 1994; Caldwell et al., 1995; Miwa, 1995). The information generated from these studies is helpful in understanding the outcomes of the safety studies (e.g., toxic and reactive metabolites, covalent binding to macromolecules, species-specific toxicity) and in some instances the outcomes of pharmacology studies (e.g., the presence of active metabolites, distribution into target organ) (Wiltshire et al., 1997; Bischoff et al., 1998; Mutlib et al., 2000). These studies are also immensely helpful in understanding the potential behavior of a drug candidate in humans. Hence, a comprehensive nonclinical ADME program has become a crucial component of the drug development package for regulatory submissions.
The goals of the present studies were to conduct ADME studies in animal models to complement the toxicological and pharmacological evaluation of cinacalcet in preclinical species. Furthermore, the disposition of cinacalcet was also evaluated in healthy human volunteers after a single oral dose of [14C-CF3]cinacalcet.
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Materials and Methods
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[14C-2-CH2]Cinacalcet (58.1 mCi/mmol) and [14C-CF3]cinacalcet (47.6 mCi/mmol) as a free base were synthesized by ChemSyn (Lenexa, KS). Cinacalcet hydrochloride was synthesized by Sumika Fine Chemicals (Osaka, Japan). The animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. For the human disposition study, written informed consent from the volunteers and approval by the Covance Clinical Research Unit Institutional Review Board (Madison, WI) were obtained.
Tissue distribution of radioactivity after a single 10 mg/kg oral dose of [14C-2-CH2]cinacalcet was examined in Sprague-Dawley rats (eight male, eight female) by quantitative whole-body autoradiography. At predetermined time points (4, 12, 24, and 48 h), two rats per sex were sacrificed and blood was collected. The carcass was frozen and embedded in 2% carboxymethyl cellulose, frozen at approximately -70°C, and prepared for sectioning. Using a Leica CM3600 cryomicrotome (Leica Microsystems Nussloch GmbH, Nussloch, Germany), approximately 40-µm-thick sections were prepared at five levels of interest in the sagittal plane, representing all major tissues, organs, and fluids. The radioactivity in the sections and externally applied 14C standards was analyzed by exposing them to PhosphorImager screens for 4 days. The exposed screens were scanned using a 445 SI PhosphorImager (Amersham Biosciences, Piscataway, NJ). Tissue concentrations of radioactivity were interpolated from the standard curve generated from 14C standards as µg Eq of [14C]cinacalcet/g of tissue.
[14C-CF3]Cinacalcet was administered to CD-1 mice, Sprague-Dawley rats, and cynomolgus monkeys in 0.5% methyl cellulose via oral gavage. Additionally, bile duct-cannulated male monkeys were dosed via oral gavage. Excreta, bile, and blood samples were collected over extended periods of time.
An open-label study of the disposition of [14C-CF3]cinacalcet after a single oral administration was conducted in healthy male subjects. Each subject (five nonsmoking and five smoking healthy male volunteers, age range 22-49 years, body weight range 73-116 kg; four Caucasians and one African-American in each group) received a single, target 75-mg (approximately 100 µCi) oral dose of [14C-CF3]cinacalcet via a capsule. Blood and excreta were collected up to 8 days postdose.
Radioactivity in plasma, urine, and bile was measured by liquid scintillation analysis. Radioactivity in fecal homogenates was measured by combustion, followed by liquid scintillation analysis. To examine the presence of glucuronide conjugates, aliquots of urine and bile samples were incubated with or without ß-glucuronidase (1000 units of ß-glucuronidase, EC 3.2.1.31
[EC]
, from Escherichia coli; Sigma-Aldrich, St. Louis, MO) for 8 h at 37°C in pH 5.0 buffer. Radioactivity was extracted from fecal homogenates by sequential extraction with organic solvents (acetonitrile and methanol). Urine and bile samples were diluted with mobile phase before analysis. Supernatant resulting from protein precipitation of plasma samples with acetonitrile or methanol was used to determine metabolite profiles in plasma.
Gradient chromatography was performed using a YMC Basic column (2.1 mm x 250 mm) and an Agilent HP 1100 HPLC module (Agilent Technologies, Palo Alto, CA) connected to a flow scintillation analyzer (ß-RAM radiometric detector; IN/US Systems, Tampa, FL). LC-MS/MS analyses for structural determination were performed using an LCQ (Thermo Finnigan, San Jose, CA) or a Micromass VG Quattro (Waters, Milford, MA) mass spectrometer with electrospray source and operated in positive or negative ionization mode. For NMR analysis, metabolites were isolated by fraction collection and were dissolved in deuterated methanol. Proton and carbon-13 resonance spectra were acquired using a Bruker drx-500 NMR spectrometer (Bruker BioSpin Corp., Rheinstetten, Germany). The metabolite characterization in this study was based on the assignments made from the extensive studies done with cinacalcet and its dihydrodiol metabolites (M2), generated in vitro in human liver microsomes, by one-dimensional proton, two-dimensional correlation spectroscopy, two-dimensional total correlation spectroscopy, two-dimensional nuclear Overhauser effect spectroscopy, heteronuclear multiple-bond correlation spectroscopy, and, in some cases, diffusion-edited spectra (L. Poppe, personal communication).
Quantitation of cinacalcet in monkey and human plasma was performed by LC-MS/MS using electrospray ionization with heptadeuterated cinacalcet as an internal standard. Extraction was performed using Oasis MCX 96-well extraction plates (Waters), and chromatography was performed using a Keystone Inertsil Silica column (Thermo Hypersil, Keystone Scientific Operations, Bellefonte, PA) (2 x 50 mm, 5-µm particle size). The mobile phase was 90:10 acetonitrile/water with 1% formic acid. Noncompartmental analysis was performed using WinNonlin (v3.1; Pharsight, Mountain View, CA).
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Results
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Tissue Distribution. The [14C-2-CH2]cinacalcet-derived radioactivity levels in select tissues in rats are summarized in Table 1. For all other tissues examined, including the central nervous system, skeletal muscle, testes, abdominal fat, and the eye, the tissue to plasma ratios (T/P) were low (<0.5). The total radioactivity levels in blood were lower than those in plasma (blood/plasma ratio <0.4), suggesting that there was minimal uptake of cinacalcet-derived radioactivity into the cellular elements of the blood. Radioactivity was widely distributed into the tissues, with no apparent gender-related differences. The decline in radioactivity levels in tissues over the 48-h period examined was approximately parallel to that in plasma. Relatively higher levels of radioactivity compared with plasma were observed in highly perfused organs such as kidney, liver, lung, and pancreas. As expected following oral dosing, the highest levels of radioactivity were associated with the contents of the gastrointestinal tract, with AUC(48 h) of radioactivity for the contents of stomach, small and large intestines, and cecum ranging from 1140 to 1510 mg Eq · h/g (Table 1).
Pharmacokinetics. In monkeys, after 10 mg/kg dosing of [14CCF3]cinacalcet, the maximum concentrations of radioactivity as well as cinacalcet levels were attained at a similar time (Table 2; Fig. 1). The absorption of [14C-CF3]cinacalcet-derived radioactivity was at least 74%, based on the recovery of radioactivity in the urine and bile (Table 3). There was an approximately dose-proportional increase in AUC of both total plasma radioactivity and cinacalcet between 10 and 100 mg/kg doses. Based on the comparison of AUCs of unchanged cinacalcet and total plasma radioactivity, less than 0.5% of the cinacalcet-derived radioactivity in plasma was due to the unchanged parent in monkeys. The apparent shorter terminal half-life of cinacalcet compared with total radioactivity is probably an artifact due to levels of cinacalcet below the limit of quantitation beyond 24 h postdose.
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TABLE 2 Pharmacokinetics of cinacalcet and total plasma radioactivity in monkeys and humans after a single oral dose
Values are mean ± S.D.
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FIG. 1. Mean plasma concentrations of radioactivity, cinacalcet and its metabolites in male cynomolgus monkey plasma after a single 10 mg/kg oral dose of [14CCF3]cinacalcet.
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After oral administration of [14C-CF3]cinacalcet to humans, maximum concentrations of cinacalcet as well as radioactivity occurred within 3 h after administration (Fig. 2). In humans, the absorption of cinacalcet-derived radioactivity was high (at least approximately 80%), based on total urinary excretion of radioactivity (Table 3). The plasma profile of cinacalcet and total radioactivity suggests that metabolites were not longer-lived than cinacalcet in humans. The AUC0-t ratio of total radioactivity in human blood and plasma was approximately 0.44 (Table 2), suggesting that there was no significant uptake of cinacalcet-derived radioactivity into blood cells. However, in an in vitro experiment conducted with human blood, the blood to plasma ratio of cinacalcet was approximately 1 (Dr. S. Surapaneni, personal communication), indicating that whereas cinacalcet itself distributes into blood cells, its metabolites probably do not. Based on the AUC ratio of total plasma radioactivity to unchanged cinacalcet, less than 0.5% of the cinacalcet-derived radioactivity in plasma was due to unchanged parent in humans. Metabolites of cinacalcet constitute the predominant portion of the cinacalcet-derived circulating radioactivity in both humans and animal models. No marked differences in the pharmacokinetics of cinacalcet were observed between nonsmoking and smoking subjects.
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FIG. 2. Plasma concentrations of cinacalcet and total radioactivity in nonsmoking human volunteers after a single 75-mg oral dose of [14C-CF3]cinacalcet.
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Excretion of Radioactivity. The excretion of radioactivity in animals and humans is summarized in Table 3. In mice, radioactivity was rapidly excreted, with the majority being excreted during the first 24 h after dosing. The excretion of radioactivity in urine accounted for 50.6% and 69.0% of the dose in male and female mice, respectively. Another 37.2% and 27.3% of the dose was excreted in the feces of male and female mice, respectively. Radioactivity was rapidly excreted by rats, with a mean total recovery of 91% of the radioactive dose excreted within 4 days of dosing, and a predominant portion being excreted during the first 24 h postdose. Approximately equal amounts of radioactivity were excreted in urine (46.5%) and feces (44.5%). The residual radioactivity in tissue and carcass accounted for <0.4% of the administered radioactivity, indicating a virtually complete elimination of radioactivity from rats within 4 days of dosing. In male cynomolgus monkeys, radioactivity was cleared via both urinary and hepatobiliary routes. In bile duct-cannulated animals, 95.8% of the dose was excreted (including cage wash/debris), with 35.9%, 19.1%, and 38.4% excreted in urine, feces, and bile, respectively. The main route of excretion of [14C-CF3]cinacalcet-derived radioactivity after oral dosing to humans was via urine (80-83%) (Fig. 3; Table 2). Excretion in the feces accounted for 13 to 17% of the radioactive dose. The mean recovery of radioactivity in excreta was 96 to 97% of the dose. There were no marked differences in the excretion of radioactivity between smoking and nonsmoking subjects.
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FIG. 3. Cumulative excretion of radioactivity in nonsmoking human volunteers after a single 75-mg oral dose of [14C-CF3]cinacalcet.
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Metabolite Identification. Cinacalcet. The protonated molecular ion of cinacalcet was observed at m/z 358 (MH+) (Fig. 4). The major fragment ions from cinacalcet were observed at m/z 204 (corresponding to m-CF3-phenyl-CH2-CH2-CH2-NH) and m/z 155 (corresponding to naphthyl-CH-CH3). Fragment ion m/z 204 further fragmented due to loss of HF, leading to m/z 184. The proton NMR data for cinacalcet are summarized in Fig. 5.
M2 and M2-Glu. The protonated molecular ion of metabolite M2-Glu was observed at m/z 568 (MH+) (Fig. 4). The major fragment ions from M2-Glu were observed at m/z 392 (parent - 176; loss of glucuronide), m/z 374 (parent - H2O-Glu), m/z 204 (corresponding to m-CF3-phenyl-CH2-CH2-CH2-NH), m/z 189 (corresponding to dihydrodiol of naphthyl-CH-CH3), and m/z 171 (corresponding to oxidized naphthyl-CH-CH3; loss of water from m/z 189). Fragment ion m/z 204 yielded m/z 184 due to loss of HF. The molecular ion of the corresponding aglycone M2 from glucuronidase-treated urine and bile was observed at m/z 392 (MH+), which is 34 atomic mass units greater than cinacalcet. The major fragment ions from aglycone M2 were similar to those observed with M2-Glu, being m/z 374, m/z 204 (fragmentation of which yielded m/z 184 and m/z 164), m/z 189, and m/z 171. This fragmentation pattern indicates that the aglycone M2 is a dihydrodiol metabolite of cinacalcet with oxidation occurring on the naphthalene ring system.
Metabolites M2-Glu and M2 were observed as several distinct peaks. This is probably due to the existence of several regioisomers of this metabolite. The major diastereomeric M2-Glu metabolite, M2a-Glu, was characterized by NMR. The proton NMR spectral data of M2a-Glu and cinacalcet are compared in Fig. 5. The most significant change in the chemical shifts occurred for protons 5 and 6 of the naphthalene ring system, indicating these locations as the sites of hydroxylation. The chemical shifts of these protons to 4.95 and 4.61 ppm suggest that these two carbons lost their aromaticity. The stereochemistry of the dihydrodiol group of M2a-Glu was established by comparisons made to the assignments of in vitro generated aglycone M2a via extensive nuclear Overhauser effect spectroscopy experiments (L. Poppe, personal communication). The site of glucuronidation, as proposed in Fig. 5, was based on the change in the chemical shift of H-4 compared with the aglycone M2a (Fig. 5). Another major form of M2-Glu, M2b-Glu, showed a shift in resonances corresponding to the b ring protons of naphthalene (data not shown), indicating that both rings of the naphthalene group of cinacalcet are subject to oxidative transformation.
M7-Gly. The protonated molecular ion of M7-Gly was observed at m/z 274 (Fig. 6). The major fragment ions were m/z 256 (due to loss of water), m/z 228 (parent - COOH), and m/z 199 (parent - NHCH2-COOH). Based on the fragmentation pattern, M7-Gly was identified as the glycine conjugate of a carboxy metabolite of cinacalcet. The structure of this metabolite was confirmed by NMR analysis (Fig. 5). The major changes in proton chemical shifts in M7-Gly were for H-10/10' (2.03 ppm of cinacalcet compared with 6.84 ppm of M7-Gly) and H-11/11' (2.74 ppm of cinacalcet compared with 7.61 ppm of M7-Gly). Furthermore, the multiplicity of these resonances simplified from multiplet in cinacalcet to doublet in M7-Gly. These data indicate that the CH2-CH2 bond of C10-C11 carbons of cinacalcet was converted to a CH=CH bond in M7-Gly. A new resonance was observed in M7-Gly with a chemical shift of 4.01 ppm, which corresponds to the methylene group of glycine. The carbon-13 NMR chemical shifts (data not shown) for the two proposed carboxy carbons C-9 (166.3 ppm) and C-7G (172.6 ppm) were also in the expected region, further confirming the presence of the glycine moiety in M7-Gly. Based on these data, M7-Gly was identified as the glycine conjugate of m-trifluoromethyl cinnamic acid.
M8-Gly. The deprotonated molecular ion of M8-Gly was observed at m/z 246 (M - H)- (Fig. 6). In the negative ion mode, the major fragment ions from m/z 246 were m/z 202 (parent - COO) and m/z 145 (m-CF3-phenyl). Fragment m/z 202 further fragmented due to sequential loss of HF, leading to m/z 182 and m/z 162. Based on the fragmentation pattern, M8-Gly was identified as the glycine conjugate of the m-trifluoromethyl-benzoic metabolite of cinacalcet. The identity of this metabolite was further confirmed by NMR analysis (Fig. 5). The major changes in chemical shifts in M8-Gly were for aromatic proton H-12 (7.46 ppm in cinacalcet compared with 8.21 ppm in M8-Gly) and H-15 (7.39 ppm of cinacalcet compared with 8.15 ppm of M8-Gly) (Fig. 5). These data indicate that a modification has occurred on the carbon between C-12 and C-15 of M8-Gly. Additionally, a new resonance was observed with a chemical shift of 4.13 ppm, which corresponds to the methylene group of glycine (carbon 10G). The carbon-13 NMR chemical shifts (data not shown) for the two proposed carboxy carbons C-11 (167.2 ppm) and C-9G (172.1 ppm) were also in the expected region, further confirming the presence of glycine moiety in M8-Gly. Based on these data, M8-Gly was identified as the glycine conjugate of m-trifluoromethyl benzoic acid.
M1-Glu. Metabolite M1-Glu (m/z 550) was observed as two peaks in urine and bile of monkeys. There were two distinct patterns of fragmentation of M1-Glu (Fig. 7). The corresponding aglycones, M1, were observed in monkey feces. Based on the fragment ions, these metabolites were identified as glucuronide conjugates of monohydroxylated products of cinacalcet. M1a and M1a-Glu yielded two major fragments with m/z 204 (corresponding to m-CF3-phenyl-CH2-CH2-CH2-NH) and m/z 171 (corresponding to oxidized naphthyl-CHCH3), indicating that the oxidation site in this molecule is on the naphthyl ring of the molecule, whereas M1b and M1b-Glu yielded two major fragments, m/z 220 (corresponding to oxidized m-CF3-phenyl-CH2-CH2-CH2-NH), m/z 202 (loss of water from m/z 220), and m/z 155 (corresponding to naphthyl-CH-CH3), indicating that the oxidation site is not on the naphthyl side of the molecule.
M3-Glu. The protonated molecular ion of minor metabolites M3-Glu, present in monkey bile and urine, was observed at m/z 584 (M + H)+ (Fig. 7). M3-Glu, present as two peaks, yielded two distinct fragment patterns. The corresponding aglycones, M3, were observed in mouse feces. The major diagnostic fragments from these two metabolites were m/z 204 (corresponding to m-CF3-phenyl-CH2-CH2-CH2-NH) and m/z 220 (corresponding to oxidized m-CF3-phenyl-CH2-CH2-CH2-NH). These data indicated that metabolites M3-Glu are further oxidation products of M2-Glu, with oxidation occurring on either the naphthalene ring system (M3a-Glu) or the m-CF3-phenyl-CH2-CH2-CH2-NH portion (M3b-Glu) of the molecule.
M5, M6, and M7. LC-MS/MS analysis of mouse plasma in the negative ion mode yielded two coeluting signals with m/z 215 and m/z 217. Based on their fragmentation patterns, the metabolites with m/z 215 and m/z 217 were identified as m-trifluoromethyl cinnamic acid (M7) and m-trifluoromethyl hydrocinnamic acid (M5), respectively. Their identity was further confirmed by comparison of HPLC retention times and fragmentation patterns with authentic standards of m-trifluoromethyl cinnamic acid and m-trifluoromethyl hydrocinnamic acid. Another metabolite M6, a hydroxy derivative of M5 (m/z 233) was present in small quantities in monkey and human plasma. Based on the fragmentation pattern, this metabolite was identified as a hydroxy derivative of m-trifluoromethyl hydrocinnamic acid.
Composition of Excreta and Plasma Radioactivity. Metabolites of cinacalcet were the predominant components of excreta radioactivity in mice (Fig. 8; Table 4). The major metabolite in urine was M7-Gly (glycine conjugate of 3'-trifluoromethyl cinnamic acid). In feces, the major metabolite was M2a (dihydrodiol of cinacalcet). It is likely that the M2-Glu, excreted via bile, was hydrolyzed to its aglycone in the gastrointestinal tract. In mouse feces, M4, a metabolite corresponding to an authentic standard of m-CF3-phenyl-CH2-CH2-CH2-NH2 (m/z 204) was observed. The major components of plasma radioactivity in mice were M5 and M7 (>78%; present in a 3:1 ratio of M5/M7), with small amounts of cinacalcet and M2a-Glu.
Cinacalcet was extensively metabolized in monkey, with oxidation of the naphthalene ring and N-dealkylation leading to carboxy metabolites as primary metabolic pathways. In urine, the major metabolites were M2-Glu and M7-Gly. The major metabolite in feces was M1, with significant amounts of unchanged cinacalcet. In bile, the major metabolites were M2-Glu and M1-Glu. The major metabolites in plasma were M5/M7 (CF3-cinnamic and CF3-hydrocinnamic acids, respectively, present in an approximately 1:1 ratio) and M2-Glu. Based on the AUC ratio of total plasma radioactivity and unchanged cinacalcet in monkeys, less than 0.5% of the cinacalcet-derived radioactivity in plasma was due to cinacalcet, with M2-Glu (59%) and M5/M7 (32%) as the predominant components (Fig. 1).
Urinary elimination was the predominant route of excretion of cinacalcet-derived radioactivity in humans. The major routes of metabolism were identified as oxidation of the naphthalene ring system leading to dihydrodiols (eliminated as glucuronide conjugates M2a-Glu and M2b-Glu) and N-dealkylation leading to carboxy metabolites (eliminated as glycine conjugates M7-Gly and M8-Gly). At 3 h postdose, the total plasma radioactivity in nonsmoking human subjects was 2.85 ± 0.223 µg Eq/g, with M5 accounting for 73% of the radioactivity. Minor amounts of dihydrodiol-glucuronides (M2a-Glu and M2b-Glu; 11% of plasma radioactivity) and M6 (hydroxy-3'-CF3-hydrocinnamic acid; 9% of plasma radioactivity) were also observed in human plasma. A similar quantitative profile of metabolites was observed in smokers. Based on the comparison of the AUC values of total radioactivity and unchanged cinacalcet, cinacalcet accounted for <0.5% of the circulating radioactivity in humans.
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Discussion
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Cinacalcet was well absorbed and extensively metabolized in all species examined, with a broad similarity in the metabolic pathways. The proposed major metabolic pathways of cinacalcet are summarized in Fig. 9, and the quantitative analyses are summarized in Table 4. The primary routes of metabolism of cinacalcet were N-dealkylation leading to carboxylic acid derivatives and oxidation of naphthalene ring system to form dihydrodiols. The oxidative metabolites were conjugated before elimination. The extent of metabolism occurring via the N-dealkylation pathway was higher in mice compared with monkeys and humans. The carboxylic acid metabolites (M5/M6/M7) and dihydrodiol glucuronides (M2-Glu) were the predominant components of circulating radioactivity. The relative circulating levels of M2-Glu were higher in monkey and human than in mouse plasma, possibly due to more facile N-dealkylation in mice. Another noticeable difference is the greater extent of radioactive dose excreted via the urinary route in humans compared with animal models, 80% versus 35 to 69%. It is not known if the relatively higher urinary excretion of radioactivity in humans is due to the lower dose used for human study (approximately 1 mg/kg) compared with animal studies (10-100 mg/kg).
Compounds containing naphthalene and quinoline ring systems are known to undergo oxidative metabolism to form dihydrodiols (Schatz and Haber, 1986; Grossman et al., 1993; Vickers et al., 1999). In the case of terbinafine, several positional isomers of dihydrodiol metabolites were reported, with oxidation occurring on either ring of the naphthalene. Similarly, both rings of naphthalene in cinacalcet were subject to oxidation with multiple positional isomers of dihydrodiols. Unlike many other drugs with dihydrodiol metabolites, glucuronide conjugation of dihydrodiol metabolites of cinacalcet appears to be facile. There was no indication for the presence of glutathione conjugates in any of the species examined. Another major route of oxidative metabolism of cinacalcet was via N-dealkylation leading the carboxy metabolite M5. This metabolite was subject to further oxidative metabolism, akin to ß-oxidation, leading to metabolite M8, possibly via intermediate metabolites M6 and M7. As is often observed with other carboxylic acids, glycine conjugation and urinary excretion were observed in this case also.
Circulating metabolites can potentially contribute to the therapeutic and/or toxicological effects of a therapeutic drug. The importance of major circulating metabolites in safety testing and monitoring was dealt with in detail in a recent publication (Baillie et al., 2002). It is generally accepted that there should be a qualitative similarity in the profile of circulating drug-related material in human and animal models to demonstrate the appropriateness of the preclinical toxicology species. In some instances, circulating metabolites could have longer terminal half-life than does the parent (Cox et al., 2002), which could potentially be of consequence when they are either toxic or active. In the case of cinacalcet, the circulating radioactivity was primarily due to metabolites in both animal models as well as humans. However, in an in vitro pharmacology assay, the circulating metabolites of cinacalcet are either inactive (M5, M7) or markedly less active (M2-Glu, 
as active as cinacalcet) (Dr. W. Karbon, personal communication). In addition, the analysis of clinical pharmacokinetic and pharmacodynamic data suggested that within individual subjects, there is a good correlation between unchanged cinacalcet plasma levels and therapeutic effect; i.e., parathyroid hormone suppression (Dr. R. Harris, personal communication), consistent with the hypothesis that the circulating metabolites of cinacalcet are not significant contributors to the overall pharmacological activity. Since the circulating and excreted profile of cinacalcet metabolites are qualitatively similar across species, the animal models used for nonclinical safety studies were appropriate representatives of the human situation. In summary, cinacalcet was metabolized extensively via oxidative and conjugative pathways. The preclinical animal models were predictive of the human metabolism of cinacalcet.
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Acknowledgments
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We acknowledge the contributions made by Drs. G. Rishton, W. Karbon, J. T. Sullivan, and. G. Lawrence of Amgen; Drs. M. Berge and C. Whalen of Covance Laboratories; and Dr. K. Dix of Research Triangle Institute. We also thank Drs. R. Harris, M. Bajpai, S. Surapaneni, R. Arends, D. T.-W. Lau, K. Miller, and S. Turner for helpful discussions.
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Footnotes
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doi:10.1124/dmd.104.000604.
ABBREVIATIONS: AUC0-t, the area under the plasma concentration versus time curve up to the last sampling time point; ADME, absorption, distribution, metabolism, and excretion; Cmax, peak plasma concentration; LC-MS/MS, liquid chromatography-tandem mass spectrometry; Tmax, time at which Cmax was reached; t1/2, the terminal-phase elimination half-life.
Address correspondence to: Gondi N. Kumar, Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA. E-mail: gkumar{at}amgen.com
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Materials and Methods
