1. In Vitro Metabolism and Prediction of Drug-Drug Interaction of the Calcimimetic Agent Cinacalcet HCl
Manoj Bajpai, Joel Esmay, Victor Chi, Mike Hayashi, Leszek Poppe and Gondi Kumar*
Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA , USA.

2. Abstract
Cinacalcet HCl is a calcimimetic agent used for the treatment for hyperparathyroidism. In the course of preclinical development, in vitro studies were performed to understand its metabolism and to predict the potential for interactions between cinacalcet HCl and concomitant medications. Cinacalcet HCl was subject to oxidative metabolism catalyzed by cytochrome P450 enzymes, with primary sites of metabolism being the naphthalene ring and dealkylation at the secondary amine. Isoform profiling studies revealed that multiple enzymes were involved in cinacalcet HCl metabolism, with CYP 1A2 and 3A4 being predominant. Cinacalcet HCl was found to have minimal inhibitory effect on CYP 1A2, 2C9, 2C19 and 3A4 (IC5010 mM), but was a potent inhibitor of CYP2D6. Cinacalcet HCl did not induce CYP 1A2, 2C19 or 3A4 activities in cultured human hepatocytes. Based on this in vitro data, multiple clinical studies were designed and performed to assess metabolism and drug-drug interaction potential with cinacalcet HCl during poly-pharmacy. The in vitro data were predictive of in vivo observations in humans.

3. Methods Metabolite Identification
Cinacalcet HCL was incubated with human liver microsomes (HLM, 0.25 mg protein/ml).
Cinacalcet HCL and metabolites were resolved by gradient chromatography.
Radiometric detection performed on a b-RAM radiometric detector, IN/US Systems, Tampa, FL.
LC-MS/MS analyses performed on an LCQ (ThermoFinnigan, San Jose, CA) mass spectrometer with electrospray source and operated in positive ionization mode.
Isoform Profiling
Metabolic Activity by cDNA Expressed Enzymes
Cinacalcet HCL was incubated with microsomes from cells overexpresseing individual P450 isozymes.
Formation of metabolites was monitored by radiometric and LC-MS detection similar to above.
% Inhibition by isoform-selective inhibitors
Inhibition of Cinacalcet HCL turnover in HLM
Analysis by HPLC
Correlation of cinacalcet turnover with isoform-specific marker activity
Effect of cinacalcet on isoform-specific CYP catalytic activities
Isoform specific substrates were co-incubated in HLM with or without Cinacalcet HCL at varying concentrations (Table 1.)
Reaction rates for isoform specific biotransformation analyzed by measuring product formation (table 1.)
Hepatocyte Incubations
Cinacalcet was incubated in fresh human hepatocytes from 3 donors for 48 hours
P450 activities were measured with CYP450 specific substrates

4. Methods (continued)
Table 1. Methods for evaluating the effect of cinacalcet on isoform-specific CYP catalytic activities in Human Liver Microsomes

5. Results of Metabolite Identification
Cinacalcet HCL is subject to oxidative metabolism including hydroxylation and dealkylation pathways (figure 1.)
The primary sites of oxidative metabolism were the naphthalene ring system and dealkylation of the secondary amine. (Figure 2.)
Overall, the in vitro Phase I metabolism of cinacalcet was predictive of the in vivo metabolism.
Conjugation of the oxidative and dealkylation metabolites was observed in in vivo pre clinical models and in humans (data not shown).

6. Results (continued)
Figure 1. Extracted ion chromatogram of Cinacalcet HCL and metabolites identified in human liver microsomes
NL: 1.78E5 5 10 15 20 25 30 35 40 45 50 55 60 65
Time (min) 70 75 80 85 90 95
100
Relative Abundance
m/z 204
m/z 392
m/z 358
Cinacalcet
m/z 374

7. Results (continued)
Figure 2. Mass spectral fragmentation pattern of a major metabolite of cinacalcet

8. Results of Isoform Profiling
Multiple enzymes are involved in Cinacalcet HCl metabolism (Table 2).
Cytochromes P450 1A2 and 3A4 are the predominant enzymes responsible for Cinacalcet HCL metabolism in vitro (Table 3).

9. Table 2. Cinacalcet HCL Metabolism by cDNA Expressed Enzymes

10. Figure 3. Effect of CYP isoform-selective chemical inhibitors on human liver microsomal metabolism of cinacalcet (n=3, mean ± SD)
* Cinacalcet HCL was run above its Km and hence CYP2D6 contribution could not be accurately characterized.

11. Figure 4. Correlation of cinacalcet metabolism with CYP1A2, CYP2D6 and CYP3A4 dependent catalytic activities in a panel of human liver microsomes
Clapp (% of mean mL/min/mg)
Clapp (% of mean mL/min/mg)
Clapp (mL/min.mg)
Clapp (mL/min.mg)
Clapp (% of mean mL/min/mg)
Clapp (mL/min.mg)

12. Table 3. Results of Three distinct approaches to Isoform Profiling
Cytochrome P450 isoform
Metabolic Activity by cDNA Expressed Enzyme
% Inhibition by isoform-selective inhibitors
Correlation coefficient
with isoform-specific activity
1A2 +
~ 28% a
0.59
2A6 - ND
0.70b
2C9
~ 23% a
0.17
2C19
~ 5%
0.22
2D6
0.63
2E1
0.49b
3A4
~ 30% a
0.88
+ denotes metabolism, where as – denotes no apparent metabolism.
a p-value < 0.05
b due to secondary correlation between 2D6/2E1 (R=0.66, P<0.01) and 3A4/2A6 (R=0.62, P<0.01) in this panel of human liver microsomes, a significant correlation was found between cinacalcet HCL metabolism and 2A6/2E1 activities, the correlation observed in this study is probably coincidental.
ND – not detected

13. Results of Studies investigating the Effect of Cinacalcet HCL on isoform-specific CYP catalytic activities
Cinacalcet HCL has minimal inhibitory effect on CYP’s 1A2, 2C9, 2C19 and 3A4 in vitro (IC50 > 10 mM, figure 4).
Only modest interaction observed between Cinacalcet HCL and Ketoconazole, a marker substrate for CYP3A (D. Phadi et al, ISSX 2005 abstract #234).
Cinacalcet is a potent inhibitor of CYP2D6 with an IC50 of 70 nM (figure 4.)
Selective inhibition of CYP 2D6 by Cinacalcet HCL was observed in vivo (R. Harris eta al, ISSX 2005 abstract #214)
Concurrent administration of 25 mg or 100 mg cinacalcet with 50 mg amitriptyline increased amitriptyline exposure and nortriptyline (active metabolite) exposure by approximately 20% in CYP2D6 extensive metabolizers. (Amgen Inc. prescribing information, 2004)

14. Figure 4. Prediction of drug-drug interactions potential of Cinacalcet HCL
Cmax Conc.
Manoj Bajpai, Ph.D.
In-vitro Metabolism Lab

15. Results of Incubation of Cinacalcet in Human Hepatocytes
Cinacalet HCL did not induce CYP2C19 (data not shown) CYP1A2 or CYP3A4-dependent activities in cultured hepatocytes isolated from three human donors (Table 4).
The responsiveness of the hepatocyte preparations to CYP inducers was verified using known inducers of CYP 1A2 and 3A4

16. Table 4. Effect of Cinacalcet HCL on the CYP1A2 and CYP3A Dependent Catalytic Activities in Cultured Primary Human Hepatocytes
Treatment (mM)
Donor A
Donor B
Donor C
Mean SD
CYP1A2 (acetaminophen formation)
Vehicle (0.1% DMSO)a
Rateb
24.8±1.3
13.5±1.3
9.4±0.7
Omeprazole (50 mM)
%VCc
1510%
2004%
2120%
1878%
324%
Cinacalcet HCl (0.02 mM)
%VC
96%
76%
68%
80%
15%
Cinacalcet HCl (0.2 mM)
90%
84%
108%
94%
13%
Cinacalcet HCl (2 mM)
110%
102%
131%
114%
CYP3A (6b-hydoxytestosterone formation)
Vehicle (0.1% DMSO)
7.88±0.32
37.4±1.57
18.4±0.52
Rifampin (25 mM)
4696%
860%
1274%
2277%
2105%
109%
99%
101% 7%
116%
97%
107%
10%
147%
112%
104%
121%
23%
a mean of three incubations
b pmol of formed/minute/million hepatocytes
c %VC: percent of vehicle control

17. Summary
Cinacalcet HCL is oxidatively metabolized in human liver microsomes to N-dealkylated form and hydroxylation of the naphalene ring
Multiple P450 isoforms metabolize cinacalcet HCL in vitro with CYP 3A4 and 1A2 being predominant.
Cinacalcet HCL is a potent inhibitor of CYP2D6 but not CYPs 1A2, 2C9, 2C19 or 3A4.
Cinacalcet did not induce CYP1A2, 2C19 or 3A4 activity in hepatocytes from 3 human donors.
The in vitro data was essential in guiding clinical studies.

18. Conclusions
Preclinical in vitro studies provided early understanding of the metabolism of Cinacalcet HCL in humans.
A variety of in vitro techniques were employed to effectively predict or rule out potential for drug – drug interactions with Cinacalcet HCL.
The clinically relevant potency for inhibition of CYP 2D6 was realized promptly by in vitro methods and confirmed by clinical experience.
The in vitro data were predictive of in vivo observations in humans