Here we report the first highly selective CYP3A4 inhibitor optimized from an initial lead with ≈30-fold selectivity over CYP3A5 to yield a series of compounds with greater than 1000-fold selectivity. During our medicinal chemistry and biology effort pursued under our existing LRH1 inverse agonist Extended Characterization (EC) project, we identified several compounds belonging to the imidazopyridine scaffold with activity against the cytochrome enzymes. As a result we have explored how these compounds act on the CYP enzymes described within our approved EC proposal. Associated toxicities due to CYP3A4 related drug–drug interactions have led to clinical failure and withdrawal from market of previously approved pharmaceuticals. Mibefradil (Posicor), a potent inhibitor of CYP3A4, was withdrawn from the market after numerous reports of serious drug–drug interactions. Terfenadine (Seldane) and Cisapride (Propulsid) were withdrawn after patients taking the recommended dose along with CYP3A4 inhibitors such as ketoconazole developed heart arrhythmia leading to heart attack or death. To date, no substrates have been identified that are metabolized exclusively by only one of the enzymes. However, using recombinantly expressed enzymes, several substrates have been shown to have higher catalytic efficiency for either CYP3A4 or CYP3A5. Similarly, no highly selective inhibitors have been identified, but many do show two to fivefold preference for one of the two enzymes. Many of these have been associated with time-dependent inactivation where CYP3A4 appears to be more susceptible to time-dependent inactivation than does CYP3A5. The probe identified herein was tested by the assay provider's lab and the Scripps team using microsomal-based and biochemical assays. These probes will provide useful tools to elucidate the roles of this enzyme in cell metabolism, and will enable studies to differentiate the actions of the diverse members of the CYP family of enzymes.

Probe Structure & Characteristics

Recommendations for scientific use of the probe

• What limitations in current state of the art is the probe addressing?

The lack of proper chemical tools to differentiate the activity of CYP3A4 and CYP3A5 has led to the longstanding, yet erroneous, convention of treating the two enzymes as if they were one. Sometimes activity has been expressed as CYP3A to point out that the results are not specific for either CYP3A4 or CYP3A5. However, too often all activity has been contributed to CYP3A4. Furthermore, the practice of pooling tissue from multiple donors to generate an “average human” has given rise to the perception that CYP3A5 has less significance than CYP3A4. While it is certainly true that the abundance of CYP3A4 exceeds that of CYP3A5 in a multi-donor pool, this is not representative of actual patients. The concentrations of the two enzymes have been reported to be roughly equal in individuals that express CYP3A5 (1).

• What will the probe be used for?

The identified probe and analogs will be useful for drug discovery projects, particularly safety pharmacology where the metabolic profile of new drugs are reported as part of the package submitted to FDA for new drug applications.

• Who in the research community will use the probe?

The probe is of significant use to the pharmaceutical industry and to researchers doing translational research. We have already sent the probe or the synthetic protocol to scientists at Pfizer, Bristol Meyers Squibb and Vanderbilt University who are now using the probe in their research.

• What is the relevant biology to which the probe can be applied?

The probe can be used to differentiate CYP3A4 and CYP3A5 activity in biological samples. This can be a source of significant variability because CYP3A5 has three common genetic alleles (2). CYP3A5*1 leads to the expression of active, full length CYP3A5. The CYP3A5*3 (22893A→G) allele in intron 3 leads to a frame shift resulting in the majority of the CYP3A5 mRNA yielding inactive protein and loss of CYP3A5 expression. Analysis by Western blot and RT-PCR demonstrates that individuals homozygous for CYP3A5*3 have low levels of correctly spliced CYP3A5 mRNA and this corresponds to sharply lower CYP3A5 protein levels (2). A second allele, CYP3A5*6 (30597G→A) on exon 7 causes the deletion of exon 7 from the splice variant and is associated with lower CYP3A5 catalytic activity. The clinical relevance of CYP3A5 genotype is seen with the immunosuppressant tacrolimus which is metabolized by CYP3A5 3-times more efficiently than by CYP3A4 (3). In order to maintain the required tacrolimus trough concentrations of 5 to 15 ng/mL, patients that express active CYP3A5 (*1/*1 and *1/*3 genotype) require approximately twice the dose as *3/*3 (inactive splice variant) patients (4). Additionally, CYP3A5 genotype has been implicated in vincristine metabolism where CYP3A5 low expressers were found to have increased risk of vincristine-induced neurotoxicity (5-8).

1. Introduction

The lack of proper chemical tools to differentiate the activity of CYP3A4 and CYP3A5 has led to the long-standing, yet erroneous, convention of treating the two enzymes as if they were one. Sometimes activity has been expressed as CYP3A to point out that the results are not specific for either CYP3A4 or CYP3A5. However, too often all activity has been attributed to CYP3A4. Furthermore, the practice of pooling tissue from multiple donors to generate an “average human” has given rise to the perception that CYP3A5 has less significance than CYP3A4. Although it is certainly true that the abundance of CYP3A4 exceeds that of CYP3A5 in a multidonor pool, this is not representative of actual patients. The concentrations of the two enzymes have been reported to be roughly equal in individuals who express CYP3A5 (9). Enzyme-selective chemical inhibitors are commonly used in reaction phenotyping studies to determine the contribution of individual cytochrome P450 isoforms (10). Knowledge of the metabolic pathway for a candidate compound allows more accurate predictions of potential drug-drug interactions. Quality chemical tools are available to determine the activity and inhibition of most of the major cytochromes P450 involved in xenobiotic metabolism. The commonly used CYP3A4 and CYP3A5 inhibitors inhibit both enzymes, precluding differentiation or activity in complex samples. Although no highly selective CYP3A4 or CYP3A5 inhibitors have been reported in the literature, several compounds have been shown to have modest 3- to 10-fold selectivity over CYP3A5 (11,12). This degree of selectivity is not sufficient to inhibit 90% of CYP3A4 without significant CYP3A5 inhibition. Despite the inability of these mildly selective inhibitors to serve as in vitro tools for the isolation of CYP3A5 activity, they demonstrate that a degree of selectivity is achievable across numerous structural classes. In general, CYP3A4 appears to be more susceptible to irreversible inactivation in the presence of compounds that are metabolized to reactive metabolites, and many of the published selective inhibitors are time dependent inhibitors (13-15). It should be stressed that many of the selective inactivators are not selective inhibitors because they display significant competitive inhibition of CYP3A5 despite their lack of time-dependent inactivation. CYP3A5 has three common genetic alleles (16). CYP3A5*1 leads to the expression of active, full-length CYP3A5. The CYP3A5*3 (22893A3G) allele in intron 3 leads to a frameshift, resulting in the majority of the CYP3A5 mRNA yielding inactive protein and loss of CYP3A5 expression. Analysis by Western blot and RT-PCR demonstrates that individuals homozygous for CYP3A5*3 have low levels of correctly spliced CYP3A5 mRNA, and this corresponds to sharply lower CYP3A5 protein levels (16). A second allele, CYP3A5*6 (30597G3A), on exon 7 causes the deletion of exon 7 from the splice variant and is associated with lower CYP3A5 catalytic activity. Selective inhibitors will refine the current prediction models for pharmacokinetic drug-drug interactions in which the catalytic efficiency of CYP3A4 and CYP3A5 can be accounted for and the influence of genetic polymorphisms can be incorporated in future models. A better understanding of both enzymes is important to make accurate clearance predictions before compounds are moved into human trials. The clinical relevance of the CYP3A5 genotype is seen with the immunosuppressant tacrolimus, which is metabolized by CYP3A5 3 times more efficiently than by CYP3A4 (17). To maintain the required tacrolimus trough concentrations of 5 to 15 ng/ml, patients who express active CYP3A5 (*1/*1 and *1/*3 genotypes) require approximately twice the dose as patients with the *3/*3 (inactive splice variant) genotype (18). In addition, the CYP3A5 genotype has been implicated in vincristine metabolism, and CYP3A5 low expressers were found to have an increased risk of vincristine-induced neurotoxicity (19-22). The current article details a highly selective CYP3A4 inhibitor suitable for isolation of CYP3A5 activity in human liver microsomes. We have published details of the structure-activity relationship of SR-9186 and a number of related analogs for selective CYP3A4 inhibition (23). In this study, we put special emphasis on the use of the inhibitor under conditions encountered in conducting reaction phenotyping studies, such as high concentrations of microsomal protein and long incubation times. Specific recommendations are made for the use of this new tool compound.

2. Materials and Methods

All chemical reagents and solvents were acquired from commercial vendors. All assay protocols are reported in the relevant PubChem AIDs, provided below. Solubility, stability, and glutathione reactivity analyses were conducted in accordance with NIH guidelines. CYP450 inhibition and microsome stability analyses were performed as previously described.

2.2. Probe Chemical Characterization

¹H NMR (700 MHz, DMSO-d₆) δ (ppm) 9.05 (s, 1H), 9.02 (s, 1H), 8.49 (s, 1H), 8.09-8.06 (m, 3H), 7.90-7.86 (m, 4H), 7.81 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 8.6 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H);

¹³C NMR (176 MHz, DMSO-d₆) δ (ppm) 152.3, 151.0, 144.3, 144.0, 140.5, 140.1, 132.9, 132.8, 131.3, 127.6, 127.2, 126.8, 119.0, 118.5, 118.2, 114.6, 109.2; LC-MS (M+H): 431.11.

Synthetic route. Probe chemical structure including stereochemistry. Separation of diastereomers (if necessary). Structure verification with 1H NMR and LCMS results. Solubility measure in phosphate buffered saline (PBS) at room temperature (23°C). PBS by definition is 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4. Stability measured at room temperature (23ºC) in PBS (no antioxidants or other protectants and DMSO concentration below 0.1%).

Compound Stability - ML368 (SR-9186) was relatively stable in PBS. Compound levels decreased slightly over the 48 hours tested. There was a small decrease which corresponded to a 97-hour half-life. To test conditions similar to cell based incubations, stability was also demonstrated in DMEM media supplemented with 10% fetal bovine serum where a half-life of 121 hours was calculated. Based on these results it is not expected that there would be an issue working with the probe, but stock solutions should be freshly prepared. While the data is not shown, two 10 mM DMSO stock solutions (one approximately one year old and the second 2-months old) had equivalent levels of ML368 to a freshly prepared stock. PBS stability of SR-7377, -8893, -8889, -8890, and -9038 were also tested and all had half-lives in PBS exceeding 48 hours.

Figure 1Stability of ML368

Compound Solubility – Solubility of ML368 (SR-9186) was determined to be 1.84 µM in PBS. Additionally the probe was found to extensively bind to plastic. For this reason intermediate dilution plates should be avoided. An example of an intermediate dilution plate would be where a small amount of compound is transferred from a DMSO stock (e.g. 1 µl) into a larger volume of an aqueous solution (e.g. 99 µl) to form an intermediate solution which is then transferred to the final incubation. Efforts should be made to make all dilutions in organic, which is directly added to the final incubation.

Glutathione Reactivity – ML368 (SR-9186) was stable when 10 µM compound was incubated with 50 µM reduced glutathione over six hours. There was not difference between samples with and without glutathione.

Figure 2Glutathione Reactivity of ML368

2.3. Probe Preparations

1-(4-(3H-imidazo[4,5-b]pyridin-7-yl)phenyl)-3-(4′-cyano-[1,1′-biphenyl]-4-yl)urea

A solution of 4′-amino-[1,1′-biphenyl]-4-carbonitrile (0.097 g, 0.5 mmol) in EtOAc (3 mL) was added slowly over 10 min to a solution of triphosgene (0.148 g, 0.5 mmol) in EtOAC (3 mL) at 0> °C. The mixture was stirred at room temperature for 1 h and then heated to reflux for 4 h. The precipitate was filtered off and the solvent was evaporated to dryness to afford 4′-Isocyanato-[1,1′-biphenyl]-4-carbonitrile in quantitative yield.

To a solution of 4′-isocyanato-[1,1′-biphenyl]-4-carbonitrile (0.132 g, 0.6 mmol) in 1,2-dichloroethane (5 mL) was added 4-(3H-imidazo[4,5-b]pyridin-7-yl)aniline (0.105 g, 0.5 mmol) in DMSO (1 mL) dropwise. The reaction was heated to reflux overnight, cooled to room temperature, and solvent was evaporated. The residue was dissolved in DMF (5 mL) and submitted to preparative HPLC. The title compound was obtained as a brownish yellow solid (37%).

The following related analogs were synthesized following the same standard protocol as described for ML#368 using 4-(3H-imidazo[4,5-b]pyridin-7-yl)aniline and the appropriate aryl isocyanate.

3-(3-(4-(3H-Imidazo[4,5-b]pyridin-7-yl)phenyl)ureido)-N-phenylbenzamide (SR-9038)

¹H NMR (400 MHz, Methanol-d₄) δ (ppm) 9.34 (s, 1H), 8.32 (d, J = 8.7 Hz, 1H), 8.16-8.13 (m, 3H), 8.05 (t, J = 1.9 Hz, 1H), 7.74-7.67 (m, 5H), 7.61 (dt, J = 8.1, 1.4 Hz, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.41-7.37 (m, 2H), 7.19-7.15 (m, 1H). LC-MS (M+H)⁺: 449.12.

3-(3-(4-(3H-Imidazo[4,5-b]pyridin-7-yl)phenyl)ureido)-N-isopropylbenzamide (SR-7377)

¹H NMR (400 MHz, Methanol-d₄) δ (ppm) 9.26 (br s, 1H), 8.33 (br s, 1H), 8.17-8.11 (m, 3H), 7.93-7.91 (m, 1H), 7.71-7.67 (m, 3H), 7.50-7.41 (m, 2H), 4.23 (sep, J = 6.6 Hz, 1H), 1.29 (d, J = 6.6 Hz, 6H). LC-MS (M+H)⁺: 415.15.

1-(4-(3H-Imidazo[4,5-b]pyridin-7-yl)phenyl)-3-(3-chlorophenyl)urea (SR-8889)

¹H NMR (400 MHz, Methanol-d₄) δ (ppm) 9.38 (br s, 1H), 8.33 (d, J = 8.3 Hz, 1H), 8.15-8.11 (m, 3H), 7.68-7.63 (m, 3H), 7.30-7.26 (m, 2H), 7.03 (dt, J = 6.7, 2 Hz, 1H). LC-MS (M+H)⁺: 364.12.

Ethyl 3-(3-(4-(3H-Imidazo[4,5-b]pyridin-7-yl)phenyl)ureido)benzoate (SR-8893)

¹H NMR (400 MHz, Methanol-d₄) δ (ppm) 9.26 (s, 1H), 8.29 (d, J = 8.7 Hz, 1H), 8.15 (t, J = 2.0 Hz, 1H), 8.14-8.10 (m, 3H), 7.74-7.69 (m, 2H), 7.66-7.63 (m, 2H), 7.24 (t, J = 8.0 Hz, 1H), 4.38 (q, J = 7.2 Hz, 2H), 1.41 (t, J = 7.2 Hz, 3H). LC-MS (M+H)⁺: 402.12.

1-(4-(3H-Imidazo[4,5-b]pyridin-7-yl)phenyl)-3-(4-chlorophenyl)urea (SR-8890)

¹H NMR (400 MHz, Methanol-d₄) δ (ppm) 9.27 (br s, 1H), 8.30 (d, J = 8.6 Hz, 1H), 8.13-8.10 (m, 3H), 7.64-7.62 (m, 2H), 7.48-7.45 (m, 2H), 7.31-7.28 (m, 2H). LC-MS (M+H)⁺: 364.10.

3. Results

Figure 3Selectivity of ML368

Selectivity under phenotyping conditions. The effect of 1 µM ketoconazole or 2.5 µM ML368 (SR-9186) was evaluated in incubations containing 1 mg/ml HLM protein (150 donor mixed gender) using the marker substrates and substrate concentrations indicated in Table 1. Incubation time was reduced to 1 minute. Inhibition of midazolam hydroxylation was similarly evaluated (B) in CYP3A5 *1/*1, *1/*3, and *3/*3 pooled microsomes (four individual donors mixed 1:1:1:1).

Table 1

P450 inhibition of substituted phenyl urea analogs.

Figure 4Inhibition of midazolam hydroxylation by ML368 (SR-9186) in genotyped individual donor human liver microsomes

Inhibition of midazolam hydroxylation by ML368 (SR-9186) in genotyped individual donor human liver microsomes. Triplicate incubations using individual donor microsomes were evaluated for formation of 1′-hydroxymidazolam. Data corresponding to *1/*1 donors (blue with a narrow dashed line), *1/*3 donors (orange with wider dashed line), and *3/*3 donors (black solid lines) were analyzed for changes in the observed IC₅₀ (Panel B) and for maximal inhibition (Panel C). Statistical analysis is a two-tailed unpaired t-test assuming unequal variance.

3.2. Dose Response Curves for Probe

IC₅₀ values for inhibition of midazolam→1′hydroxymidazolam, testosterone→6β-hydroxytestosterone, and vincristine→vincristine M1 9, 4, and 38 nM, respectively. Selectivity was demonstrated using recombinant CYP3A5 where ML368 was a much weaker inhibitor. ML368 did not inhibit any of the reactions by 50 percent when tested at concentrations up to 60 µM. When the CYP3A5 inhibition data was curve fit to a sigmoidal one-site competition model, inhibition constants of 7.4, 0.36, and 1.5 µM were calculated. The data is presented graphically.

Figure 5Dose Response Curves for ML368

Selective inhibition of CYP3A4 vs. CYP3A5 by ML368 in recombinant P450. Triplicate incubations with ML368 (SR-9186), 4 nM to 60 µM, were co-incubated with 1 mM NADPH, and 1 nM recombinant P450 in 100 mM phosphate buffer, pH 7.4. Probe substrates were 2.5 µM midazolam, 75 µM testosterone or 20 µM vincristine.

3.3. Scaffold/Moiety Chemical Liabilities

An initial set of analogs meant to explore what portions of the molecule were amenable to modification resulted in the discovery that the corresponding 4-substituted phenyl analogs SR8890 and 4 showed similar IC₅₀'s for CYP3A4 inhibition but had reduced CYP3A5 inhibition leading to increased selectivity (Table 1). Given the improved CYP3A4/CYP3A5 selectivity in the phenyl urea series, structure activity relationship studies (SAR) were initiated on this scaffold. Compounds were easily synthesized as described in Scheme 2. The bromo imidazopyridine (1) was first protected with a p-methoxybenzyl group. This gave an inseparable mixture of protected regioisomers (only one shown for clarity), but the mixture could be carried forward without incident. The protected imidazopyridine underwent smooth Suzuki coupling to yield intermediate 2. TFA deprotection and exposure to a variety of phenyl isocyanates afforded final products as solids.

Scheme 2

(a) PMB-Br, K₂CO₃, DMAC; (b) 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, Pd(Ph₃P)₄, K₂CO₃, THF; (c) TFA; (d) RCNO, toluene.

Table 1 summarizes the effects of substitution of the terminal phenyl ring upon inhibition of CYP3A4 and CYP3A5 activity using recombinant enzymes. ⁶ Substitution at all positions of the phenyl ring led to potent CYP3A4 inhibitors with varying degrees of selectivity against CYP3A5. 2- and 3- substituted analogs (8-11) showed reduced selectivity whereas larger 3-substituted analogs (14,15) showed improvement in selectivity for CYP3A4. 4-substituted analogs (16,17) also showed good levels of selectivity, however for 17, precipitation at higher concentrations was observed. Given the good potency and nice selectivity profile of ester 15, this compound was further analoged as shown in Scheme 2.

While esters 15 and 18 exhibited strong selectivity and potency for CYP3A4 vs CYP3A5, they were not pursued because of stability concerns. When tested in incubations containing 1 mg/ml human liver microsomes or human hepatic S9, the compounds had a half-life of approximately 8 minutes with or without the addition of NADPH indicating probably hydrolysis by hepatic esterases making 15 and 18 poor in vitro probes. Hence, amides were investigated as more stable surrogates. The parent ester was easily saponified, and a variety of amines were coupled to form the amides (20-29, Table 1).

Several of the analogs were highly selective for the inhibition of CYP3A4 vs CYP3A5. A wide range of substituents were acceptable; however, larger substituents particularly with the disubstituted amides exhibited increased CYP3A5 inhibition, thus decreasing the fold-selectivity.

In order to evaluate the necessity of the imidazopyridine group, a variety of compounds were generated using compound SR7377 as a template (Table 2). Surprisingly, only SR 33 and SR

43 showed some CYP inhibition, however they were no longer selective. None of the other heterocycles investigated exhibited any appreciable CYP inhibition.

The most potent CYP3A4 inhibitor identified within the series was compound 17; however, compound 17 suffered from poor solubility. SR 17 also wasn't the most selective inhibitor identified from this series, however it warranted further investigation. Hence, a series of substituted 4-phenyl analogs were prepared using similar strategies as in Scheme 1 (30-36, Table 1). 4-substituted biphenyl analogs (30-32) showed very good CYP3A4 inhibition and exquisite selectivity over CYP3A5. SR9186 was the most potent CYP3A4 inhibitor identified and the most selective over CYP3A5 as well, and it if for this reason SR9186 was chosen as the probe.

Scheme 1

Synthesis of ML368 (SR-9186).

3.4. SAR Tables

20-30 analogs derivatized at more than two distinct positions of the molecule) to improve potency/selectivity and provide initial SAR that substantiates the specific probe activities on the target/pathway. A graphic, showing sample SAR Table (including Label and Title), is shown in Table 1.

Table 2Imidazopyridine derivatives of compound SR7377

View in own window

				enzyme IC50 (µM)a		Fold-Selective
SR#	SID	CID	R	CYP3A4	CYP3A5
7377	162008145	71295820		0.090	41%	>650
7127	162008199	71295831		0.56	34	61
7734	162008208	71295828		2.9	25%	>21
7454	162008203	71295826		22	60	3
7681	162008207	71295833		4.6	8.1	2
7453	162008202	71295830		14	44	3
7485	162008204	71295822		36%	21%	None
7128	162008200	71295824		28%	7%	None
7376	162008201	71295834		33%	8%	None
7126	43	43		0.13	0.98	8

a

Values are means of two or more experiments. The error in these values is within ±30% of the mean.

4. Discussion

Cytochrome P450 are the major family of enzymes responsible for the oxidative metabolism of pharmaceuticals and xenobiotics. CYP3A4 and CYP3A5 have been shown to have overlapping substrate and inhibitor profiles and their inhibition has been demonstrated to be involved in numerous pharmacokinetic drug-drug interactions. Here we report the first highly selective CYP3A4 inhibitor optimized from an initial lead with ≈30-fold selectivity over CYP3A5 to yield a series of compounds with greater than 1000-fold selectivity. The lack of proper chemical tools to differentiate the activity of CYP3A4 and CYP3A5 has led to the longstanding, yet erroneous, convention of treating the two enzymes as if they were one. Sometimes activity has been expressed as CYP3A to point out that the results are not specific for either CYP3A4 or CYP3A5. However, too often all activity has been contributed to CYP3A4. Furthermore, the practice of pooling tissue from multiple donors to generate an “average human” has given rise to the perception that CYP3A5 has less significance than CYP3A4. While it is certainly true that the abundance of CYP3A4 exceeds that of CYP3A5 in a multi-donor pool, this is not representative of actual patients. The concentrations of the two enzymes have been reported to be roughly equal in individuals that express CYP3A5 (1). The current manuscript details a highly selective CYP3A4 inhibitor suitable for isolation of CYP3A5 activity in human liver microsomes. We have published details of the structure activity relationship of ML368 and a number of related analogs for selective CYP3A4 inhibition (23).

5. References

1.

Lin YS, Dowling AL, Quigley SD, Farin FM, Zhang J, Lamba J, Schuetz EG, Thummel KE. Co-regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism. Mol Pharmacol. 2002;62:162–172. [PubMed: 12065767]

2.

Leskela S, Honrado E, Montero-Conde C, Landa I, Cascon A, Leton R, Talavera P, Cozar JM, Concha A, Robledo M, Rodriguez-Antona C. Cytochrome P450 3A5 is highly expressed in normal prostate cells but absent in prostate cancer. Endocr Relat Cancer. 2007;14:645–654. [PubMed: 17914095]

3.

Barry A, Levine M. A systematic review of the effect of CYP3A5 genotype on the apparent oral clearance of tacrolimus in renal transplant recipients. Ther Drug Monit. 2010;32:708–714. [PubMed: 20864901]

4.

Zhao Y, Song M, Guan D, Bi S, Meng J, Li Q, Wang W. Genetic polymorphisms of CYP3A5 genes and concentration of the cyclosporine and tacrolimus. Transplant Proc. 2005;37:178–181. [PubMed: 15808586]

5.

Dennison JB, Jones DR, Renbarger JL, Hall SD. Effect of CYP3A5 expression on vincristine metabolism with human liver microsomes. J Pharmacol Exp Ther. 2007;321:553–563. [PubMed: 17272675]

6.

Dennison JB, Kulanthaivel P, Barbuch RJ, Renbarger JL, Ehlhardt WJ, Hall SD. Selective metabolism of vincristine in vitro by CYP3A5. Drug Metab Dispos. 2006;34:1317–1327. [PubMed: 16679390]

7.

Dennison JB, Mohutsky MA, Barbuch RJ, Wrighton SA, Hall SD. Apparent high CYP3A5 expression is required for significant metabolism of vincristine by human cryopreserved hepatocytes. J Pharmacol Exp Ther. 2008;327:248–257. [PubMed: 18650247]

8.

Egbelakin A, Ferguson MJ, MacGill EA, Lehmann AS, Topletz AR, Quinney SK, Li L, McCammack KC, Hall SD, Renbarger JL. Increased risk of vincristine neurotoxicity associated with low CYP3A5 expression genotype in children with acute lymphoblastic leukemia. Pediatr Blood Cancer. 2011;56:361–367. [PMC free article: PMC3020258] [PubMed: 21225912]

9.

Lin YS, Dowling AL, Quigley SD, Farin FM, Zhang J, Lamba J, Schuetz EG, Thummel KE. Co-regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism. Mol Pharmacol. 2002;62:162–172. [PubMed: 12065767]

10.

Clarke SE. In vitro assessment of human cytochrome P450. Xenobiotica. 1998;28:1167–1202. [PubMed: 9890158]

11.

Rendic S. Summary of information on human CYP enzymes: human P450 metabolism data. Drug Metab Rev. 2002;34:83–448. [PubMed: 11996015]

12.

Soars MG, Grime K, Riley RJ. Comparative analysis of substrate and inhibitor interactions with CYP3A4 and CYP3A5. Xenobiotica. 2006;36:287–299. [PubMed: 16684709]

13.

Khan KK, He YQ, Correia MA, Halpert JR. Differential oxidation of mifepristone by cytochromes P450 3A4 and 3A5: selective inactivation of P450 3A4. Drug Metab Dispos. 2002;30:985–990. [PubMed: 12167563]

14.

Stresser DM, Broudy MI, Ho T, Cargill CE, Blanchard AP, Sharma R, Dandeneau AA, Goodwin JJ, Turner SD, Erve JC, et al. Highly selective inhibition of human CYP3Aa in vitro by azamulin and evidence that inhibition is irreversible. Drug Metab Dispos. 2004;32:105–112. [PubMed: 14709627]

15.

Pearson JT, Wahlstrom JL, Dickmann LJ, Kumar S, Halpert JR, Wienkers LC, Foti RS, Rock DA. Differential time-dependent inactivation of P450 3A4 and P450 3A5 by raloxifene: a key role for C239 in quenching reactive intermediates. Chem Res Toxicol. 2007;20:1778–1786. [PubMed: 18001057]

16.

Leskelä S, Honrado E, Montero-Conde C, Landa I, Casco´n A, Leto´n R, Talavera P, Co´zar JM, Concha A, Robledo M, et al. Cytochrome P450 3A5 is highly expressed in normal prostate cells but absent in prostate cancer. Endocr Relat Cancer. 2007;14:645–654. [PubMed: 17914095]

17.

Barry A, Levine M. A systematic review of the effect of CYP3A5 genotype on the apparent oral clearance of tacrolimus in renal transplant recipients. Ther Drug Monit. 2010;32:708–714. [PubMed: 20864901]

18.

Zhao Y, Song M, Guan D, Bi S, Meng J, Li Q, Wang W. Genetic polymorphisms of CYP3A5 genes and concentration of the cyclosporine and tacrolimus. Transplant Proc. 2005;37:178–181. [PubMed: 15808586]

19.

Dennison JB, Jones DR, Renbarger JL, Hall SD. Effect of CYP3A5 expression on vincristine metabolism with human liver microsomes. J Pharmacol Exp Ther. 2007;321:553–563. [PubMed: 17272675]

20.

Dennison JB, Kulanthaivel P, Barbuch RJ, Renbarger JL, Ehlhardt WJ, Hall SD. Selective metabolism of vincristine in vitro by CYP3A5. Drug Metab Dispos. 2006;34:1317–1327. [PubMed: 16679390]

21.

Dennison JB, Mohutsky MA, Barbuch RJ, Wrighton SA, Hall SD. Apparent high CYP3A5 expression is required for significant metabolism of vincristine by human cryopreserved hepatocytes. J Pharmacol Exp Ther. 2008;327:248–257. [PubMed: 18650247]

22.

Egbelakin A, Ferguson MJ, MacGill EA, Lehmann AS, Topletz AR, Quinney SK, Li L, McCammack KC, Hall SD, Renbarger JL. Increased risk of vincristine neurotoxicity associated with low CYP3A5 expression genotype in children with acute lymphoblastic leukemia. Pediatr Blood Cancer. 2011;56:361–367. [PMC free article: PMC3020258] [PubMed: 21225912]

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