Carnosic acid is a phenolic diterpene isolated from rosemary (Rosmarinus officinalis), which may have anticancer, antiadipogenic, and anti-inflammatory properties. Recently, carnosic acid was shown ...to prevent weight gain and hepatic steatosis in a mouse model of obesity and type II diabetes. Based on these results, carnosic acid has been suggested as a potential treatment for obesity and nonalcoholic fatty liver disease; however, little is known about the safety of carnosic acid at doses needed to elicit a pharmacological effect. For this reason, hepatotoxicity and cytochrome P450 inhibition and induction studies were performed in primary human hepatocytes and microsomes. Measuring cellular ATP, carnosic acid showed a dose-dependent increase in hepatotoxicity with an EC(50) value of 94.8 ± 36.7 μM in three human hepatocyte donors without a concurrent increase in the apoptosis markers caspase-3/7. In human liver microsomes, carnosic acid did not exhibit significant time-dependent inhibition for any of the cytochrome P450 enzymes investigated, although it did inhibit CYP2C9- and CYP3A4-catalyzed reactions with K(i) values of 9.2 and 4.3 μM, respectively. Carnosic acid also induced CYP2B6 and CYP3A4 mRNA and enzyme activity in a dose-dependent manner. At 10 μM, carnosic acid increased CYP2B6 enzyme activity 61.6 and 49.3% in two donors compared with phenobarbital, and it increased CYP3A enzyme activity 82.6 and 142% compared with rifampicin. These results indicate the potential for drug interactions with carnosic acid and illustrate the need for an appropriate safety assessment before being used as a weight loss supplement.
Drug-drug interactions (DDIs) between therapeutic proteins (TPs) and small-molecule drugs have recently drawn the attention of regulatory agencies, the pharmaceutical industry, and academia. TP-DDIs ...are mainly caused by proinflammatory cytokine or cytokine modulator-mediated effects on the expression of cytochrome P450 enzymes. To build consensus among industry and regulatory agencies on expectations and challenges in this area, a working group was initiated to review the preclinical state of the art. This white paper represents the observations and recommendations of the working group on the value of in vitro human hepatocyte studies for the prediction of clinical TP-DDI. The white paper was developed following a "Workshop on Recent Advances in the Investigation of Therapeutic Protein Drug-Drug Interactions: Preclinical and Clinical Approaches" held at the Food and Drug Administration White Oak Conference Center on June 4 and 5, 2012. Results of a workshop poll, cross-laboratory data comparisons, and the overall recommendations of the in vitro working group are presented herein. The working group observed that evaluation of TP-DDI for anticytokine monoclonal antibodies is currently best accomplished with a clinical study in patients with inflammatory disease. Treatment-induced changes in appropriate biomarkers in phase 2 and 3 studies may indicate the potential for a clinically measurable treatment effect on cytochrome P450 enzymes. Cytokine-mediated DDIs observed with anti-inflammatory TPs cannot currently be predicted using in vitro data. Future success in predicting clinical TP-DDIs will require an understanding of disease biology, physiologically relevant in vitro systems, and more examples of well conducted clinical TP-DDI trials.
Pharmacogenomics in the age of personalized medicine Dickmann, Leslie J.; Ware, Joseph A.
Drug discovery today. Technologies,
September-December 2016, 2016 Sep - Dec, 2016-09-00, 20160901, Volume:
21-22
Journal Article
Peer reviewed
The aim of personalized medicine is to offer the right treatment to the right person at the right dose, thus maximizing efficacy and minimizing toxicity for each individual patient. Pharmacogenomic ...approaches attempt to refine the aim of personalized medicine by utilizing an individual's germline and somatic DNA signatures to guide treatment. In this review, we highlight the current use of pharmacogenomic based biomarker information in drug labeling. We also present several case studies on the implementation of pharmacogenomic strategies in drug discovery and development. Lastly, we comment on current challenges to implementing pharmacogenomic based testing in the clinic.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Retinoic acid (RA) is a critical signaling molecule that performs multiple functions required to maintain cellular viability. It is also used in the treatment of some cancers. Enzymes in the CYP26 ...family are thought to be responsible for the elimination of RA, and CYP26A1 appears to serve the most critical functions in this family. In spite of its importance, CYP26A1 has neither been heterologously expressed nor characterized kinetically. We expressed the rCYP26A1 in baculovirus-infected insect cells and purified the hexahistidine tagged protein to homogeneity. Heme incorporation was determined by carbon monoxide difference spectrum and a type 1 spectrum was observed with RA binding to CYP26A1. We found that RA is a tight binding ligand of CYP26A1 with low nM binding affinity. CYP26A1 oxidized RA efficiently (depletion
K
m 9.4
±
3.3
nM and
V
max 11.3
±
4.3
pmoles
min
−1
pmole
P450
−1) when supplemented with P450 oxidoreductase and NADPH but was independent of cytochrome b5. 4-Hydroxy-RA (4-OH-RA) was the major metabolite produced by rCYP26A1 but two other primary products were also formed. 4-OH-RA was further metabolized by CYP26A1 to more polar metabolites and this sequential metabolism of RA occurred in part without 4-OH-RA leaving the active site of CYP26A1. The high efficiency of CYP26A1 in eliminating both RA and its potentially active metabolites supports the major role of this enzyme in regulating RA clearance in vivo. These results provide a biochemical framework for CYP26A1 function and offer insight into the role of CYP26A1 as a drug target as well as in fetal development and cell cycle regulation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
In this work, we assessed the ability of fluorophotometry to measure the vitreal pharmacokinetics (PK) of fluorescently-labeled ranibizumab in the rabbit after intravitreal injection. We compared ...these values to those obtained using enzyme-linked immunosorbent assays (ELISA). Data obtained in this study were also compared to historical ranibizumab ocular PK data, either measured in-house or previously published.
Three individual in vivo studies were performed in New Zealand White rabbits to assess the feasibility of using fluorophotometry to measure rabbit ocular PK of ranibizumab; explore the dynamic range of dosing fluorescently-labeled ranibizumab; and directly compare ranibizumab concentrations and calculated PK parameters measured by vitreal fluorophotometry to those measured using ELISA.
In direct comparisons between fluorophotometry and ELISA, the calculated clearance (CL) values were 0.26 and 0.21 mL/day, the volumes of distribution at steady state (Vss) were 0.80 and 0.94 mL, the half-lives (t₁/₂) were 3.1 and 2.9 days and the dose normalized areas under the curve (AUC/D) were 4.7 and 3.9 μg·day/mL/μg, respectively. These values fell within the ranges of 0.13 to 0.44 mL/day for CL, 0.5 to 1.8 mL for Vss, 2.8 to 3.5 days for t1/2, and 2.3 to 7.9 μg·day/mL/μg for AUC/D that have been either measured previously in-house or published elsewhere.
Although not suitable for measuring retinal concentrations, fluorophotometry is a valuable, noninvasive method to measure vitreous concentrations of protein therapeutics after intravitreal injection.
CYP2C9 is a polymorphic gene for which there are four known allelic variants; CYP2C9*1 , CYP2C9*2 , CYP2C9*3 , and CYP2C9*4 . In the present study, DNA from 140 European Americans and 120 African ...Americans was examined by single-strand conformational
polymorphism and restriction fragment length polymorphism analyses, resulting in the identification of a new CYP2C9 variant,
CYP2C9*5 . This variant is derived from a C1080G transversion in exon 7 of CYP2C9 that leads to an Asp360Glu substitution in the encoded protein. The CYP2C9*5 variant was found to be expressed only in African Americans, such that approximately 3% of this population carries the CYP2C9*5 allele. The variant was expressed in, and purified from, insect cells infected with a recombinant baculovirus. Comparative
kinetic studies using the purified wild-type protein CYP2C9*1; the Ile359Leu variant, CYP2C9*3; and the Asp360Glu variant,
CYP2C9*5 were carried out using ( S )-warfarin, diclofenac, and lauric acid as substrates. The major effect of the Asp360Glu mutation was to increase the K
m value relative to that of CYP2C9*1 for all three substrates: 12-fold higher for ( S )-warfarin 7-hydroxylation, 5-fold higher for the 4â²-hydroxylation of diclofenac, and 3-fold higher for the Ï-1 hydroxylation
of lauric acid. V
max values differed less than K
m values between the CYP2C9*1 and CYP2C9*5 proteins. In vitro intrinsic clearances for CYP2C9*5, calculated as the ratio of V
max / K
m , ranged from 8 to 18% of CYP2C9*1 values. The corresponding ratio for CYP2C9*3 was 4 to 13%. Accordingly, the in vitro data
suggest that carriers of the CYP2C9*5 allele would eliminate CYP2C9 substrates at slower rates relative to persons expressing the wild-type protein.
•UGT protein interactions in hepatocytes were studies using siRNA down regulation.•Selective siRNA down regulation of UGT1A9 and UGT2B7 expression was achieved.•UGT1A9 activity (but not expression) ...also decreased with UGT2B7 down regulation•Data suggests potential for protein interactions between UGT1A9 and UGT2B7•Expression of UGT2B7 may impact glucuronidation of selective UGT1A9 substrates.
Previous experiments performed in recombinant systems have suggested that protein–protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein–protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In this study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. To our knowledge, this represents the first piece of evidence that UGT protein–protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Changes in cytochrome P450 expression incurred by inflammatory disease were studied in a murine collagen antibody induced arthritis (CAIA) model and compared to bacterial lipopolysaccharide-treated ...mice and to cytochrome P450 changes in primary mouse hepatocytes following combination treatments with cytokines IL-1β, IL-6, or TNFα. CAIA in female mice increased serum IL-1β, IL-6 and hepatic serum amyloid A (SAA) mRNA and significantly altered cytochrome P450 mRNA and activity levels. Most cytochrome P450 isoforms were down-regulated, although some, such as Cyp3a13, were up-regulated. Cytokine effects on cytochrome P450 levels in mouse hepatocytes were compared at in vitro cytokine concentrations similar to those measured in CAIA mouse serum in vivo. In vivo and in vitro cytochrome P450 suppression by cytokines was congruent for some cytochrome P450 isoforms (Cyp1a2, Cyp2c29, and Cyp3a11) but not for others (cytochrome P450 oxidoreductase (POR) and Cyp2e1). In mouse hepatocytes, IL-6 and IL-1β in combination in vitro caused a synergistic increase in SAA mRNA expression, but not in cytochrome P450 suppression. IL-1β and IL-6 were equipotent in the suppression of cytochrome P450 gene expression, while TNFα caused mild suppression only at the highest concentrations used. TNFα in combination with IL-1β, IL-6, or both had a protective effect against IL-1β and IL-6-mediated cytochrome P450 suppression. When IL-1β or IL-6 was combined with low concentrations of TNFα, several P450 isoforms were induced rather than suppressed. These data highlight the complexities of performing in vitro–in vivo comparisons using disease models for cytochrome P450 regulation by cytokines.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The increasing number of transgenic or gene knockout mouse models generated for use in drug metabolism studies has meant that a greater understanding of the function and substrate specificities of ...murine cytochromes P450 (P450s) has become essential, particularly with the recent advances in "humanized" mouse models. In this study, we have heterologously expressed nine murine P450s--Cyp1a1, Cyp1a2, Cyp1b1, Cyp2a4, Cyp2b20, Cyp2c29, Cyp2d22, Cyp2e1, and Cyp3a11--individually with human P450 oxidoreductase to generate functional monooxygenase systems in Escherichia coli. We have identified a suitable fluorogenic probe for each P450 and determined the apparent kinetic parameters. These probes have enabled the screening of a panel of 31 test compounds classified as "drugs," "natural compounds," "endogenous compounds," and "pesticides" by measurement of IC(50), thus allowing the comparison of binding affinities. Human P450s CYP2C9, CYP2D6, and CYP3A4 were also included in the study to enable direct comparisons to be made with the mouse enzymes. Although there were general similarities between human and mouse P450s, perhaps the most significant finding in this study was the observation that, despite 77% amino acid identity, Cyp2d22 and CYP2D6 were remarkably dissimilar in a range of enzymatic properties, with potentially serious implications for pharmacokinetic studies using CYP2D substrates. The data presented in this study provide a solid foundation with which to assess the degree of similarity (or difference) between mouse and human P450s involved in xenobiotic metabolism and can be used as a basis for further studies.
The role of C239 as the active-site residue responsible for forming the covalent linkage with raloxifene during P450 3A4 time-dependent inactivation (TDI) was recently identified. The corresponding ...residue in CYP3A5 is S239, and when the potential for TDI in P450 3A5 was investigated, only reversible inhibition was observed against midazolam and testosterone, with median inhibitory concentration (IC50) values of 2.4 and 2.9 µM, respectively. In a similar fashion, when C239 was replaced with alanine in P450 3A4, TDI was successfully engineered out, and the reversible inhibition was characterized by IC50 values of 3.7 and 3.5 µM against midazolam and testosterone, respectively. Metabolism studies confirmed that the reactive diquinone methide intermediate required for P450 3A4 inactivation formed in all of the P450 3A enzymes investigated. Furthermore, the absence of TDI in P450 3A5 led to an increase in the formation of GSH-related adducts of raloxifene compared with that for P450 3A4. Consequently, the absence of the nucleophilic cysteine leads to differential TDI and generation of reactive metabolites in the P450 3A enzyme, providing the foundation for pharmacogenetics that contributes to individual differences in susceptibility to adverse drug reactions.
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IJS, KILJ, NUK, PNG, UL, UM
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