High-Fat Diet-Induced Functional and Pathologic Changes in Lacrimal Gland.

The lacrimal gland is critical for maintaining the homeostasis of the ocular surface microenvironment through secreting aqueous tears in mammals. Many systemic diseases such as Sjögren syndrome, rheumatoid arthritis, and diabetes can alter the lacrimal gland function, eventually resulting in aqueous tear-deficient dry eye. Here, a high-fat diet (HFD) experimental mouse model was used to clarify how hyperlipidemia affects lacrimal gland function. Aqueous tear secretion fell about 50% after 1 month on a HFD. Lipid droplets accumulated in the matrix and acinar cells of the lacrimal gland after this period, along with changes in the lipid metabolism, changes in gene expression levels, and disruption of fatty acid oxidative activity. Immune cell infiltration and rises in the gene expression levels of the inflammation-related cytokines Il1ß, Tnfα, Tsg6, Il10, Mmp2, and Mmp9 were found. HFD also induced mitochondrial hypermegasoma, increased apoptosis, and decreased lacrimal gland acinar cell proliferation. Replacement of the HFD with the standard diet partially reversed pathologic changes in the lacrimal gland. Similarly, supplementing the HFD with fenofibrate also partially reversed the inhibited tear secretion and reduced lipid accumulation, inflammation, and oxidative stress levels. The authors conclude that a HFD induces pathophysiological changes and functional decompensation of the lacrimal gland. Therefore, ingestion of a HFD may be a causative factor of dry eye disease.

Hyperlipidemia Affects Tight Junctions and Pump Function in the Corneal Endothelium.

Hyperlipidemia impacts on various diseases, such as atherosclerosis, hypertension, and diabetes mellitus. However, its influence, if any, on ocular tissues is largely unknown. Herein, we developed hyperlipidemic murine models by feeding 4-week-old male wild-type mice with a high-fat diet and apolipoprotein E knockout (ApoE-/-) mice with a high-fat diet or standard diet to investigate the corneal endothelial change under hyperlipidemic conditions. Oil Red O staining showed an accumulation of lipid droplets in corneal endothelial cells (CECs) of hyperlipidemic mice. Other manifestations included a reduced cell density and distorted cell morphology, a disruption of the endothelial cell tight junctions and adhesion junctions, a reduced number of surface microvilli, down-regulation of Na+-K+-ATPase expression and function, activation of oxidative stress, changes in mitochondrial ultrastructure, and increased apoptosis. CEC recovery after injury, moreover, was diminished in hyperlipidemic mice; and high palmitate levels were found in the aqueous humor. In vitro hyperlipemia model, moreover, was found to be associated with dose-dependent CEC cytotoxicity, altered cell morphology, reduced pump function, and an induction of oxidative stress, leading to functional and pathologic changes in the corneal endothelium.

Ectodysplasin A protein promotes corneal epithelial cell proliferation.

The EDA gene encodes ectodysplasin A (Eda), which if mutated causes X-linked hypohidrotic ectodermal dysplasia (XLHED) disease in humans. Ocular surface changes occur in XLHED patients whereas its underlying mechanism remains elusive. In this study, we found Eda was highly expressed in meibomian glands, and it was detected in human tears but not serum. Corneal epithelial integrity was defective and the thickness was reduced in the early postnatal stage of Eda mutant Tabby mice. Corneal epithelial cell proliferation decreased and the epithelial wound healing was delayed in Tabby mice, whereas it was restored by exogenous Eda. Eda exposure promoted mouse corneal epithelial wound healing during organ culture, whereas scratch wound assay showed that it did not affect human corneal epithelial cell line migration. Epidermal growth factor receptor (EGFR), phosphorylated EGFR (p-EGFR), and phosphorylated ERK1/2 (p-ERK) were down-regulated in Tabby mice corneal epithelium. Eda treatment up-regulated the expression of Ki67, EGFR, p-EGFR, and p-ERK in human corneal epithelial cells in a dose-dependent manner. In conclusion, Eda protein can be secreted from meibomian glands and promotes corneal epithelial cell proliferation through regulation of the EGFR signaling pathway. Eda release into the tears plays an essential role in the maintenance of corneal epithelial homeostasis.

Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic transporter inhibition and as a translatable model to predict pharmacokinetic interactions with statins in humans.

Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can cause drug-drug interactions (DDIs). Determining the impact of perpetrator drugs on the plasma exposure of endogenous substrates for OATP1B could be valuable to assess the risk for DDIs early in drug development. As OATP1B orthologs are well conserved between human and monkey, we assessed in cynomolgus monkeys the endogenous OATP1B substrates that are potentially suitable to assess DDI risk in humans. The effect of rifampin (RIF), a potent inhibitor for OATP1B, on plasma exposure of endogenous substrates of hepatic transporters was measured. From the 18 biomarkers tested, RIF (18 mg/kg, oral) caused significant elevation of plasma unconjugated and conjugated bilirubin, which may be attributed to inhibition of cOATP1B1 and cOATP1B3 based on in vitro to in vivo extrapolation analysis. To further evaluate whether cynomolgus monkeys are a suitable translational model to study OATP1B-mediated DDIs, we determined the inhibitory effect of RIF on in vitro transport and pharmacokinetics of rosuvastatin (RSV) and atorvastatin (ATV). RIF strongly inhibited the uptake of RSV and ATV by cOATP1B1 and cOATP1B3 in vitro. In agreement with clinical observations, RIF (18 mg/kg, oral) significantly decreased plasma clearance and increased the area under the plasma concentration curve (AUC) of intravenously administered RSV by 2.8- and 2.7-fold, and increased the AUC and maximum plasma concentration of orally administered RSV by 6- and 10.3-fold, respectively. In contrast to clinical findings, RIF did not significantly increase plasma exposure of either intravenous or orally administered ATV, indicating species differences in the rate-limiting elimination pathways.

[Comparing the safety of vaccination on vastus lateralis muscle and deltoid muscle of infants with haemophilus influenzae type b conjugate vaccine].

OBJECTIVE: To compare the safety of haemophilus influenzae type b (Hib) vaccine vaccination on vastus lateralis muscle and deltoid muscle of infant. METHODS: A total of 408 3-4 months old infants were divided into vastus lateralis muscle group and deltoid muscle group in Beijing, 2014. They were divided into the vastus lateralis muscle group (204) and deltoid muscle group (204) by extracting random number. Each observation object was given 3 doses of Hib vaccine according to the program. Collected systemic and local reactions after vaccination and calculated the incidence of adverse reactions. RESULTS: A total of 61 infants were quitted during the study, 1 132 doses were observed. The total reactions incidence of Vastus lateralis muscle group and Deltoid muscle group were 33.0% (186/564) and 27.6% (157/568) with no statistical differences (χ² = 3.818, P = 0.059). The two groups incidence at the same day of vaccination (day 0) which the highest were 23.2% (131/564) and 20.6% (117/568), then declined with time (linear trend test vastus lateralis muscle group χ² = 36.600, P < 0.001,deltoid muscle group χ² = 29.947, P < 0.001), day 1 were 20.4% (115/564) and 17.6% (100/568), day 2 were 16.0% (90/564) and 13.4% (76/568), day 3 were 10.3% (58/564) and 10.6% (60/568), day 4-7 were 11.2% (63/564) and 11.3% (64/568). No serious adverse events (SAE) were reported during the study. The local reactions incidence of two groups were 7.1% (40/564) and 7.7% (44/568)with no statistical differences (χ² = 0.176, P = 0.675). The systemic reactions incidence of two groups were 25.9% (146/564) and 20.6% (117/568) with obvious statistical differences (χ² = 4.437, P = 0.035). The fever incidence of vastus lateralis muscle group (11.5% (65/564)) was higher than Deltoid muscle group (4.4% (25/568)) with obvious statistical differences (χ² = 4.868, P = 0.027). The 1st dose incidence of fever and abnormal crying of vastus lateralis muscle group (fever 11.3% (23/204), abnormal crying 19.1% (39/204)) was higher than deltoid muscle group (fever 4.4% (9/204), abnormal crying 11.8% (24/204)) and the 2nd dose of diarrhea of deltoid muscle group (11.6% (22/190)) was higher than vastus lateralis muscle group (5.9% (11/187)) with obvious statistical differences (fever χ² = 15.288, P < 0.001, abnormal crying χ² = 4.224, P = 0.040, diarrhea χ² = 3.829, P = 0.046). CONCLUSION: Both vastus lateralis muscle group and deltoid muscle group had lower incidence of adverse reactions after vaccination. No serious adverse events were associated with vaccination. Vastus lateralis muscle vaccination as well as deltoid muscle vaccination demonstrated safe.

Pitavastatin is a more sensitive and selective organic anion-transporting polypeptide 1B clinical probe than rosuvastatin.

AIMS: Rosuvastatin and pitavastatin have been proposed as probe substrates for the organic anion-transporting polypeptide (OATP) 1B, but clinical data on their relative sensitivity and selectivity to OATP1B inhibitors are lacking. A clinical study was therefore conducted to determine their relative suitability as OATP1B probes using single oral (PO) and intravenous (IV) doses of the OATP1B inhibitor rifampicin, accompanied by a comprehensive in vitro assessment of rifampicin inhibitory potential on statin transporters. METHODS: The clinical study comprised of two separate panels of eight healthy subjects. In each panel, subjects were randomized to receive a single oral dose of rosuvastatin (5 mg) or pitavastatin (1 mg) administered alone, concomitantly with rifampicin (600 mg) PO or IV. The in vitro transporter studies were performed using hepatocytes and recombinant expression systems. RESULTS: Rifampicin markedly increased exposures of both statins, with greater differential increases after PO vs. IV rifampicin only for rosuvastatin. The magnitudes of the increases in area under the plasma concentration-time curve were 5.7- and 7.6-fold for pitavastatin and 4.4- and 3.3-fold for rosuvastatin, after PO and IV rifampicin, respectively. In vitro studies showed that rifampicin was an inhibitor of OATP1B1 and OATP1B3, breast cancer resistance protein and multidrug resistance protein 2, but not of organic anion transporter 3. CONCLUSIONS: The results indicate that pitavastatin is a more sensitive and selective and thus preferred clinical OATP1B probe substrate than rosuvastatin, and that a single IV dose of rifampicin is a more selective OATP1B inhibitor than a PO dose.

In vitro assessment of drug-drug interaction potential of boceprevir associated with drug metabolizing enzymes and transporters.

The inhibitory effect of boceprevir (BOC), an inhibitor of hepatitis C virus nonstructural protein 3 protease was evaluated in vitro against a panel of drug-metabolizing enzymes and transporters. BOC, a known substrate for cytochrome P450 (P450) CYP3A and aldo-ketoreductases, was a reversible time-dependent inhibitor (k(inact) = 0.12 minute(-1), K(I) = 6.1 µM) of CYP3A4/5 but not an inhibitor of other major P450s, nor of UDP-glucuronosyltransferases 1A1 and 2B7. BOC showed weak to no inhibition of breast cancer resistance protein (BCRP), P-glycoprotein (Pgp), or multidrug resistance protein 2. It was a moderate inhibitor of organic anion transporting polypeptide (OATP) 1B1 and 1B3, with an IC(50) of 18 and 4.9 µM, respectively. In human hepatocytes, BOC inhibited CYP3A-mediated metabolism of midazolam, OATP1B-mediated hepatic uptake of pitavastatin, and both the uptake and metabolism of atorvastatin. The inhibitory potency of BOC was lower than known inhibitors of CYP3A (ketoconazole), OATP1B (rifampin), or both (telaprevir). BOC was a substrate for Pgp and BCRP but not for OATP1B1, OATP1B3, OATP2B1, organic cation transporter, or sodium/taurocholate cotransporting peptide. Overall, our data suggest that BOC has the potential to cause pharmacokinetic interactions via inhibition of CYP3A and CYP3A/OATP1B interplay, with the interaction magnitude lower than those observed with known potent inhibitors. Conversely, pharmacokinetic interactions of BOC, either as a perpetrator or victim, via other major P450s and transporters tested are less likely to be of clinical significance. The results from clinical drug-drug interaction studies conducted thus far are generally supportive of these conclusions.

Effects of Chengqi Decoction on Complications and Prognosis of Patients with Pneumonia-Derived Sepsis: Retrospective Cohort Study.

PURPOSE: A specific and efficacious method for treatment of pneumonia-derived sepsis is lacking. Chengqi decoction has been used for treatment of pneumonia-derived sepsis, but a clinical trial on patients with pneumonia-derived sepsis is lacking, a gap in the literature that we sought to fill. Patients and Methods. 282 patients with pneumonia-derived sepsis admitted to the intensive care unit of our hospital were selected. They were divided into the treatment group (141 cases) and control group (141 cases). Both groups underwent conventional treatment, but Chengqi decoction (in the form of enema) was given to the treatment group. Mortality, morbidity (abdominal distension and gastrointestinal bleeding), duration of antibiotic use, and use of vasoactive agents were documented 28 days after the drug was used. RESULTS: The treatment group reduced mortality and morbidity (abdominal distension) (P < 0.05). After adjustment for significant covariates, 28-day survival was similar for the whole group (hazard ratio (HR): 0.48; 95% confidence interval (CI): 0.23-0.97; P=0.037), for the subgroup (n = 120) with Acute Physiology and Chronic Health Evaluation II score ≥25 (HR: 0.180; 95% CI: 0.032-0.332; P=0.039) and for the subgroup (n = 66) with N-terminal B-type natriuretic peptide <1800 (0.059, 0.004-0.979, and 0.019). There was no difference between the two groups for the duration of antibiotic use, major bleeding, or use of vasoactive drugs. CONCLUSIONS: Chengqi decoction improved 28-day survival and reduced the prevalence of abdominal distension in patients with pneumonia-derived sepsis.

Hyperlipidemia induces meibomian gland dysfunction.

PURPOSE: To investigate the pathological changes of the meibomian gland (MG) and ocular surface in Apolipoprotein E knockout (ApoE-/-) mice and to investigate the association of meibomian gland dysfunction (MGD) with hyperlipidemia. METHODS: Total plasma cholesterol was measured in different ages of ApoE-/- and wild type (WT) mice, whilst the ocular surfaces were observed by slit-lamp biomicroscopy. MG sections were subjected to H&E staining, Oil Red O staining, TUNEL assay and immunostaining. Quantitate RT-PCR and Western blot analyses were performed to detect the relative gene expression in MGs. The 5-month-old ApoE-/- mice were administered with rosiglitazone or GW9662 + rosiglitazone via oral gavage for 2 months to determine their effect on MG pathological change. RESULTS: We found eyelid abnormality, MG dropout, abnormal MG acinar morphology, dilated MG duct and plugging of the MG orifice in ApoE-/- mice. MG acini in ApoE-/- mice showed exaggerated lipid accumulation. Abnormal keratinization increased in MG duct, accompanied with decreased proliferation and increased apoptosis in ApoE-/- mice. Inflammatory cells infiltrated into the surrounding microenvironment of MG acini, and the NF-κB signaling pathway was activated in MG acinar cells. Oxidative stress was evident in MG acinar cells of ApoE-/- mice. Further investigation showed downregulation of PPAR-γ in MG acinar cells of ApoE-/- mice. PPAR-γ agonist rosiglitazone treatment reduced the morbidity of eyelid, as well as corneal pathological changes and MG inflammation in ApoE-/- mice. CONCLUSION: MGD and hyperlipidemia are closely associated in ApoE-/- mice, which represent a new model to study the pathophysiology of MGD related to dyslipidemia.

Therapeutic Effect of MK2 Inhibitor on Experimental Murine Dry Eye.

Purpose: To investigate the role of mitogen-activated protein kinase-activated protein kinase-2 (MK2) in ocular surface damage of dry eye. Methods: MK2 inhibition was performed in mice subjected to desiccating stress (DS) by topical application of MK2 inhibitor (MK2i) or vehicle eye drops. The total and phosphorylated MK2 in conjunctiva were detected by Western blot. The phenol red cotton test was used to measure tear production, and Oregon green dextran staining was performed to assess corneal epithelial barrier function. PAS staining was used to quantify conjunctival goblet cells. Immunofluorescent staining and quantitative RT-PCR were used to assess the expression of matrix metalloproteinase (MMP)-3 and -9 in corneal epithelium. Apoptosis in ocular surface was assessed by TUNEL and immunofluorescent staining for activated caspase-3 and -8. Inflammation was evaluated by CD4+ T-cell infiltration and production of T helper (Th) cytokines, including IFN-γ, IL-13, and IL-17A in conjunctiva. Results: DS promoted MK2 activation in conjunctiva. Compared with vehicle control mice, MK2i-treated mice showed increased tear production, decreased goblet cell loss, and improved corneal barrier function. Topical MK2 inhibition decreased the expression of MMP-3 and -9 in corneal epithelium, and suppressed cell apoptosis in ocular surface under DS. Topical MK2 inhibition decreased CD4+ T-cell infiltration, with decreased production of IFN-γ and IL-17A and increased production of IL-13 in conjunctiva. Conclusions: Topical MK2 inhibition effectively alleviated ocular surface damage via suppressing cell apoptosis and CD4+ T-cell-mediated inflammation in ocular surface of dry eye.

Metabolism and renal elimination of gaboxadol in humans: role of UDP-glucuronosyltransferases and transporters.

PURPOSE: Gaboxadol, a selective extrasynaptic agonist of the delta-containing gamma-aminobutyric acid type A (GABAA) receptor, is excreted in humans into the urine as parent drug and glucuronide conjugate. The goal of this study was to identify the UDP-Glucuronosyltransferase (UGT) enzymes and the transporters involved in the metabolism and active renal secretion of gaboxadol and its metabolite in humans.Methods. The structure of the glucuronide conjugate of gaboxadol in human urine was identified by LC/MS/MS. Human recombinant UGT isoforms were used to identify the enzymes responsible for the glucuronidation of gaboxadol. Transport of gaboxadol and its glucuronide was evaluated using cell lines and membrane vesicles expressing human organic anion transporters hOAT1 and hOAT3, organic cation transporter hOCT2, and the multidrug resistance proteins MRP2 and MRP4.Results. Our study indicated that the gaboxadol-O-glucuronide was the major metabolite excreted in human urine. UGT1A9, and to a lesser extent UGT1A6, UGT1A7 and UGT1A8, catalyzed the O-glucuronidation of gaboxadol in vitro. Gaboxadol was transported by hOAT1, but not by hOCT2, hOAT3, MRP2, and MRP4. Gaboxadol-O-glucuronide was transported by MRP4, but not MRP2.Conlusion. Gaboxadol could be taken up into the kidney by hOAT1 followed by glucuronidation and efflux of the conjugate into urine via MRP4.

Transport of the dipeptidyl peptidase-4 inhibitor sitagliptin by human organic anion transporter 3, organic anion transporting polypeptide 4C1, and multidrug resistance P-glycoprotein.

Sitagliptin, a selective dipeptidyl peptidase 4 inhibitor recently approved for the treatment of type 2 diabetes, is excreted into the urine via active tubular secretion and glomerular filtration in humans. In this report, we demonstrate that sitagliptin is transported by human organic anion transporter hOAT3 (Km=162 microM), organic anion transporting polypeptide OATP4C1, and multidrug resistance (MDR) P-glycoprotein (Pgp), but not by human organic cation transporter 2 hOCT2, hOAT1, oligopeptide transporter hPEPT1, OATP2B1, and the multidrug resistance proteins MRP2 and MRP4. Our studies suggested that hOAT3, OATP4C1, and MDR1 Pgp might play a role in transporting sitagliptin into and out of renal proximal tubule cells, respectively. Sitagliptin did not inhibit hOAT1-mediated cidofovir uptake, but it showed weak inhibition of hOAT3-mediated cimetidine uptake (IC50=160 microM). hOAT3-mediated sitagliptin uptake was inhibited by probenecid, ibuprofen, furosemide, fenofibric acid, quinapril, indapamide, and cimetidine with IC50 values of 5.6, 3.7, 1.7, 2.2, 6.2, 11, and 79 microM, respectively. Sitagliptin did not inhibit Pgp-mediated transport of digoxin, verapamil, ritonavir, quinidine, and vinblastine. Cyclosporine A significantly inhibited Pgp-mediated transport of sitagliptin (IC50=1 microM). Our data indicate that sitagliptin is unlikely to be a perpetrator of drug-drug interactions with Pgp, hOAT1, or hOAT3 substrates at clinically relevant concentrations. Renal secretion of sitagliptin could be inhibited if coadministered with OAT3 inhibitors such as probenecid. However, the magnitude of interactions should be low, and the effects may not be clinically meaningful, due to the high safety margin of sitagliptin.

Characterization of mice lacking the multidrug resistance protein MRP2 (ABCC2).

The multidrug resistance protein Mrp2 is an ATP-binding cassette (ABC) transporter mainly expressed in liver, kidney, and intestine. One of the physiological roles of Mrp2 is to transport bilirubin glucuronides from the liver into the bile. Current in vivo models to study Mrp2 are the transporter-deficient and Eisai hyperbilirubinemic rat strains. Previous reports showed hyperbilirubinemia and induction of Mrp3 in the hepatocyte sinusoidal membrane in the mutant rats. In addition, differences in liver cytochrome P450 and UGT1a levels between wild-type and mutant rats were detected. To study whether these compensatory mechanisms were specific to rats, we characterized Mrp2(-/-) mice. Functional absence of Mrp2 in the knockout mice was demonstrated by showing increased levels of bilirubin and bilirubin glucuronides in serum and urine, a reduction in biliary excretion of bilirubin glucuronides and total glutathione, and a reduction in the biliary excretion of the Mrp2 substrate dibromosulfophthalein. To identify possible compensatory mechanisms in Mrp2(-/-) mice, the expression levels of 98 phase I, phase II, and transporter genes were compared in liver, kidney, and intestine of male and female Mrp2(-/-) and control mice. Unlike in Mrp2 mutant rats, no induction of Mrp3 in Mrp2(-/-) mice was detected. However, Mrp4 mRNA and protein in liver and kidney were increased approximately 6- and 2-fold, respectively. Phenotypic analysis of major cytochrome P450-mediated activities in liver microsomes did not show differences between wild-type and Mrp2(-/-) mice. In conclusion, Mrp2(-/-) mice are a new valuable tool to study the role of Mrp2 in drug disposition.

Validation of (-)-N-3-benzyl-phenobarbital as a selective inhibitor of CYP2C19 in human liver microsomes.

(-)-N-3-Benzyl-phenobarbital (NBPB) was reported to be a potent and selective inhibitor of CYP2C19. To validate the selectivity of NBPB toward CYP2C19 in human liver microsomes, the inhibitory effects on major cytochrome P450 isoform-specific reactions were evaluated in the present study. In human liver microsomes, NBPB showed potent competitive inhibition on CYP2C19-mediated S-mephenytoin 4'-hydroxylation with an IC(50) value of 0.25 microM and K(i) value of 0.12 microM, whereas weak inhibition was observed for CYP1A2-, CYP2A6-, CYP2B6-, CYP2C8-, CYP2C9-, CYP2D6-, and CYP3A4-mediated reactions with IC(50) values >100, >100, 62, 34, 19, >100, and 89 microM, respectively. Importantly, its selectivity toward CYP2C19 among the CYP2C subfamily was demonstrated. Therefore, NBPB can be used as a potent and selective inhibitor to establish the relative contribution of CYP2C19 for in vitro reaction phenotyping studies. This compound can also serve as a positive control inhibitor of CYP2C19 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source.

Cytochrome P450 3A4-mediated bioactivation of raloxifene: irreversible enzyme inhibition and thiol adduct formation.

Raloxifene is a selective estrogen receptor modulator which is effective in the treatment of osteoporosis in postmenopausal women. We report herein that cytochrome P450 (P450)3A4 is inhibited by raloxifene in human liver microsomal incubations. The nature of the inhibition was irreversible and was NADPH- and preincubation time-dependent, with K(I) and k(inact) values estimated at 9.9 microM and 0.16 min(-1), respectively. The observed loss of P450 3A4 activity was attenuated partially by glutathione (GSH), implying the involvement of a reactive metabolite(s) in the inactivation process. Subsequently, GSH adducts of raloxifene were identified in incubations with human liver microsomes; substitution with GSH occurred at the 5- or 7-position of the benzothiophene moiety or at the 3'-position of the phenol ring, with the 7-glutathionyl derivative being most abundant based on LC/MS and NMR analyses. These adducts are postulated to derive from addition of GSH to raloxifene arene oxides followed by dehydration and aromatization. Alternatively, raloxifene may be oxidized to an extended quinone intermediate, which then is trapped by GSH conjugation. The bioactivation of raloxifene most likely is catalyzed by P450 3A4, since the formation of GSH adducts was almost abolished when liver microsomes were pretreated with ketoconazole or with an inhibitory anti-P450 3A4 IgG. The GSH adducts also were detected in incubations of raloxifene with rat or human hepatocytes, while the corresponding N-acetylcysteine adducts were identified in the bile and urine from rats treated orally with the drug at 5 mg/kg. Taken together, these data indicate that P450 3A4-mediated bioactivation of raloxifene in vitro is accompanied by loss of enzyme activity. The significance of these findings with respect to the clinical use of raloxifene remains to be determined.