Dexamethasone increases expression and activity of multidrug resistance transporters at the rat blood-brain barrier.

Brain edema is an important factor leading to morbidity and mortality associated with primary brain tumors. Dexamethasone, a synthetic glucocorticoid, is routinely prescribed with antineoplastic agents to alleviate pain associated with chemotherapy and reduce intracranial pressure. We investigated whether dexamethasone treatment increased the expression and activity of multidrug resistance (MDR) transporters at the blood-brain barrier. Treatment of primary rat brain microvascular endothelial cells with submicromolar concentrations of dexamethasone induced significantly higher levels of drug efflux transporters such as breast cancer resistance protein (abcg2), P-glycoprotein (P-gp; abcb1a/abcb1b), and MDR protein 2 (Mrp2; abcc2) as indicted by protein and mRNA levels as well as by functional activity. The effect of dexamethasone on transporter function was significant within 6 h of treatment, was dose dependent, and was reversible. Dexamethasone-induced upregulation of Bcrp and P-gp expression and function was partially abrogated by the glucocorticoid receptor (GR) antagonist RU486. In contrast, RU486 had no effect on the dexamethasone-induced upregulation of Mrp2, suggesting a GR-independent regulation of Mrp2, and a GR-dependent regulation of P-gp and Bcrp. In addition to the dexamethasone-induced upregulation of MDR transporters, we measured a dose-dependent and reversible increase in the expression of the nuclear transcription factor pregnane xenobiotic receptor (PXR). Administering dexamethasone to rats caused increased expression of PXR in brain microvessels within 24 h. These results suggest that adjuvant therapy with corticosteroids such as dexamethasone in the treatment of brain tumors may increase the expression of MDR transporters at the blood-brain barrier through pathways involving GR and PXR.

Janus kinase 3 regulates interleukin 2-induced mucosal wound repair through tyrosine phosphorylation of villin.

Janus kinase 3 (Jak3) is a non-receptor tyrosine kinase known to be expressed in hematopoietic cells. Studies of whole organ homogenates show that Jak3 is also expressed in the intestines of both human and mice. However, neither its expression nor its function has been defined in intestinal epithelial enterocytes. The present studies demonstrate that functional Jak3 is expressed in human intestinal enterocytes HT-29 Cl-19A and Caco-2 and plays an essential role in the intestinal epithelial wound repair process in response to interleukin 2 (IL-2). Exogenous IL-2 enhanced the wound repair of intestinal enterocytes in a dose-dependent manner. Activation by IL-2 led to rapid tyrosine phosphorylation and redistribution of Jak3. IL-2-stimulated redistribution of Jak3 was inhibited by the Jak3-specific inhibitor WHI-P131. IL-2 also induced Jak3-dependent redistribution of the actin cytoskeleton in migrating cells. In these cells Jak3 interacted with the intestinal and renal epithelial cell-specific cytoskeletal protein villin in an IL-2-dependent manner. Inhibition of Jak3 activation resulted in loss of tyrosine phosphorylation of villin and a significant decrease in wound repair of the intestinal epithelial cells. Previously, we had shown that tyrosine phosphorylation of villin is important for cytoskeletal remodeling and cell migration. The present study demonstrates a novel pathway in intestinal enterocytes in which IL-2 enhances intestinal wound repair through mechanisms involving Jak3 and its interactions with villin.

Sulfasalazine-induced reduction of glutathione levels in breast cancer cells: enhancement of growth-inhibitory activity of Doxorubicin.

BACKGROUND: We previously showed that the anti-inflammatory drug, sulfasalazine (salicylazosulfapyridine, SASP), can arrest proliferation of MCF-7 and MDA-MB-231 mammary cancer cells by inhibiting uptake of cystine via the x(c-) cystine/glutamate antiporter. Here we examined SASP with regard to reduction of cellular glutathione (GSH) levels and drug efficacy-enhancing ability. METHODS: GSH levels were measured spectrophotometrically. Cellular drug retention was determined with 3H-labeled methotrexate, and drug efficacy with a colony formation assay. RESULTS: Incubation of the mammary cancer cells with SASP (0.3-0.5 mM) led to reduction of their GSH content in a time- and concentration-dependent manner. Similar to MK-571, a multidrug resistance-associated protein inhibitor, SASP increased intracellular accumulation of methotrexate. Preincubation of cells with SASP (0.3 mM) significantly enhanced the potency of the anticancer agent doxorubicin (2.5 nM). CONCLUSIONS: SASP-induced reduction of cellular GSH levels can lead to growth arrest of mammary cancer cells and enhancement of anticancer drug efficacy.

Simultaneous measurement of in vivo P-glycoprotein and cytochrome P450 3A activities.

Digoxin and midazolam are routinely used as probe drugs to measure in vivo activity of P-glycoprotein (P-gp) and cytochrome P450 3A4/5 (CYP3A), respectively. We investigated whether digoxin and midazolam could be coadministered to simultaneously determine P-gp and CYP3A activity without a significant pharmacokinetic interaction. In a randomized crossover design, digoxin (0.5 mg oral) or midazolam (2.0 mg oral) was administered individually or in combination (digoxin 1 hour after midazolam) to 14 healthy volunteers. Blood and urine samples were collected for up to 48 hours. Pharmacokinetic parameters of digoxin, midazolam and 1'-OH midazolam were evaluated to determine the presence of an interaction. The geometric mean ratios of all measured pharmacokinetic parameters of digoxin and midazolam were not significantly affected by coadministration. Coadministration of digoxin and midazolam can be used to simultaneously phenotype P-gp and CYP3A activity without a significant pharmacokinetic interaction.

Pharmacokinetic profiling and bioequivalence assessment of two marketed brands of nevirapine tablets in healthy Indian volunteers.

Nevirapine (CAS 129618-40-2), a non-nucleoside reverse transcriptase inhibitor, has been effectively used for treatment of HIV-infected patients. A randomized, two-way, crossover study was conducted in 24 fasting, healthy, Indian male subjects to compare plasma pharmacokinetic profile and single-dose tolerability of a new nevirapine tablet formulation (test, T) with that of a reference (R) tablet. Each volunteer received T and R formulations separated by at least 19 days of drug free wash-out period. Plasma concentrations of nevirapine, determined up to 288 h post dose by a sensitive and validated HPLC assay, were utilized to assess pharmacokinetic parameters such as the maximum observed plasma concentration (Cmax), time to Cmax (tmax), and area under plasma concentration curve (AUCinfinity). The primary plasma pharmacokinetic parameters of anti-retroviral substances, Cmax and AUCinfinity, were comparable for either of the formulations. Oral absorption of nevirapine was almost complete within 5 h. Geometric mean ratios (% reference) of AUCinfinity and Cmax and their 90% confidence intervals were 96.9 [93.69-100.24] and 100.8 [94.61-107.4], respectively. As the 90% confidence intervals of the geometric mean ratio were entirely within 80 to 125% for log-transformed parameters, the two formulations were considered bioequivalent in the extent and rate of absorption. Both formulations exhibited similar tolerability under fasting conditions.

Pharmacokinetic profiling and bioequivalence evaluation of 2 lamivudine tablet formulations after single oral administration in healthy human Indian volunteers.

The present study compared pharmacokinetic (PK) profile and single-dose tolerability of 2 marketed brands of lamivudine (3TC) 150-mg tablets, Lamivir (Cipla, Mumbai, India) and Epivir (GSK, Basingstoke, UK). The randomized, 2-treatment study was conducted in 24 fasting, healthy, Indian male subjects. Each subject received Epivir and Lamivir formulation separated by 7 days of drug-free washout period. Plasma concentrations of 3TC were used to estimate PK parameters such as maximum observed plasma concentration (Cmax) and area under plasma concentration-time curve (AUCinfinity). Geometric mean ratios (relative to Epivir) and resultant 90% CI of 3TC for Cmax and AUCinfinity were 1.00 (0.89-1.12) and 1.01 (0.94-1.07), respectively. As 90% CIs were entirely within 0.80-1.25 for log-transformed data, the 2 formulations were considered bioequivalent in the extent (AUCinfinity) and rate of absorption (Cmax and time to Cmax [tmax]). The means of primary PK parameters of 3TC, Cmax, and AUCinfinity in Indian subjects were comparable to those previously reported in the literature. Both formulations exhibited similar tolerability under fasting conditions.

A combined-formulation tablet of lamivudine/nevirapine/stavudine: bioequivalence compared with concurrent administration of lamivudine, nevirapine, and stavudine in healthy Indian subjects.

Generic fixed-dose combinations of antiretrovirals are frequently prescribed for the treatment of human immunodeficiency virus infection. A randomized, 2-way study was conducted in 24 fasting, healthy, Indian male subjects to assess bioequivalence between a single combination tablet containing lamivudine, stavudine, and nevirapine (treatment A) with respect to separate marketed tablets administered simultaneously (treatment B). Each subject received treatments A and B separated by 19 days of a drug-free washout period. Plasma concentrations of antiretrovirals, determined by a validated liquid chromatography/tandem mass spectrometry assay, were used to assess pharmacokinetic parameters such as maximum observed plasma concentration and area under the plasma concentration curve. Pharmacokinetic parameters were comparable for either treatment. As geometric mean ratios (% treatment A/treatment B) of log-transformed parameters of area under the plasma concentration curve and plasma concentration, as well as their resultant 90% confidence intervals, were within 80% to 125% and 75% to 133%, respectively, 2 treatments were considered bioequivalent in the extent and rate of absorption. Both treatments exhibited similar tolerability under fasting conditions.

Bioequivalence evaluation of two marketed brands of stavudine 40 mg capsules in healthy human South African volunteers.

Stavudine (d4T), a thymidine nucleoside analogue has been effectively used for treatment of patients infected with HIV. A randomized, two-way, crossover study was conducted in 24 fasting, healthy, Caucasian male volunteers to compare plasma pharmacokinetic (PK) profile and single-dose tolerability of a new d4T formulation (Stavir, Cipla Ltd, India; 40 mg capsule, test, T) with that of reference (R) formulation (Zerit), Bristol-Myers Squib, NJ, USA; capsule, 40 mg). Each volunteer received T and R formulation separated by at least 10 days of drug free wash-out period. Plasma concentrations of d4T, determined upto 24h post-dose by a validated LC-MS/MS assay were utilized to assess PK parameters such as maximum observed plasma concentration (Cmax), time to Cmax (tmax), and area under plasma concentration curve (AUC(infinity)). The primary plasma PK parameters, Cmax, and AUC(infinity), of anti-retroviral were comparable for either of the formulations. tmax was achieved within an hour suggesting rapid absorption of d4T from both formulations. Geometric mean ratios (GMR) (percentage reference) of AUC(infinity) and Cmax, and their 90% confidence intervals (CI) were 106.32 [102.52-110.26] and 102.32 [90.25-116.00], respectively. As the 90% CI of GMR were entirely within 80-125% for log-transformed parameters, two formulations were considered bioequivalent, in the extent and rate of absorption. Both formulations exhibited similar tolerability under fasting conditions.

Suppression of cystine uptake by sulfasalazine inhibits proliferation of human mammary carcinoma cells.

BACKGROUND: Malignant progression of lymphoma cells is associated with acquisition of the cystine/glutamate antiporter, xc-, enhancing cystine uptake. Recently, we showed that sulfasalazine (SASP) is a specific xc- inhibitor. Here, we investigated xc- in mammary cancer cell lines. MATERIALS AND METHODS: Expression and function of xc- were evaluated by RT-PCR and 35S-cystine uptake analysis. RESULTS: Xc- expression was elevated 4-fold (p < 0.001) in cells of the most malignant line, MDA-MB-231, associated with increased 35S-cystine uptake (p < 0.001). Proliferation was inhibited by 0.2-0.5 mM SASP. 2-Mercaptoethanol (60 microM), a cystine uptake enhancer, completely prevented SASP-mediated growth inhibition in MDA-MB-231 cultures, but only partially in 184A1 and MCF-7 cultures. SASP-induced growth arrest was reversible and not cell cycle-specific. CONCLUSION: The results suggest: (i) malignant progression of human mammary cancer may be associated with acquisition of xc- expression potentially leading to increased growth autonomy and drug resistance, (ii) xc- may act as a therapeutic target.