Absence Of P-glycoprotein Research Articles

Revealing innovative JAK1 and JAK3 inhibitors: a comprehensive study utilizing QSAR, 3D-Pharmacophore screening, molecular docking, molecular dynamics, and MM/GBSA analyses.

The heterocycle compounds, with their diverse functionalities, are particularly effective in inhibiting Janus kinases (JAKs). Therefore, it is crucial to identify the correlation between their complex structures and biological activities for the development of new drugs for the treatment of rheumatoid arthritis (RA) and cancer. In this study, a diverse set of 28 heterocyclic compounds selective for JAK1 and JAK3 was employed to construct quantitative structure-activity relationship (QSAR) models using multiple linear regression (MLR). Artificial neural network (ANN) models were employed in the development of QSAR models. The robustness and stability of the models were assessed through internal and external methodologies, including the domain of applicability (DoA). The molecular descriptors incorporated into the model exhibited a satisfactory correlation with the receptor-ligand complex structures of JAKs observed in X-ray crystallography, making the model interpretable and predictive. Furthermore, pharmacophore models ADRRR and ADHRR were designed for each JAK1 and JAK3, proving effective in discriminating between active compounds and decoys. Both models demonstrated good performance in identifying new compounds, with an ROC of 0.83 for the ADRRR model and an ROC of 0.75 for the ADHRR model. Using a pharmacophore model, the most promising compounds were selected based on their strong affinity compared to the most active compounds in the studied series each JAK1 and JAK3. Notably, the pharmacokinetic, physicochemical properties, and biological activities of the selected compounds (As compounds ZINC79189223 and ZINC66252348) were found to be consistent with their therapeutic effects in RA, owing to their non-toxic, cholinergic nature, absence of P-glycoprotein, high gastrointestinal absorption, and ability to penetrate the blood-brain barrier. Furthermore, ADMET properties were assessed, and molecular dynamics and MM/GBSA analysis revealed stability in these molecules.

Immunotoxin in the Absence of P-Glycoprotein allows Selective Target Cell Killing in the Semi-Direct Antigen Presentation Pathway Following Organ Transplantation

Immunotoxin in the Absence of P-Glycoprotein allows Selective Target Cell Killing in the Semi-Direct Antigen Presentation Pathway Following Organ Transplantation

Differential role of P-glycoprotein and breast cancer resistance protein in drug distribution into brain, CSF and peripheral nerve tissues in rats

1. This study was designed to evaluate how the absence of P-glycoprotein (Pgp, Mdr1a), breast cancer-resistance protein (Bcrp, Abcg2) or both affects drug distribution into sciatic nerves, brain and cerebrospinal fluid (CSF) in rats.2. Pgp substrate (loperamide), BCRP substrates (dantrolene and proprietary compound X) and dual substrates (imatinib and proprietary compound Y) were well distributed into sciatic nerves with comparable nerve to plasma concentration ratios between wild-type and knockout (KO) rats.3. Brain exposure increased substantially in Mdr1a(−/−) rats for loperamide and in Mdr1a(−/−)/Abcg2(−/−) rats for imatinib and compound Y, but minimally to modestly in Abcg2(−/−) rats for dantrolene and compound X. The deletion of Mdr1a or Abcg2 alone had little effect on brain distribution of compound Y.4. While CSF to unbound brain concentration ratio remained ≥3 in the KO animals for dantrolene, compounds X and Y, it was reduced to 1 in the Mdr1a(−/−)/Abcg2(−/−) rats for imatinib.5. The data indicate that Pgp and Bcrp do not play significant roles in drug distribution into peripheral nerve tissues in rats, while working in concert to regulate brain penetration. Our results further support that CSF concentration may not be a good surrogate for unbound brain concentration of efflux substrates.

Moxidectin has a lower neurotoxic potential but comparable brain penetration in P‐glycoprotein‐deficient CF‐1 mice compared to ivermectin

The anti-parasitic drugs ivermectin (IVM) and moxidectin (MOX) normally show limited brain penetration in vertebrates because of effective drug efflux at the blood-brain barrier by P-glycoprotein, encoded by the multi-drug resistance (MDR1) gene. However, dogs with homozygous nt230(del4) mutation in the MDR1 gene do not express a functionally active P-glycoprotein and show increased brain penetration of these drugs, resulting in neurological toxicity to different degrees. Thus, whereas IVM provokes neurological toxicity at 0.1 mg/kg, MOX is tolerated at this dosage. To investigate whether this difference is attributable to lower brain penetration of MOX in the absence of P-glycoprotein or to their neurotoxic potential, we applied IVM and MOX to P-glycoprotein-deficient CF-1 mice and comparatively analysed the absolute drug concentrations in the brain. Furthermore, we quantified drug-induced neurotoxicity by measuring the walking performance of the mice on a rotarod setup. We found that at a dosage of 0.2 mg/kg, representing 0.23 μmol/kg IVM and 0.31 μmol/kg MOX, the absolute drug concentrations in the brain were comparable with 100.8 pmol/g and 140.2 pmol/g, respectively. However, MOX induced the same degree of neurotoxicosis at the higher dosage of 1.09 μmol/kg (0.7 mg/kg) compared with IVM at 0.40 μmol/kg (0.35 mg/kg), demonstrating the 2.7-fold lower neurotoxic potential of MOX compared to IVM. This could be explained by a lower binding affinity or lower intrinsic activity of MOX at the relevant central nervous system receptors compared with IVM.

Effects of P-glycoprotein and Mrp2 inhibitors on the hepatobiliary disposition of Rhodamine 123 and its glucuronidated metabolite in isolated perfused rat livers

The hepatobiliary disposition of rhodamine 123 (RH-123) and its glucuronidated (RH-Glu) and deacylated (RH-110) metabolites were studied in an isolated perfused rat liver (IPRL) model in the presence and absence of P-glycoprotein (P-gp) and Mrp2 inhibitors. A single dose (180 µg) of RH-123 was added to a recirculating perfusate in the absence (Control) or presence of cyclosporine A (CyA) or dibromosulfophthalein (DBSP) in the perfusate. Serial (0–90 min) perfusate and bile and terminal liver samples were collected for analysis by HPLC. In the Control livers, 25.4 ± 2.2% (mean ± SD) of the dose was recovered as RH-123 (11.7 ± 2.0%) and RH-Glu (13.2 ± 0.9%) in the bile. Whereas CyA substantially (90%) reduced (p < 0.001) the biliary excretion of RH-123 without affecting the excretion of RH-Glu, DBSP reduced the biliary excretion of RH-Glu by >80% (p < 0.001) with no effect on the biliary excretion of RH-123. Mass balance studies showed that DBSP, in addition to reducing the biliary clearance of RH-Glu, also strongly inhibited the glucuronidation of RH-123, an effect that was confirmed in vitro using the glucuronidation marker umbelliferone. It is concluded that the use of RH-123 in an IPRL model may serve as a dual marker for the determination of the altered functions of P-gp and/or Mrp2. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:455–466, 2010

P-glycoprotein deficient mouse in situ blood–brain barrier permeability and its prediction using an in combo PAMPA model

The purpose of the study was to assess the permeability of mouse blood–brain barrier (BBB) to a diverse set of compounds in the absence of P-glycoprotein (Pgp) mediated efflux, to predict it using an in combo PAMPA model, and to explore its role in brain penetration classification (BPC). The initial brain uptake ( K in) of 19 compounds in both wild-type and Pgp mutant [mdr1a(−/−)] CF-1 mice was determined by the in situ brain perfusion technique. PAMPA measurements were performed, and the values were used to develop an in combo model, including Abraham descriptors. Published rodent K in values were used to enhance the dataset and validate the model. The model predicted 92% of the variance of the training set permeability. In all, 182 K in values were considered in this study, spanning four log orders of magnitude and where Pgp decreased brain uptake by as much as 14-fold. The calculated permeability-surface area (PS) values along with literature reported brain tissue binding were used to group molecules in terms of their brain penetration classification. The in situ BBB permeability can be predicted by the in combo PAMPA model to a satisfactory degree, and can be used as a lower-cost, high throughput first-pass screening method for BBB passive permeability.

Brain penetration of ivermectin and selamectin inmdr1a,bP‐glycoprotein‐ andbcrp‐ deficient knockout mice

P-glycoprotein, which is encoded by the multi-drug resistance gene (MDR1), highly restricts the entry of ivermectin into the brain by an ATP-driven efflux mechanism at the blood-brain barrier. In dogs with a homozygous MDR1 mutation though, ivermectin accumulates in the brain and provokes severe signs of neurotoxicosis and even death. In contrast to ivermectin, selamectin is safer in the treatment of MDR1 mutant dogs, suggesting that selamectin is transported differently by P-glycoprotein across the blood-brain barrier. To test this, we applied selamectin to mdr1-deficient mdr1a,b(-/-) knockout mice and wild-type mice. Brain penetration, organ distribution, and plasma kinetics were analyzed after intravenous, oral, and dermal spot-on application in comparison with ivermectin. We found that in vivo both macrocyclic lactone compounds are substrates of P-glycoprotein and that these strongly accumulate in the brain of mdr1a,b(-/-) knockout mice compared with wild-type mice at therapeutic doses of 12 mg/kg selamectin and 0.2 mg/kg ivermectin. However, selamectin accumulates to a much lesser degree (5-10 times) than ivermectin (36-60 times) in the absence of P-glycoprotein. This could explain the broader margin of safety of selamectin in MDR1 mutant dogs. In liver, kidney, and testes, ivermectin and selamectin accumulated less than four times as much in mdr1a,b mutant mice as in wild-type mice. Breast cancer resistance protein (Bcrp)-deficient bcrp(-/-) knockout mice were also included in the application studies, but showed no differences in brain concentrations or organ distribution of either ivermectin or selamectin compared with wild-type mice. This indicates that Bcrp is not a relevant efflux carrier for these macrocyclic lactone compounds in vivo at the blood-brain barrier.

W1196 Stability of Gut Microbiota Over Time in Crohn's Disease Patients Compared to Healthy Relatives

well plates. BMDC were treated with media alone, E. coli LPS (1 μg/mL) or murine CpG (20 μM) for 24 hours, after which culture supernatant was harvested for cytokine ELISA. BMDC were stained for cell surface markers of activation and analyzed by flow cytometry. Results: LPS treated FVB.mdr1a-/BMDC cultures had greater amounts of IL-12 (679±149 vs. 488±73, pg/mL) but similar levels of TNF-α compared to FVB.WT. FVB.mdr1a-/BMDC cultured with CpG also produced more IL-12 (98±28 pg/mL vs. non-detectable) and TNFα (418±91 vs. 207±40, pg/mL). The percentage and absolute number of CD11c+CD11b+ cells were greater in CpG treated FVB.mdr1a-/BMDC. The levels of BMDC activation as determined as being CD11c+ CD80+ and CD11c+ CD80+, were greater in the LPS and CpG treated FVB.mdra1-/BMDC. Conclusions: TLR4 and TLR9 ligand treatment results in a greater activation of BMDC from FVB.mdra1-/-. Since these TLRs are present in the intracellular compartment the absence of p-glycoprotein (an ATP-dependent pump) to expel LPS or CpG from intestinal dendritic cells could contribute to the mechanism of colitis in FVB.mdra1-/mice.

Intrinsic chemotherapy resistance to the tubulin‐binding antimitotic agents in renal cell carcinoma

Renal cancer is one of the most chemoresistant tumor types. Using a panel of 10 established renal cancer cell lines that have not been subjected to prior drug selection, the range of functional resistance phenotypes to the tubulin-binding agents paclitaxel, vinblastine, vincristine and patupilone (epothilone B, EPO906) was determined, together with expression of P-glycoprotein (PgP), multidrug resistance associated protein-2 (MRP2) and major vault protein (MVP) proteins. The IC(50) values for vincristine correlated positively with PgP expression (r = 0.73; p = 0.031), with values for paclitaxel and vinblastine just failing to reach significance. A significant positive correlation was observed for sensitivity to paclitaxel and MRP2 expression only (r = 0.8; p = 0.013). MVP expression did not correlate with sensitivity to any of the drugs examined. All cell lines exhibited much greater sensitivity to patupilone, demonstrating for the first time the potential use of patupilone in this cancer. In tissue samples from chemotherapy-naive renal cell carcinoma (RCC) patients, marked downregulation or absence of PgP in many tumor cells with expression levels more similar to sensitive cell lines rather than the resistant lines was seen. Similarly, MRP2 was absent or only weakly present in tumor cells, whereas MVP was very strongly upregulated in most tumor samples. This study illustrating discrepancies between results exclusively based on studies in cell lines and findings in vivo suggests that the role of PgP and MRP2 in intrinsic resistance in RCC in vivo may be less than expected from the in vitro findings and supports a potential role for MVP on the basis of in vivo expression studies.

Structural determinants of P-glycoprotein-mediated transport of glucocorticoids.

The aim of this study was to determine requisite structural features for P-glycoprotein-mediated transport of a series of structurally related glucocorticoids (GCs). Transport experiments were conducted in wild-type and stably transfected MDRI LLC-PK cell line. Transport efficiency (Teff = Peff, B-->A / Peff, A-->B) in both cell lines was compared as a measure of passive diffusion and P-glycoprotein-mediated transepithelial transport for each steroid. Three-dimensional structure-activity relationships were built to determine how specific structural features within the steroids affect their P-gp-mediated efflux. Mean (+/- SD) Teff in LLC-PK cells was 1.1 +/- 0.17, indicating that differences in structure and partition coefficient did not affect drug flux in the absence of P-glycoprotein. Teff in L-MDRI cells ranged from 3.6 to 26.6, demonstrating the importance of glucocorticoid structure to P-glycoprotein transport. The rank order of Teff in MDR1 cells was: methylprednisolone> prednisolone > betamethasone > dexamethasone/prednisone > cortisol. There was no correlation between individual Teff values and partition coefficient. 3D-QSAR models were built using CoMFA and CoMSIA with a q2 (r2) of 0.48 (0.99) and 0.41 (0.95), respectively. Nonpolar bulky substituents around the C-6alpha position, as well as a hydrogen-bond donor at position C-11, enhance P-glycoprotein affinity and cellular efflux, whereas bulky substituents at C-16 diminish transporter affinity.

Multiple mechanisms confer different drug-resistant phenotypes in pancreatic carcinoma cells.

Drug-resistant phenotypes of cancer cells may be caused by complex multimodal mechanisms of resistance. In order to gain further insighte into these mechanisms, a P-glycoprotein-mediated multidrug-resistant phenotype induced by daunorubicin-selection and an alternative drug resistance due to treatment with mitoxantrone were investigated in pancreatic carcinoma-derived cells. For assessing cross-resistance against various drugs, cell proliferation assays were performed. Drug accumulation was measured by flow cytometry. Messenger RNA expression was analyzed by Northern blot and RT-PCR, whereas protein expression was determined by Western blot. Catalytic activity of DNA-topoisomerases (Topo) II was determined by the decatenation assay. In mitoxantrone-selected EPP85-181RNOV cells a decreased accumulation of mitoxantrone and daunorubicin was observed in the absence of P-glycoprotein, multidrug resistance protein or breast cancer resistance protein over-expression. An approximately twofold decrease of DNA topoisomerase II catalytic activity could be observed in both drug-resistance-exhibiting cell lines. The reduction of Topo II catalytic activity was reflected by decreased expression of Topo IIalpha and IIbeta mRNAs and proteins. The decreased drug accumulation in EPP85-181RNOV cells indicates that alternative transport events are occurring. The decreased catalytic activity and expression of Topo II indicate that modulation of Topo II catalytic activity contributes to both drug-resistant phenotypes in pancreatic carcinoma cells.

The subpopulation of CF-1 mice deficient in P-glycoprotein contains a murine retroviral insertion in the mdr1a gene.

A subpopulation of the CF-1 mouse strain is sensitive to neurotoxicity following exposure to avermectins, a family of structurally related antiparasitic agents. This unusual sensitivity is the result of a deficiency in the mdr1a P-glycoprotein that normally contributes to a functional blood-brain barrier. Previous studies demonstrated a correlation between P-glycoprotein levels in the brain, intestine, testis, and placenta with an restriction fragment length polymorphism (RFLP) pattern from DNA isolated from the animals. We have demonstrated that only P-glycoprotein derived from the mdr1a gene is deficient in these mice. In this article, we describe the genetic defect in the subpopulation of CF-1 mice resulting in an absence of P-glycoprotein. The data presented describes a reverse transcription--polymerase chain reaction (RT-PCR) protocol that specifically amplifies mdr1a mRNA from tissue and confirms that the P-glycoprotein defect results from a truncated mRNA with a deleted exon 23. Genomic amplification and sequencing of the intron between exon 22 and 23 in Pgp-deficient animals reveals an insertion of approximately 8.35 kb of DNA at the exon 23 intron--exon junction corresponding to a murine leukemia virus. This insertion results in the aberrant splicing of the mRNA and the loss of exon 23 during RNA processing.

Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. I. Regulatory volume decrease.

Exposure of NIH/3T3 fibroblasts not expressing P-glycoprotein to 50, 30, 20, and 10% hyposmotic solutions led to cell volume increases of 70, 32, 21, and 12%, respectively. After swelling, NIH/3T3 cells exhibited regulatory volume decrease (RVD), attaining complete volume recovery after 30 min except in 50% hyposmotic solution, in which volume recovery was 76%. RVD was accelerated by gramicidin and inhibited by the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid and by the K channel, blocker quinidine. RVD was reduced 15% by removal of extracellular Ca. The pathway opened by hypotonicity was highly permeable to K and Rb and only partly permeable to other cations. Most anions were able to permeate, with a permeability ranking of nitrate > benzoate = iodide > thiocyanate > chloride > > gluconate. The pathway was permeable to neutral amino acids, with a permeability ranking of glycine > alanine > glutamate > taurine > gamma-aminobutyric acid > glutamine. The pathway was not permeable to basic amino acids. These results show that, despite the absence of P-glycoprotein, NIH/3T3 cells exhibit RVD with properties similar to those expressed in most cell types.

Decreased drug accumulation in a mitoxantrone-resistant gastric carcinoma cell line in the absence of P-glycoprotein.

An established gastric-carcinoma cell line, EPG85-257P, is extremely sensitive to mitoxantrone (IC50, 0.12 ng/ml). Stepwise selection with mitoxantrone for 3 years resulted in a cell line (EPG85-257RN) that is 7,056-fold resistant to mitoxantrone (IC50, 846 ng/ml) and displays cross-resistance to the topoisomerase(topo)-II poisons ametantrone (411x), etoposide (112x) and teniposide (60x) as well as the topo-I poisons 7-ethyl-10-hydroxycamptothecin (331x) and topotecan (58x). We now show that this resistance is multifactorial. Western blotting revealed a 5-fold decrease in topo-IIalpha polypeptide in the mitoxantrone-resistant cells. Immunohistochemistry and Western blotting failed to demonstrate P-glycoprotein overexpression. Formation of trapped topo-II-DNA complexes in the resistant cells required higher mitoxantrone concentrations than in parental cells, even though nuclei isolated from the EPG85-257RN cells formed cleavage complexes normally. In agreement with these observations, which suggest the possibility of a defect in mitoxantrone accumulation, examination of mitoxantrone accumulation in both cell lines by confocal laser microscopy revealed that the EPG85-257RN cells accumulate less mitoxantrone at steady state. From these results, we propose that mitoxantrone accumulation, along with alterations in topo-IIalpha expression, contribute to the resistance to mitoxantrone observed in these cells.

Clinical Significance of Multi-Drug Resistance Associated Protein and P-Glycoprotein in Patients with Bladder Cancer

The clinical significance of multi-drug resistant proteins, such as multi-drug resistance associated protein and P-glycoprotein, in terms of prognostic value was determined in patients with bladder cancer. The expression of multi-drug resistance associated protein and P-glycoprotein was investigated immunohistochemically before and after chemotherapy. The relationship between expression of these multi-drug resistant proteins and clinical outcome assessed by tumor recurrence rate, cystectomy rate and 5-year survival rate was also investigated in 33 patients with bladder cancer. Before chemotherapy multi-drug resistance associated protein expression was observed in 1 of 28 patients (4%) while P-glycoprotein expression was observed in 22 of 33 (67%). Multi-drug resistance associated protein induction by chemotherapy was observed in 6 of 28 patients (21%), whereas P-glycoprotein induction was noted in 4 (14%). Multi-drug resistance associated protein in this disease is induced more frequently by high dose (more than 300 mg.) than low dose (less than 300 mg.) anthracyclines (p < 0.01). Immunohistochemical analysis also revealed co-expression of multi-drug resistance associated protein and P-glycoprotein in 5 of 28 patients (18%) after chemotherapy. However, there was no significant correlation between positive P-glycoprotein expression before chemotherapy and clinical outcome. Multi-drug resistance associated protein as well as P-glycoprotein mediated multi-drug resistance may be induced after chemotherapy for bladder tumors. However, the presence of P-glycoprotein before chemotherapy does not predict clinical outcome in patients with bladder cancer.

Cross-resistance and collateral sensitivity to natural product drugs in cisplatin-sensitive and -resistant rat lymphoma and human ovarian carcinoma cells.

The cytotoxicity of mitotic spindle poisons, vinca alkaloids and the anthracycline, adriamycin, against cisplatin-sensitive and -resistant rat lymphoma and human ovarian carcinoma cell lines was investigated. Interestingly, it was found that all cell lines were more sensitive to the mitotic spindle poisons, vincristine and vinblastine. Adriamycin was the least effective and taxol had intermediate activity. The Walker rat lymphoma cell line resistant to cisplatin (WR) exhibited the multiple drug resistance phenotype since it showed collateral resistance to all drugs (ranging from twofold to taxol, colcemid and colchicine and sixfold to the vinca alkaloids). Verapamil potentiated the cytotoxic activity of adriamycin and vincristine in a striking fashion with the Walker cells. P-glycoprotein was found to be present in the plasma membranes of the Walker cells with approximately a 2.5-fold increase in the WR as compared to the sensitive (WS) cells. Glutathione levels were elevated in all of the cisplatin-resistant cell lines when compared to the cisplatin-sensitive parental cell lines. A profound effect of buthionine sulfoximine pretreatment on adriamycin cytotoxicity was observed. Glutathione S-transferase (pi) was present in all the human cell lines but the WS cells had markedly lower levels (almost negligible) when compared to the WR cells. These observations imply that cisplatin-resistant cells may be more sensitive to mitotic spindle poisons and vinca alkaloids, irrespective of the mechanism of platinum resistance, and that the cytotoxicity of vinca alkaloids could be further modulated by verapamil, irrespective of the presence or absence of P-glycoprotein.

Expression of the MDR1 gene (P-glycoprotein) in breast cancer.

Of the many types of drug resistance reported in the literature, one of the best characterized is classic multidrug resistance, a genetically determined type of resistance to chemotherapy [1,2]. Cancers exhibiting intrinsic multidrug resistance mediated by P-glycoprotein, the product of the MDR1 gene, do not respond to chemotherapy upon primary exposure to “natural product” lipophilic drugs such as colchicine, vinblastine, and doxorubicin. Characteristics of multidrug resistance in cancer cells include: (a) increased levels of P-glycoprotein, a 170-kDa membrane transporter, (b) decreased drug accumulation and enhanced drug efflux, (c) chemoresistance to multiple natural product agents, and (d) chemosensitization by transporter inhibitors (e.g., verapamil). Other cancers that respond initially to drugs may subsequently become resistant to these and other drugs to which the patient has had no previous exposure. This is multidrug resistance in the acquired form. Tumors with either intrinsic or acquired multidrug resistance mediated by P-glycoprotein usually are not cross-resistant to alkylating agents, most antimetabolites, or heavy metals. Often, intrinsic multidrug resistance occurs in tumors derived from organs that normally express P-glycoprotein, such as intestine, liver, kidney, and adrenal [3]. The multidrug resistance phenotype also occurs in the absence of P-glycoprotein, reflecting the multiplicity of mechanisms that produce this phenomenon [4].