Integrated twin-screw wet granulation, continuous vibrational fluid drying and milling: A fully continuous powder to granule line.

Highly homogeneous low-dose (50 µg) tablets were produced incorporating perfectly free-flowing granules prepared by a fully integrated Continuous Manufacturing (CM) line. The adopted CM equipment consisted of a Twin-Screw Wet Granulator (TSWG), a Continuous Fluid Bed Dryer (CFBD) and a Continuous Sieving (CS) unit. Throughout the experiments a pre-blend of lactose-monohydrate and corn starch was gravimetrically dosed with 1 kg/h into the TSWG, where they were successfully granulated with the drug containing water-based PVPK30 solution. The wet mass was subsequently dried in the CFBD on a vibratory conveyor belt and finally sieved in the milling unit. Granule production efficiency was maximized by determining the minimal Liquid-to-Solid (L/S) ratio (0.11). Design of Experiments (DoE) were carried out in order to evaluate the influence of the drying process parameters of the CFBD on the Loss-on-Drying (LOD) results. The manufactured granules were compressed into tablets by an industrial tablet rotary press with excellent API homogeneity (RSD < 3%). Significant scale-up was realized with the CM line by increasing the throughput rate to 10 kg/h. The manufactured granules yielded very similar results to the previous small-scale granulation runs. API homogeneity was demonstrated (RSD < 2%) with Blend Uniformity Analysis (BUA). The efficiency of TSWG granulation was compared to High-Shear Granulation (HSG) with the same L/S ratio. The final results have demonstrated that both the liquid distribution and more importantly API homogeneity was better in case of the TSWG granulation (RSD 1.3% vs. 4.5%).

Poor sleep quality predicts psychotic-like symptoms: an experience sampling study in young adults with schizotypal traits.

OBJECTIVE: Psychotic-like experiences (PLEs) are unusual experiences such as perceptual abnormalities and delusional-like thoughts that resemble the symptoms of psychosis at the sub-clinical level. PLEs are associated with sleep complaints in healthy and clinical samples; however, evidence for day-to-day associations between poor sleep and subsequent PLEs under naturalistic conditions is scarce. We hypothesized that poor sleep quality would predict next days' PLEs, and vice versa, daytime PLEs would be associated with worse subsequent sleep quality. METHOD: Seventy-three university students with moderate to high levels of positive schizotypy participated in an experience sampling study. Participants rated their sleep each morning, as well as PLEs and affective states during the day over 3 weeks. RESULTS: Multilevel regression models indicated that poor sleep quality predicted increased PLEs the following day. Poor sleep was linked to negative daytime mood that partially mediated the associations between sleep quality and next days' PLEs. Furthermore, PLEs were enhanced in the evening as compared to daytime reports. The prediction of poor sleep quality by previous days' PLEs was negligible. CONCLUSIONS: The results are consistent with the position that sleep-related interventions might reduce the risk of psychosis, especially in individuals that tend to experience psychotic-like phenomena and negative affect.

Efficient middle-infrared generation in LiGaS2by simultaneous spectral broadening and difference-frequency generation: erratum.

In this erratum, we correct the abstract and introduction of Opt. Lett.43, 1742 (2018)OPLEDP0146-959210.1364/OL.43.001742 to not include a wrong name for the crystal.

Efficient middle-infrared generation in LiGaS2 by simultaneous spectral broadening and difference-frequency generation.

We report a surprisingly broadband and efficient midinfrared pulse generation in LiGaS2 (Langasite, LGS) by invoking a simultaneous interplay of intrapulse difference-frequency generation, self-phase modulation, and dispersion. This cascaded mechanism expands the output bandwidth and output power at the same time. With 30-fs driving pulses centered at 1030-nm wavelength we obtain a broadband middle-infrared spectrum of 8-11 µm with an LGS crystal as thick as 4 mm, which is eight times longer than the walk-off length.

Rapid single-cell detection and identification of pathogens by using surface-enhanced Raman spectroscopy.

For the successful treatment of infections, real-time analysis and enhanced multiplex capacity, sensitivity and cost-effectiveness of the developed detection method are critical. In this work, surface-enhanced Raman scattering (SERS) was employed with the final aim of identification and discrimination of pathogenic bacteria, based on their detected SERS fingerprint at the single-cell level. Several genera of bacteria that are found in most of the isolated infections in bacteraemia were successfully identified in less than 5 minutes without the use of antibodies or other specific receptors. The key element of the SERS direct detection platform is the SERS substrate, which combines easy production at low costs with a high enhancement enabling single-cell detection. The innovative approach of detection required the in situ synthesis of silver nanoparticles (NPs), ensuring an intimate contact with the bacterial membrane. This protocol provided a good reproducibility of the single-cell SERS spectra and was successfully applied both on Gram-negative and Gram-positive microorganisms (E. coli, M. morganii, E. lactis, L. casei). Thus, a label-free SERS-based biosensor for pathogen detection was developed with low costs, minimal sample preparation, high-accuracy and a very short analysis time of less than 5 min, which is crucial for infection diagnosis.

Controlled reperfusion decreased reperfusion induced oxidative stress and evoked inflammatory response in experimental aortic-clamping animal model.

UNLABELLED: Revascularization after long term aortic ischaemia in vascular surgery induces reperfusion injury accompanied with oxidative stress and inflammatory responses. The hypothesis of this study was that the aortic occlusion followed by controlled reperfusion (CR) can reduce the ischaemia-reperfusion injury, the systemic and local inflammatory response induced by oxidative stress.Animal model was used. CONTROL GROUP: animals underwent a 4-hour infrarenal aortic occlusion followed by continuous reperfusion. Treated group: animals were treated with CR: after a 4-hour infrarenal aortic occlusion we made CR for 30 minutes with the crystalloid reperfusion solution (blood: crystalloid solution ratio 1:1) on pressure 60 Hgmm. Blood samples were collected different times. The developing oxidative stress was detected by the plasma levels of malondialdehyde, reduced glutathion, thiol groups and superoxide dismutase. The inflammatory response was measured by phorbol myristate acetate-induced leukocyte reactive oxygen species production and detection of change in myeloperoxidase levels. The animals were anaesthetized one week after terminating ligation and biopsy was taken from quadriceps muscle and large parenchymal organs.CR significantly reduced the postischaemic oxydative stress and inflammatory responses in early reperfusion period. Pathophysiological results: The rate of affected muscle fibers by degeneration was significantly higher in the untreated animal group. The infiltration of leukocytes in muscle and parenchymal tissues was significantly lower in the treatedgroup.CR can improve outcome after acute lower-limb ischaemia. The results confirm that CR might be also a potential therapeutic approach in vascular surgery against reperfusion injury in acute limb ischaemia. Supported by OTKA K108596.

Differential roles of inflammation and apoptosis in initiation of mid-gestational abortion in malaria-infected C57BL/6 and A/J mice.

INTRODUCTION: Plasmodium chabaudi AS-infection in pregnant A/J and C57BL/6J mice results in mid-gestational pregnancy loss. Although associated with increased systemic and placental pro-inflammatory responses and coagulopathy, the molecular mechanisms that underlie poor pregnancy outcomes in these mice are not yet fully understood. This study investigates the relationships between inflammation, apoptosis and malaria-induced pregnancy loss. METHODS: Infection with P. chabaudi AS in early murine pregnancy and term human placental tissues from an endemic setting were assessed by histology, immunohistochemistry, TUNEL staining, real-time PCR, flow cytometry, western blot, and ELISA. RESULTS: Quantitative PCR reveals accumulation of lymphocytes and monocytes and upregulation of chemokines that attract these cell types in malaria-exposed mid-gestational A/J conceptuses. Monocyte accumulation is confirmed by flow cytometry and placental immunohistochemistry. Concurrent with initiation of malaria-induced abortion, markers of apoptosis are evident in the junctional zone, but not the labyrinth, of A/J placentae. In contrast, mid-gestation conceptuses in infected C57BL/6J lack evidence for monocyte accumulation, exhibiting low or no in situ placental staining despite trophoblast immunoreactivity for the monokine, CCL2. Additionally, placental apoptosis is not consistently observed, and when evident, appears after malaria-induced abortion typically initiates. Similarly, trophoblast apoptosis in term human placental malaria is not observed. Of those studied, a sole common feature of malaria-induced abortion in A/J and C57BL/6J mice is elevation of plasma tumor necrosis factor. DISCUSSION: Consistent with our previous observations, tumor necrosis factor is likely to be a central driver of malaria-induced pregnancy loss in both strains, but likely operates through mechanisms distinct from placental apoptosis in C57BL/6J mice.

Increased Helicobacter pylori-associated gastric cancer risk in the Andean region of Colombia is mediated by spermine oxidase.

Helicobacter pylori infection causes gastric cancer, the third leading cause of cancer death worldwide. More than half of the world's population is infected, making universal eradication impractical. Clinical trials suggest that antibiotic treatment only reduces gastric cancer risk in patients with non-atrophic gastritis (NAG), and is ineffective once preneoplastic lesions of multifocal atrophic gastritis (MAG) and intestinal metaplasia (IM) have occurred. Therefore, additional strategies for risk stratification and chemoprevention of gastric cancer are needed. We have implicated polyamines, generated by the rate-limiting enzyme ornithine decarboxylase (ODC), in gastric carcinogenesis. During H. pylori infection, the enzyme spermine oxidase (SMOX) is induced, which generates hydrogen peroxide from the catabolism of the polyamine spermine. Herein, we assessed the role of SMOX in the increased gastric cancer risk in Colombia associated with the Andean mountain region when compared with the low-risk region on the Pacific coast. When cocultured with gastric epithelial cells, clinical strains of H. pylori from the high-risk region induced more SMOX expression and oxidative DNA damage, and less apoptosis than low-risk strains. These findings were not attributable to differences in the cytotoxin-associated gene A oncoprotein. Gastric tissues from subjects from the high-risk region exhibited greater levels of SMOX and oxidative DNA damage by immunohistochemistry and flow cytometry, and this occurred in NAG, MAG and IM. In Mongolian gerbils, a prototype colonizing strain from the high-risk region induced more SMOX, DNA damage, dysplasia and adenocarcinoma than a colonizing strain from the low-risk region. Treatment of gerbils with either α-difluoromethylornithine, an inhibitor of ODC, or MDL 72527 (N(1),N(4)-Di(buta-2,3-dien-1-yl)butane-1,4-diamine dihydrochloride), an inhibitor of SMOX, reduced gastric dysplasia and carcinoma, as well as apoptosis-resistant cells with DNA damage. These data indicate that aberrant activation of polyamine-driven oxidative stress is a marker of gastric cancer risk and a target for chemoprevention.

Cetuximab, gemcitabine and capecitabine in patients with inoperable biliary tract cancer: a phase 2 study.

PURPOSE: Biliary tract cancer is rare and has dismal prognosis. Chemotherapy has its role in inoperable disease but the role of targeted agents like cetuximab remains to be defined. On the basis of high epidermal growth factor receptor expression of biliary tract cancers this study aims to investigate the efficacy of cetuximab, gemcitabine and capecitabine in an exploratory phase 2 trial. PATIENTS AND METHODS: Inoperable biliary tract cancer patients were treated with the combination of gemcitabine (1000 mg/m(2) on day 1 and 8), capecitabine (1300 mg/m(2)/d on day 1-14) and weekly cetuximab (400mg/m(2) loading and 250 mg/m(2) maintenance dose) in 21-d cycles until progression or the appearance of intolerable side-effects. RESULTS: Out of 34 patients (mean age 59.7 years) accrued in this study 16 had intrahepatic, eight extrahepatic cholangiocarcinoma and 10 gall bladder cancer. The best overall response rate was 17.6% (two complete responses and four partial responses) and the clinical benefit rate was 76.5%. After a median of 15.4 months follow-up the median progression free survival was 34.3 weeks and the median overall survival was 62.8 weeks. The performance status and chemotherapy efficacy were independent and significant markers of survival. Only moderate side-effects were registered in this study. KRAS mutation was evaluable in 24 tumours, all of these were of wild type. CONCLUSION: The efficacy of cetuximab, gemcitabine and capecitabine combination is encouraging and a well tolerated treatment of inoperable biliary tract cancers.

Centrally administered urocortin 2 decreases gorging on high-fat diet in both diet-induced obesity-prone and -resistant rats.

OBJECTIVE: Obesity is a costly, deadly public health problem for which new treatments are needed. Individual differences in meal pattern have been proposed to have a role in obesity risk. The present study tested the hypothesis that (i) the microstructure of chronic high-fat diet intake differs between genetically selected diet-induced obesity (DIO) and diet-resistant (DR) rats, and (ii) central administration of urocortin 2 (Ucn 2), a corticotropin-releasing factor type 2 agonist, decreases high-fat diet intake not only in lean DR rats, but also in obese DIO rats. DESIGN: Male, selectively bred DIO and DR rats (n=10/genotype) were chronically fed a high-fat diet. Food and water intake as well as ingestion microstructure were then compared under baseline conditions and following third intracerebroventricular injection of Ucn 2 (0, 0.1, 0.3, 1, 3 µg). RESULTS: Irrespective of genotype, Ucn 2 reduced nocturnal food intake with a minimum effective dose of 0.3 µg, suppressing high-fat diet intake by ∼40% at the 3 µg dose. Ucn 2 also made rats of both genotypes eat smaller and briefer meals, including at doses that did not reduce drinking. Obese DIO rats ate fewer but larger meals than DR rats, which they ate more quickly and consumed with two-third less water. CONCLUSIONS: Unlike leptin and insulin, Ucn 2 retains its full central anorectic efficacy to reduce high-fat diet intake even in obese, genetically prone DIO rats, which otherwise show a 'gorging' meal pattern. These results open new opportunities of investigation toward treating some forms of DIO.

The use of high dose d,l-leucovorin in first-line bevacizumab+mFOLFIRI treatment of patients with metastatic colorectal cancer may enhance the antiangiogenic effect of bevacizumab.

The role of d,l-leucovorin (d,l-LV) dose on efficacy and toxicity of first-line bevacizumab+mFOLFIRI or mFOLFIRI treatment has never been investigated in patients with metastatic colorectal cancer. This study was an investigator initiated retrospective observational investigation performed on 450 consecutive patients. The mFOLFIRI regimen consisted of irinotecan (180 mg/m(2)), d,l-LV low (200 mg/m(2)) or high (400 mg/m(2)) dose and bolus 5-fluorouracil (5-FU) (400 mg/m(2)), followed by a 46-h infusion of 5-FU (2400 mg/m(2)). The bevacizumab+mFOLFIRI regimen consisted of bevacizumab (5 mg/kg)+mFOLFIRI. The efficacy (objective response [OR], progression-free [PFS] and overall survival [OS]) and toxicity was evaluated and compared. The use of high versus low dose d,l-LV in bevacizumab+mFOLFIRI regimen improved the OR rate (63 and 38 %, respectively; P = 0.00015), median PFS (13 and 9 months, respectively; P = 0.000005) and median OS (26 and 21 months, respectively; P = 0.0058). The efficacy of mFOLFIRI and the toxicity pattern of both bevacizumab+mFOLFIRI and mFOLFIRI regimens were independent of d,l-LV dose. Beside the d,l-LV dose the bevacizumab-related hypertension was an independent marker of longer survival. The use of high d,l-LV dose in bevacizumab+mFOLFIRI regimen would enhance the antiangiogenic effect of bevacizumab and subsequently the efficacy of treatment without increasing the number of adverse events. These findings need to be further confirmed in a randomized controlled prospective trial.

Plasmodium chabaudi AS induces pregnancy loss in association with systemic pro-inflammatory immune responses in A/J and C57BL/6 mice.

The molecular mechanisms that underlie poor birth outcomes in malaria during pregnancy remain poorly defined. To assess the role of host immune responses, mice known to respond differentially to Plasmodium chabaudi AS infection were studied. Following infection at day 0 of pregnancy, A/J mice developed significantly higher parasitemia than C57BL/6 (B6) mice and succumbed to infection. Both strains had evidence of parasite accumulation in the placenta at mid-gestation and aborted, with significantly higher embryo loss in infected A/J mice on day 9. While infection-induced systemic tumour necrosis factor (TNF) and interleukin (IL)-1ß in the latter were significantly higher at day 11, day 10 IL-10 levels were higher in B6 mice. No differences in the levels of splenic lymphocyte subsets, neutrophils or monocytes between infected pregnant A/J and B6 mice were observed, with most cell types expanding in response to infection regardless of pregnancy. Antibody ablation of TNF exacerbated infection in A/J mice and did not ameliorate pregnancy outcome. Thus, malaria induces poor pregnancy outcome in both the mouse strains in the context of quantitatively different systemic inflammatory responses. Further evaluation of the roles of soluble and cellular immune components, particularly at the uteroplacental level, will be required to define the most critical pregnancy-compromising mechanisms.

Decreased blood catalase activity is not related to specific beta-thalassemia mutations in Hungary.

INTRODUCTION: Thalassemia erythrocytes are exposed to oxidative stress especially to hydrogen peroxide, which is regulated with the enzyme catalase. The aim of this study was to examine blood catalase activity and the relationship of blood catalase and beta-thalassemia gene mutations. METHODS: Blood catalase activity, hemoglobin, HbA(2) , HbF, and beta-globin gene mutations were determined in 43 Hungarian patients with beta-thalassemia trait. RESULTS: Compared to controls, the beta-thalassemia trait patients showed a low mean (P < 0.001) of blood catalase (men: 84 ± 29 MU/L vs. sex-matched controls: 118 ± 18 MU/L and women: 74 ± 18 MU/L vs. 108 ± 114 MU/L) and a low mean of blood catalase-to-blood hemoglobin ratio (men: 0.72 ± 0.22 MU/g vs. 0.85 ± 0.12 MU/g, women: 0.77 ± 0.26 MU/g vs. 0.84 ± 0.11 MU/g). The HbA(2) determination showed high sensitivity and specificity for the detection of beta-thalassemia trait patients. Mutation analyses revealed 13 beta-thalassemia trait mutations, of which six have not been reported before in Hungarian beta-thalassemia trait patients. Each group of mutations revealed decreased (P < 0.01) mean of blood catalase and catalase-to-hemoglobin ratio. Acatalasemia mutations were not found in beta-thalassemia trait patients. CONCLUSION: The decrease in blood catalase activity might be due to the damaging effects of free radicals on the catalase protein. Consequently, these beta-thalassemia trait patients may be relatively susceptible to damage caused by oxidative stress.

Clindamycin release determined by high performance liquid chromatography from a novel low-cost local drug delivery system: a new potential treatment option for chronic osteomyelitis.

Osteomyelitis continues to be a severe problem worldwide, causing plenty of hospital admissions and entailing vast expenses. Previously, we developed a low-cost polymethyl-methacrylate (PMMA)-sorbitol based capsule system for local long-term drug delivery. In the present study we aimed to test the in vitro release of clindamycin capsules by high performance liquid chromatography. By the end of the clinically relevant period (42 days), the capsules released 70-100% of their load. Furthermore, the release kinetics suggested that an effective antimicrobial concentration may be maintained within the target area. Our findings indicate that these newly developed capsules may be a versatile device for local clindamycin delivery by providing efficient release and reducing financial burdens.

Calcein assay: a high-throughput method to assess P-gp inhibition.

Transporter mediated drug-drug interactions (tDDI) mediated by ABCB1 have been shown to be clinically relevant. Hence, the assessment of the ABCB1 tDDI potential early in the drug development process has gained interest. We have evaluated the Calcein assay as a means of assessing the ABCB1 tDDI that is amenable to high throughout and compared it with the monolayer efflux assay. We found the Calcein assay, when performed in K562MDR cells using the protocol originally published more sensitive than digoxin transport inhibition in MDCKII-MDR1 cells. Application of the Calcein assay to cell lines containing different amounts of ABCB1, yielded IC(50) values that varied 10-100-fold. The differences observed for IC(50) values for the same compounds were in the following rank order: IC(50, MDCKII-MDR1) >IC(50, K562MDR)>IC(50, hCMEC/D3). Higher IC(50) values were obtained in cells with higher ABCB1 expression. The Calcein assay is a high-throughput alternative to digoxin transport inhibition as it appears to have a comparable selectivity but higher sensitivity than previously published digoxin transport inhibition in MDCKII-MDR1 cells. In addition, it can be performed in a barrier-specific manner highlighting the dependence of ABCB1 IC(50) values on different ABCB1 expression levels.

No compelling evidence that sibutramine prolongs life in rodents despite providing a dose-dependent reduction in body weight.

OBJECTIVE: The health and longevity effects of body weight reduction resulting from exercise and caloric restriction in rodents are well known, but less is known about whether similar effects occur with weight reduction from the use of a pharmaceutical agent such as sibutramine, a serotonin-norepinephrine reuptake inhibitor. RESULTS AND CONCLUSION: Using data from a 2-year toxicology study of sibutramine in Sprague-Dawley CD rats and CD-1 mice, despite a dose-dependent reduction in food intake and body weight in rats compared with controls, and a body weight reduction in mice at the highest dose, there was no compelling evidence for reductions in mortality rate.

Identification of virus resistant tumor cell subpopulations in three-dimensional uveal melanoma cultures.

To better understand melanoma resistance to herpes simplex virus type 1 (HSV-1)-mediated oncolysis, traditional two-dimensional (2D) cultures and extracellular matrix (ECM) containing three-dimensional (3D) cultures of OCM1 and C918 uveal melanoma cells were infected with an HSV-1 strain that expresses the green fluorescent protein (GFP) marker during replication. Although 2D cultures were completely destroyed within a few days of HSV-1 inoculation, viable GFP-negative tumor cells remained detectable in 3D cultures for several weeks. Tumor cells with increased resistance to HSV-1 included cells that formed vasculogenic mimicry patterns and multicellular spheroids and cells that invaded Matrigel individually. Mechanisms of tumor resistance against HSV-1 in the 3D environment included impaired virus spread in the ECM and ECM-mediated inhibition of viral replication after viral entry into tumor cells. Observations also suggested that HSV-1 established quiescent infection in some tumor cells present in multicellular spheroids and that this could revert to productive viral infection when the tumor growth pattern changed. These findings indicate that 3D tumor cell cultures can be used to identify distinct tumor cell populations with increased resistance to HSV-1 and to explore mechanisms of ECM-mediated tumor resistance to oncolytic virotherapy.

Therapeutic potential of genetically modified adult stem cells for osteopenia.

Adult stem cells have therapeutic potential because of their intrinsic capacity for self-renewal, especially for bone regeneration. The present study shows the utility of ex vivo modified mesenchymal stem cells (MSC) to enhance bone density in an immunocompetent mouse model of osteopenia. MSC were transduced ex vivo with a recombinant adeno-associated virus 2 (rAAV2) expressing bone morphogenetic protein 2 (BMP2) under the transcriptional control of collagen type-1alpha promoter. To enrich bone homing in vivo, we further modified the cells to transiently express the mouse alpha4 integrin. The modified MSC were systemically administered to ovariectomized, female C57BL/6 mice. Effects of the therapy were determined by dual-energy X-ray absorptiometry, 3D micro-CT, histology and immunohistochemistry for up to 6 months. Results indicated that mice transplanted with MSC expressing BMP2 showed significant increase in bone mineral density and bone mineral content (P < 0.001) with relatively better proliferative capabilities of bone marrow stromal cells and higher osteocompetent pool of cells compared to control animals. Micro-CT analysis of femora and other bone histomorphometric analyses indicated more trabecular bone following MSC-BMP2 therapy. Results obtained by transplanting genetically modified MSC from green fluorescent protein transgenic mouse suggested that production of BMP2 from transplanted MSC also influenced the mobilization of endogenous progenitors for new bone formation.

Interaction of macrocyclic lactones with the multidrug transporters: the bases of the pharmacokinetics of lipid-like drugs.

Like most drugs, macrocyclic lactone endectocides (MLs) exert their antiparasitic effects within the defined target tissues where parasites are located, and whose drug concentrations correlate with those in the plasma compartment. The process of drug distribution to the active site constitutes the link in the pharmacokinetic/pharmacodynamic relationship. In the past few years it has become evident that transporter proteins play a major role in regulating the distribution, elimination and metabolism of the antiparasitic macrocyclic lactones. The efflux transporter P-glycoprotein (P-gp) has received the most attention with regards to its strong interaction with ivermectin and other MLs. P-gp has been reported to be involved in restricting the absorption of these drugs, in enhancing their intestinal elimination, in the protection against their neurotoxicity and in the ML resistance mechanisms in parasites. This review focuses on the interaction of MLs with P-glycoprotein and with other multidrug resistance transporters. Given the structural and physicochemical diversity of these drugs, they constitute models of interest to study the major molecular determinants for the interaction with transporters. We will discuss the consequences of such interactions on ML pharmacokinetics and the possibility of benefiting from of drug/drug interaction to reverse multidrug resistance in several therapeutic fights such as against parasites and tumors.

Leflunomide and its metabolite A771726 are high affinity substrates of BCRP: implications for drug resistance.

BACKGROUND: Earlier publications have suggested a possible role for the efflux transporter breast cancer resistance protein (BCRP) in acquired resistance to disease-modifying antirheumatic drugs (DMARDs) such as leflunomide and its metabolite A771726 (teriflunomide). However, there is no direct evidence that BCRP interacts with these drugs. OBJECTIVES: To characterise the interaction between BCRP transporter and leflunomide and its active metabolite A771726, with emphasis on the nature of the interaction (substrate or inhibitor) and the kinetic characterisation of the interactions. METHODS: Different in vitro membrane-based methods (ATPase and vesicular transport assay) using BCRP-HAM-Sf9 membrane preparations and cellular assays (Hoechst assay and cytotoxicity assay) were performed on PLB985-BCRP and HEK293-BCRP cell lines overexpressing BCRP. RESULTS: In all assays used, an interaction between the investigated drugs and BCRP was detected. In the vesicular transport assay, both leflunomide and its metabolite inhibited BCRP-mediated methotrexate transport. Both compounds are likely substrates of BCRP as shown by the vanadate-sensitive ATPase assay. In line with the membrane assays, leflunomide and A771726 inhibited BCRP-mediated Hoechst efflux from PLB985-BCRP cells. In the cytotoxicity assay, overexpression of BCRP conferred 20.6-fold and 7.5-fold resistance to HEK293 cells against leflunomide and A771726, respectively. The resistance could be reversed by Ko134, a specific inhibitor of BCRP. CONCLUSION: Based on these results, BCRP could play an important role in the resistance to leflunomide and A771726 via interactions with these drugs. BCRP may also mediate drug-drug interactions when leflunomide is administered with other BCRP substrate drugs such as methotrexate.