Metabolic parameters in type 2 diabetic patients with varying degrees of glycemic control during Ramadan: An observational study.

AIMS/INTRODUCTION: The changes in metabolic parameters in type 2 diabetic patients who fast during Ramadan have not been studied in Singapore. This study aimed to examine the trends of glycated hemoglobin (HbA1c), systolic blood pressure, low-density lipoprotein cholesterol, and triglycerides in diabetic patients with varying degrees of glycemic control and different types of therapeutic approaches during Ramadan. METHODS: The present retrospective study used a national electronic database to examine the metabolic parameter of Malay patients with type 2 diabetes. Eligible patients were stratified into three groups based on their mean HbA1c control before Ramadan: group 1 (HbA1c ≥10%), group 2 (HbA1c 7.1-9.9%) and group 3 (HbA1c ≤7%). Patients with a glomerular filtration rate <15 mL/min were excluded. The trends of metabolic parameters were traced before, during and after Ramadan. RESULTS: Of 13,565 patients examined, 5,172 patients (38.1%) were eligible for this study. Mean change of HbA1c varied from -1.4% to +0.2% during Ramadan, with the greatest reduction observed in group 1 (P < 0.001). A minimal systolic blood pressure reduction was observed in groups 2 and 3 (2 mmHg; P < 0.01). Low-density lipoprotein cholesterol and triglycerides changes were insignificant. A small, 0.1%, reduction in mean HbA1c was observed in patients taking oral antidiabetic agents during Ramadan (P < 0.001). CONCLUSIONS: Blood glucose was most affected during Ramadan, particularly in patients with mean baseline HbA1c ≥10%. The type of antidiabetic agent used did not seem to contribute to glycemic changes.

Effect of type and ratio of solubilising polymer on characteristics of hot-melt extruded orodispersible films.

In formulating an orodispersible film (ODF), it is important for polymer choice to strike a balance between mechanical properties and release rates. Studies have been done to study polymer combinations. However, there is a lack of a systematic study to determine key factors affecting these properties. We studied the effect of varying the ratios of a solubilising polymer (Kollidon(®) VA 64 or Soluplus(®)) to a film forming polymer, hydroxypropyl cellulose (HPC), on mechanical properties and release rates of hot-melt extruded ODFs using a 2(3) factorial design. The two drugs evaluated were chlorpheniramine and indomethacin. The main effects impacting mechanical properties were the drug and two-way interaction between drug and solubilising polymer. For dissolution, the main effects were the solubilising polymer; the drug; and the two-way interaction between solubilising polymer and ratio of solubilising to film forming polymer. Both drugs exhibited plasticising effects on the polymer matrix and had higher film ductility and lower film stiffness. Kollidon(®) VA 64-containing films performed better in terms of drug release whereas Soluplus(®)-containing films had better mechanical properties. The dissolution rate can be improved by decreasing film thickness. The findings of our study will be crucial to forming a robust ODF formulation.

Development of controlled release inhalable polymeric microspheres for treatment of pulmonary hypertension.

Pulmonary hypertension (PAH) is a condition of the lungs characterised by an elevated arterial pressure and increased vascular resistance. Existing medications have to be administered frequently, resulting in non compliance by patients. Little work has been reported to date where microspheres have been developed to control the release of drug for treatment of pulmonary hypertension. To transcend this drawback, controlled release microspheres were formulated to minimise the number of doses required for treatment of PAH. Nifedipine and polyvinyl alcohol (PVA) were used as the model drug and release modifier respectively. Microspheres were developed by varying the PVA concentration using the spray drying technique. The formulated microspheres were characterised in terms of particle size, morphology, crystallinity, interaction between PVA and nifedipine via Fourier transformed infra-red spectroscopy (FTIR) and differential scanning calorimetry (DSC), in vitro release profile by employing the United States Pharmacopeia Apparatus type II and in vitro aerosolisation profile by using multi-stage liquid impinger (MSLI). The toxicity of PVA on lung epithelial cells was tested using human alveolar basal epithelium A549 cell line. From the data, it was observed that the microspheres were within the inhalable range (1-10 µm) with spherical morphology. The X-ray diffraction demonstrated that the microspheres were amorphous. There was no interaction between PVA and nifedipine during the formation of microspheres as seen by FTIR. The in vitro release profile showed a burst release followed by controlled release. A more prolonged release can be achieved by increasing the PVA:nifedipine ratio. In vitro aerosolisation showed that the Fine Particle Fractionemitted of the microspheres is greater than 20%, which is similar to that of marketed inhalation formulations. PVA was found to have insignificant effect on cell viability after 48 h of exposure to A549 cell line. In conclusion, microspheres of nifedipine and PVA, prepared by spray drying were found to exhibit suitable properties to achieve controlled release by the inhalation route.

Metabolically functional hepatocyte-like cells from human umbilical cord lining epithelial cells.

The primary hepatocyte is the best benchmark for drug biotransformation studies. However, due to the severe shortage of primary hepatocytes, there is a need for alternative reliable cell source. This study aims to isolate multipotent epithelial cells from the umbilical cord, differentiate these cells into hepatocyte-like cells (HLCs), and investigate the potential of using the differentiated cells for in vitro drug metabolism model. Human umbilical cord lining epithelial cells (UCLECs) were subjected to hepatic induction over a period of 28 days. HepG2 and cryopreserved human hepatocytes were used as control. Immunohistological staining was carried out for α-fetoprotein (AFP), albumin, cytokeratin 18 (CK18), and 19 (CK19). Glycogen storage ability was assessed through periodic acid-Schiff stain. Reverse transcription polymerase chain reaction was performed to examine gene expression of hepatic nuclear factor 4α (HNF4α) and cytochrome P450 isozymes 1A2, 2C9, 2D6, and 3A4. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) was utilized to analyze functional metabolic ability of the HLCs, where CYP3A4 was chosen as the study focus and testosterone as the drug substrate. After 28 days of induction, the fibroblastic phenotype of UCLECs changed to rotund polygonal shape resembling that of hepatocytes. Protein expression of AFP and CK19 was negative, while albumin and CK18 expression was upregulated. Gene expression of HNF4α, CYP1A2, CYP2D6, and CYP3A4 was observed but not for CYP2C9. After 4 h of incubation with testosterone, UPLC/MS/MS detected 2α-, 6ß-, 15ß-, and 16ß-hydroxytestosterone. UCLECs are able to differentiate into HLCs that express liver-specific markers, and have functional metabolic capabilities.

Protein encapsulation in polymeric microneedles by photolithography.

BACKGROUND: Recent interest in biocompatible polymeric microneedles for the delivery of biomolecules has propelled considerable interest in fabrication of microneedles. It is important that the fabrication process is feasible for drug encapsulation and compatible with the stability of the drug in question. Moreover, drug encapsulation may offer the advantage of higher drug loading compared with other technologies, such as drug coating. METHODS AND RESULTS: In this study, we encapsulated a model protein drug, namely, bovine serum albumin, in polymeric microneedles by photolithography. Drug distribution within the microneedle array was found to be uniform. The encapsulated protein retained its primary, secondary, and tertiary structural characteristics. In vitro release of the encapsulated protein showed that almost all of the drug was released into phosphate buffered saline within 6 hours. The in vitro permeation profile of encapsulated bovine serum albumin through rat skin was also tested and shown to resemble the in vitro release profile, with an initial release burst followed by a slow release phase. The cytotoxicity of the microneedles without bovine serum albumin was tested in three different cell lines. High cell viabilities were observed, demonstrating the innocuous nature of the microneedles. CONCLUSION: The microneedle array can potentially serve as a useful drug carrier for proteins, peptides, and vaccines.

Synergistic effects of suberoylanilide hydroxamic acid combined with cisplatin causing cell cycle arrest independent apoptosis in platinum-resistant ovarian cancer cells.

Histone deacetylase inhibitors (HDACIs) belong to an emerging class of anticancer compounds. It is increasingly recognized that their unique and complementary mode of action make HDACIs valuable agents in augmenting the cytotoxicity of conventional chemotherapeutics. We examined the potential for combined use of an approved HDACI, suberoylanilide hydroxamic acid (SAHA), with cisplatin (Cddp) in platinum-resistant ovarian cancer cells, OVCAR-3 and SKOV-3. The nature of the drug interaction following combinatory therapy was assessed using median effect analysis. We found that SAHA acted synergistically with Cddp over a wide range of concentrations in both cell types, resulting in favorable dose reductions of both compounds. In particular, in the more Cddp-resistant SKOV-3 cells, more than 8-fold dose reduction of Cddp was achieved with the simultaneous use of SAHA and Cddp as compared to the dose required to elicit similar cell kill effects using Cddp alone. More importantly, therapeutic selectivity for ovarian cancer cells over normal fibroblast cells were maintained with the combinatorial therapy. We further observed that the augmentation of Cddp-induced cell death was mediated by the net increase in apoptosis and was independent of cell cycle arrest. Overall, concurrent application of SAHA and Cddp yielded the most favorable cell kill, indicating that this combination is promising for treatment of platinum-resistant ovarian tumors.

Method development and validation for rapid quantification of hydroxychloroquine in human blood using liquid chromatography-tandem mass spectrometry.

A novel and specific liquid chromatography-tandem mass spectrometric method (LC-MS/MS) was developed and validated for the quantification of hydroxychloroquine in human blood using its stable labeled isotope, hydroxychloroquine-d4 as the internal standard. Chromatographic separation of analytes was achieved using an Agilent ZORBAX Eclipse XDB - C8 analytical HPLC column (50 mm × 2.1 mm, 5 µm). The mobile phase comprising water containing 0.1% formic acid-acetonitrile (94:6, v/v) was delivered isocratically at a flow rate of 0.5 mL/min. The column effluent was detected by API 4000 triple quadrupole mass spectrometer using electrospray ionization (ESI) and monitored by multiple reaction monitoring with positive mode. The precursor to product ion transitions of m/z 336 → 247 and m/z 340 → 251 were used to measure the analyte and IS, respectively. The assay demonstrated a good linear dynamic range of 5-2000 ng/mL for hydroxychloroquine in human blood, with coefficient of determination (r(2)) of =0.9999. The values for intra-day and inter-day precisions of hydroxychloroquine were ≤ 7.86% with the accuracies ranged from 93.8% to 107.6%. The chromatographic run time was 3 min, making it possible to achieve a high throughput analysis. This method was used as a bio-analytical tool in a phase I clinical trial to quantify blood hydroxychloroquine concentrations in patients with non-small cell lung cancer receiving both hydroxychloroquine and gefitinib in their treatment.

Microarray analysis revealed dysregulation of multiple genes associated with chemoresistance to As(2)O(3) and increased tumor aggressiveness in a newly established arsenic-resistant ovarian cancer cell line, OVCAR-3/AsR.

The potential of arsenic trioxide (As(2)O(3)) for use as a novel therapy for ovarian cancer treatment has been increasingly recognized. In this study, we developed an arsenic-resistant OVCAR-3 subline (OVCAR-3/AsR) and aimed to identify the molecular mechanisms and signaling pathways contributing to the development of acquired arsenic chemoresistance in ovarian cancer. OVCAR-3/AsR cells were obtained following continual exposure of parental OVCAR-3 cells to low dose As(2)O(3) for 12months. Cytotoxicity of OVCAR-3/AsR cells to As(2)O(3), paclitaxel and cisplatin was investigated. Cell apoptosis and cell cycle distribution following As(2)O(3) treatment of OVCAR-3/AsR cells was also analyzed using flow cytometry. Subsequently, cDNA microarray analysis was performed from the RNA samples of OVCAR-3 and OVCAR-3/AsR cells in duplicate experiments. Microarray data were analyzed using Genespring® and Pathway Studio® Softwares. OVCAR-3/AsR cells showed 9-fold greater resistance to As(2)O(3) and lack of collateral resistance to cisplatin and paclitaxel. Compared with parental OVCAR-3 cells, OVCAR-3/AsR had significantly lower apoptotic rates following As(2)O(3) treatment. These cells were also arrested at both the S phase and G(2)/M phase of the cell cycle after exposure to high concentrations of As(2)O(3). Gene expression profiling revealed significant differences in expression levels of 397 genes between OVCAR-3/AsR and OVCAR-3 cells. The differentially regulated transcripts genes have functional ontologies related to continued cancer cell growth, cell survival, tumor metastasis and tumor aggressiveness. Additionally, numerous gene targets of the nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor showed elevated expression in OVCAR-3/AsR cells. Subsequent pathway analysis further revealed a gene network involving interleukin 1-alpha (IL1A) in mediating the arsenic-resistant phenotype. These results showed that changes in multiple genes and an increased in tumor aggressiveness occurred during the development of acquired chemoresistance to As(2)O(3) in ovarian cancer cells. The functional relevance of these genetic changes should be validated in future studies.

Rapid determination of gefitinib and its main metabolite, O-desmethyl gefitinib in human plasma using liquid chromatography-tandem mass spectrometry.

A novel, rapid and specific liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous quantification of gefitinib and its predominant metabolite, O-desmethyl gefitinib in human plasma. Chromatographic separation of analytes was achieved on an Alltima C18 analytical HPLC column (150 mm × 2.1 mm, 5 µm) using an isocratic elution mode with a mobile phase comprised acetonitrile and 0.1% formic acid in water (30:70, v/v). The flow rate was 300 µL/min. The chromatographic run time was 3 min. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionization (ESI) in positive mode. Linearity was demonstrated in the range of 5-1000 ng/mL for gefitinib and 5-500 ng/mL for O-desmethyl gefitinib. The intra- and inter-day precisions for gefitinib and O-desmethyl gefitinib were ≤10.8% and the accuracies ranged from 89.7 to 104.7% for gefitinib and 100.4 to 106.0% for O-desmethyl gefitinib. This method was used as a bioanalytical tool in a phase I clinical trial to investigate the possible effect of hydroxychloroquine on the pharmacokinetics of gefitinib. The results of this study enabled clinicians to ascertain the safety of the combination therapy of hydroxychloroquine and gefitinib in patients with advanced (Stage IIIB-IV) non-small cell lung cancer (NSCLC).

Differential augmentative effects of buthionine sulfoximine and ascorbic acid in As2O3-induced ovarian cancer cell death: oxidative stress-independent and -dependent cytotoxic potentiation.

The potential of arsenic trioxide (As2O3) as a novel therapy against ovarian cancer has been progressively recognized. Its prospective usefulness for treatment of this malignancy either alone or in combination with other chemotherapeutic agents has been increasingly explored. In this study, we attempted to enhance the cytotoxicity of As2O3 in ovarian cancer cells through manipulation of cellular glutathione (GSH) levels using either buthionine sulfoximine (BSO) or ascorbic acid (AA). Results from our studies showed that combinatorial therapies using As2O3 with either low dose BSO or only pharmacological doses of AA acted synergistically to enhance the cytotoxicity of As2O3 in ovarian tumor cells. With these regimens, therapeutic selectivity was observed with preferential killing of ovarian tumor cells over normal fibroblast controls. Furthermore, contrary to previous reports, enhancement of As2O3-mediated cell killing by these two agents was propagated through different effects. With BSO, apoptotic and non-apoptotic cell death enhancement were mediated through increased arsenic accumulation and GSH depletion that occurred independently of reactive oxygen species. With pharmacological doses of AA, increase in cell death proceeded through non-apoptotic routes via an oxidative stress-related pathway independent of GSH levels. Taken together, these results indicate that GSH depleting agents or pro-oxidative chemicals have capabilities of improving the utility of As2O3 in ovarian cancer management.

Comparison of the in vitro and in vivo effects of retinoids either alone or in combination with cisplatin and 5-fluorouracil on tumor development and metastasis of melanoma.

PURPOSE: Retinoids have previously been reported to inhibit proliferation of melanoma cell lines in vitro. However, the relative antimetastatic efficacy of various retinoids on melanoma in vivo is unknown. Therefore, we investigated the effects of different retinoids on the invasion and metastasis of murine melanoma B16-F10 cells in vitro and in vivo. Based on the findings, the antitumor effects of a selected retinoid either alone or in combination with cisplatin were also investigated in a preclinical mouse melanoma model. METHODS: Cell proliferation and invasion analyses of murine melanoma B16-F10 cells were assessed in the presence of different retinoids, either alone or in combination with cisplatin (CDDP) or 5-fluorouracil (5-FU). Experimental lung metastasis assay was performed in this study to investigate the antimetastatic efficacy of retinoids. Additionally, a mouse melanoma model was used to assess the antitumor efficacy of a selected retinoid in combination with cisplatin. RESULTS: Retinoids showed significant antiproliferation and anti-invasion effects on murine melanoma B16-F10 cells. Pretreatment with retinoids increased the sensitivity to CDDP but not to 5-FU in in-vitro. Moreover, the number of metastatic colonies formed in the lungs of mice injected intravenously with B16-F10 cells was significantly reduced by injecting the respective retinoid once a day for 10 days. Treatment with a combination of cisplatin and 13-cis-retinoic acid resulted in a significant reduction in primary tumor size and the number of lung metastatic nodules in melanoma-bearing mice. CONCLUSION: These results suggest that retinoids not only exhibit antimetastatic effect, but also enhance the antitumor activity of cisplatin in vivo.

Efficient intracellular delivery of functional proteins using cationic polymer core/shell nanoparticles.

Cationic core/shell nanoparticles self-assembled from biodegradable, cationic and amphiphilic copolymer poly{N-methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] sebacate}, P(MDS-co-CES), were fabricated and employed to deliver lectin A-chain, an anticancer glycoprotein. Lectin A-chain was efficiently bound onto the surfaces of the nanoparticles at high mass ratios of nanoparticles to lectin A-chain. The nanoparticle/lectin A-chain complexes had an average size of approximately 150 nm with zeta potential of about +30 mV at the mass ratio of 50 or above while the BioPorter/lectin A-chain complexes had a larger particle size and relatively lower zeta potential (150 nm vs. 455 nm; +30 mV vs. +20 mV). Therefore, the cellular uptake of nanoparticle/lectin A-chain complexes was much greater than that of BioPorter/lectin A-chain complexes. The results obtained from cytotoxicity tests show that lectin A-chain delivered by the nanoparticles was significantly more toxic against MDA-MB-231, HeLa, HepG2 and 4T1 cell lines when compared to BioPorter, and IC50 of lectin A-chain delivered by the nanoparticles was 0.2, 0.5, 10 and 50 mg/l, respectively, while that of lectin A-chain delivered by BioPorter was higher than 100 mg/l in all cell lines tested. These nano-sized particles may provide an efficient approach for intracellular delivery of biologically active proteins.

Biopharmaceutics of 13-cis-retinoic acid (isotretinoin) formulated with modified beta-cyclodextrins.

13-cis-Retinoic acid (13-cis-RA), also known as isotretinoin, is commonly used in the management of severe acne. Its clinical efficacy in oncology has also been documented. As a vitamin A derivative, it is not soluble in water. This solubility barrier not only affects its oral absorption but also makes parenteral delivery difficult. Recently, water-soluble formulations of 13-cis-RA have been attempted with 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and randomly methylated-beta-cyclodextrin (RM-beta-CD). In this study, the pharmacokinetic profiles of these two formulations were assessed in Sprague-Dawley rats after single intravenous or oral administration. We found that 13-cis-RA was eliminated from the body through a dose-independent process after intravenous injection of either sodium salt or the HP-beta-CD formulation within the tested dosage range (2.0-7.5mg/kg). Furthermore, HP-beta-CD did not alter the kinetic profile of 13-cis-RA after intravenous administration in comparison with 13-cis-RA sodium salt. We also found that RM-beta-CD dramatically enhanced the oral absorption of 13-cis-RA. At 10.0mg/kg, the bioavailability of 13-cis-RA formulated with RM-beta-CD was about three-fold higher than that of the control (13-cis-RA suspended in 0.5% carboxymethylcellulose (CMC)). Similarly, the oral absorption of 13-cis-RA was not saturated within our tested range (2.5-10.0mg/kg) and the bioavailability remained unchanged. These results demonstrated that HP-beta-CD and RM-beta-CD were suitable excipients for the delivery of 13-cis-RA.

St. John's wort attenuates irinotecan-induced diarrhea via down-regulation of intestinal pro-inflammatory cytokines and inhibition of intestinal epithelial apoptosis.

Diarrhea is a common dose-limiting toxicity associated with cancer chemotherapy, in particular for drugs such as irinotecan (CPT-11), 5-fluouracil, oxaliplatin, capecitabine and raltitrexed. St. John's wort (Hypericum perforatum, SJW) has anti-inflammatory activity, and our preliminary study in the rat and a pilot study in cancer patients found that treatment of SJW alleviated irinotecan-induced diarrhea. In the present study, we investigated whether SJW modulated various pro-inflammatory cytokines including interleukins (IL-1beta, IL-2, IL-6), interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) and intestinal epithelium apoptosis in rats. The rats were treated with irinotecan at 60 mg/kg for 4 days in combination with oral SJW or SJW-free control vehicle at 400 mg/kg for 8 days. Diarrhea, tissue damage, body weight loss, various cytokines including IL-1beta, IL-2, IL-6, IFN-gamma and TNF-alpha and intestinal epithelial apoptosis were monitored over 11 days. Our studies demonstrated that combined SJW markedly reduced CPT-11-induced diarrhea and intestinal lesions. The production of pro-inflammatory cytokines such as IL-1beta, IFN-gamma and TNF-alpha was significantly up-regulated in intestine. In the mean time, combined SJW significantly suppressed the intestinal epithelial apoptosis induced by CPT-11 over days 5-11. In particular, combination of SJW significantly inhibited the expression of TNF-alpha mRNA in the intestine over days 5-11. In conclusion, inhibition of pro-inflammatory cytokines and intestinal epithelium apoptosis partly explained the protective effect of SJW against the intestinal toxicities induced by irinotecan. Further studies are warranted to explore the potential for STW as an agent in combination with chemotherapeutic drugs to lower their dose-limiting toxicities.

Pharmacokinetic mechanisms for reduced toxicity of irinotecan by coadministered thalidomide.

The clinical use of irinotecan (CPT-11) is hindered by dose-limiting diarrhea and myelosuppression. Recent clinical studies indicate that thalidomide, a known tumor necrosis factor-alpha inhibitor, ameliorated the toxicities induced by CPT-11. However, the mechanisms for this are unknown. This study aimed to investigate whether combination of thalidomide modulated the toxicities of CPT-11 using a rat model and the possible role of the altered pharmacokinetic component in the toxicity modulation using in vitro models. The toxicity model was constructed by treatment of healthy rats with CPT-11 at 60 mg/kg per day by intravenous (i.v.) injection. Body weight, acute and delayed-onset diarrhea, blood cell counts, and macroscopic and microscopic intestinal damages were monitored in rats treated with CPT-11 alone or combined therapy with thalidomide at 100 mg/kg administered by intraperitoneal (i.p.) injection. Single dose and 5-day multiple-dose studies were conducted in rats to examine the effects of concomitant thalidomide on the plasma pharmacokinetics of CPT-11 and its major metabolites SN-38 and SN-38 glucuronide (SN-38G). The effect of CPT-11 on thalidomide's pharmacokinetics was also checked. Rat liver microsomes and a rat hepatoma cell line, H4-II-E cells, were used to study the in vitro metabolic interactions between these two drugs. H4-II-E cells were also used to investigate the effect of thalidomide and its hydrolytic products on the transport of CPT-11 and SN-38. In addition, the effect of thalidomide and its hydrolytic products on rat plasma protein binding of CPT-11 and SN-38 was examined. Administration of CPT-11 by i.v. for 4 consecutive days to rats induced significant body weight loss, decrease in neutrophil and lymphocyte counts, severe acute- and delayed-onset diarrhea, and intestinal damages. These toxicities were alleviated when CPT-11 was combined with thalidomide. In both single-dose and 5-day multiple-dose pharmacokinetic study, coadministered thalidomide significantly increased the area under the plasma concentration-time curve (AUC) of CPT-11, but the AUC and elimination half-life (t(1/2)) of SN-38 were significantly decreased. However, CPT-11 did not significantly alter the pharmacokinetics of thalidomide. Thalidomide at 25 and 250 microM and its hydrolytic products at a total concentration of 10 microM had no significant effect on the plasma protein binding of CPT-11 and SN-38, except for that thalidomide at 250 microM caused a significant increase in the unbound fraction (f(u)) of CPT-11 by 6.7% (P < 0.05). The hydrolytic products of thalidomide (total concentration of 10 microM), but not thalidomide, significantly decreased CPT-11 hydrolysis by 16% in rat liver microsomes (P < 0.01). The formation of both SN-38 and SN-38G from CPT-11, SN-38 glucuronidation, or intracellular accumulation of both CPT-11 and SN-38 in H4-II-E cells followed Michaelis-Menten kinetics with the one-binding site model being the best fit for the kinetic data. Coincubation or 2-hr preincubation of thalidomide at 25 microM and 250 microM and its hydrolytic products at 10 microM did not show any significant effects on CPT-11 hydrolysis and SN-38 glucuronidation. However, preincubation of H4-II-E cells with thalidomide (250 microM), its hydrolytic products (total concentration of 10 microM), or phthaloyl glutamic acid (one major thalidomide hydrolytic product, 10 microM) significantly increased the intracellular accumulation of SN-38, but not CPT-11 (P < 0.01). The dose-limiting toxicities of CPT-11 were alleviated by combination with thalidomide in rats and the pharmacokinetic modulation by thalidomide may partially explain its antagonizing effects on the toxicities of CPT-11. The hydrolytic products of thalidomide, instead of the parental drug, modulated the hepatic hydrolysis of CPT-11 and intracellular accumulation of SN-38, probably contributing to the altered plasma pharmacokinetics of CPT-11 and SN-38. Further studies are needed to explore the role of both pharmacokinetics and pharmacodynamic components in the protective effect of thalidomide against the toxicities of CPT-11.

Relationship between genotype and enzyme activity of glutathione S-transferases M1 and P1 in Chinese.

Glutathione S-transferases (GSTs) are the major detoxifying Phase II enzyme for eliminating electrophilic compounds. Mutations in GSTM1, GSTP1 and GSTT1 in Caucasian and GSTA1 in Chinese have been found to reduce enzyme activity. However, data on the impact of common genetic polymorphisms of GSTM1 and GSTP1 on enzyme activity in Chinese is lacking. This study aimed to investigate the effect of common GSTP1 and GSTM1 polymorphisms on erythrocyte GST activity in healthy Chinese (n = 196). GSTM1 null mutation (GSTM1*0) was analyzed by a PCR-Multiplex procedure, whereas GSTP1 313A-->G polymorphism (resulting in Ile105Val at codon 105) was analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis. Erythrocyte GST activity was measured using 1-chloro-2,4-dinitro-bezene (CDNB) as the model substrate. The frequency of GSTM1 null genotype was 54.3% and the frequency of GSTP1-Ile/Ile, -Ile/Val, and -Val/Val genotype was 60.7%, 35.2% and 4.1%, respectively, with a frequency of 21.7% for the 105 valine allele. Age, gender and smoking did not significantly affect the erythrocyte GST activities. The mean erythrocyte GST enzyme activity for GSTP1*-Ile/Val genotype group (3.53 +/- 0.63U/gHb) was significantly lower than that for subjects with GSTP1-Ile/Ile genotype (4.25 +/- 1.07U/gHb, P = 0.004), while subjects with the GSTP1-Val/Val genotype had the lowest enzyme activity (2.44 +/- 0.67U/gHb). In addition, the GST activity in carriers of GSTM1*0/GSTP1-Ile/Ile was significantly higher than that of subjects inherited GSTM1*0/GSTP1-Ile/Val or GSTM1*0/GSTP1-Val/Val. However, there is no association between GSTM1 null mutation and reduced enzyme activity. GSTP1 codon 105 mutation led to reduced erythrocyte GST activity in Chinese. A combined GSTP1 and GSTM1 null mutations also resulted in significantly reduced GST activity. Further studies are needed to explore the clinical implications of GSTM1 and GSTP1 polymorphisms.

Topotecan is a substrate for multidrug resistance associated protein 4.

Topotecan (TPT) is a semisynthetic water-soluble derivative of camptothecin (CPT) used as second-line therapy in patients with metastatic ovarian carcinoma, small cell lung cancer, and other malignancies. However, both dose-limiting toxicity and tumor resistance hinder the clinical use of TPT. The mechanisms for resistance to TPT are not fully defined, but increased efflux of the drug by multiple drug transporters including P-glycoprotein (PgP), multidrug resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) from tumor cells has been highly implicated. This study aimed to investigate whether overexpression of human MRP4 rendered resistance to TPT by examining the cytotoxicity profiles using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay and cellular accumulation of TPT in HepG2 cells stably overexpressing MRP4. Two kinds of cell lines, HepG2 with insertion of an empty vector plasmid (V/HepG2), HepG2 cells stably expressing MRP4 (MRP4/HepG2), were exposed to TPT for 4 or 48 hr in the absence or presence of various MRP4 inhibitors including DL-buthionine-(S,R)-sulphoximine (BSO), diclofenac, celecoxib, or MK-571. The intracellular accumulation of TPT and paclitaxel (a PgP substrate) by V/HepG2 and MRP4/HepG2 cells was determined by incubation of TPT with the cells and the amounts of the drug in cells were determined by validated HPLC methods. The study demonstrated that MRP4 conferred a 12.03- and 6.86-fold resistance to TPT in the 4- and 48-hr drug-exposure MTT assay, respectively. BSO, MK-571, celecoxib, or diclofenac sensitised MRP4/HepG2 cells to TPT cytotoxicity and partially reversed MRP4-mediated resistance to TPT. In addition, the accumulation of TPT was significantly reduced in MRP4/HepG2 cells compared to V/HepG2 cells, and one-binding site model was found the best fit for the MRP4-mediated efflux of TPT, with an estimated K(m) of 1.66 microM and V(max) of 0.341 ng/min/106 cells. Preincubation of MRP4/HepG2 cells with BSO (200 microM) for 24 hr, celecoxib (50 microM), or MK-571 (100 microM) for 2 hr significantly increased the accumulation of TPT over 10 min in MRP4/HepG2 cells by 28.0%, 37.3% and 32.5% (P < 0.05), respectively. By contrast, there was no significant difference in intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells over 120 min. MRP4 also rendered resistance to adefovir dipivoxil (bis-POM-PMEA) and methotrexate, two reported MRP4 substrates. MRP4 did not exhibit any significant resistance to other model drugs including vinblastine, vincristine, etoposide, carboplatin, cyclosporine and paclitaxel in both long (48 hr) and short (4 hr) drug-exposure MTT assays. These findings indicate that MRP4 confers resistance to TPT and TPT is the substrate for MRP4. Further studies are needed to explore the role of MRP4 in resistance to, toxicity and pharmacokinetics of TPT in cancer patients.

Monitoring of immune responses to a herbal immuno-modulator in patients with advanced colorectal cancer.

Many herbal medicines are widely used as immuno-modulators in Asian countries. Ganoderma lucidum (Lingzhi) is one of the most commonly used herbs in Asia and preclinical studies have established that the polysaccharide fractions of G. lucidum have potent immuno-modulating effects. However, clinical evidence for this is scanty. The present open-labeled study aimed to evaluate the effects of G. lucidum polysaccharides on selected immune functions in patients with advanced colorectal cancer. Forty-seven patients were enrolled and treated with oral G. lucidum at 5.4 g/day for 12 weeks. Selected immune parameters were monitored using various immunological methods throughout the study. In 41 assessable cancer patients, treatment with G. lucidum tended to increase mitogenic reactivity to phytohemagglutinin, counts of CD3, CD4, CD8 and CD56 lymphocytes, plasma concentrations of interleukin (IL)-2, IL-6 and interferon (IFN)-gamma, and NK activity, whereas plasma concentrations of IL-1 and tumor necrosis factor (TNF)-alpha were decreased. For all of these parameters, no statistical significance was observed when a comparison was conducted between baseline and those values after a 12-week treatment with G. lucidum. The changes of IL-1 were correlated with those for IL-6, IFN-gamma, CD3, CD4, CD8 and NK activity (p<0.05) and IL-2 changes were correlated with those for IL-6, CD8 and NK activity. The results indicate that G. lucidum may have potential immuno-modulating effect in patients with advanced colorectal cancer. Further studies are needed to explore the benefits and safety of G. lucidum in cancer patients.

Insights into oxazaphosphorine resistance and possible approaches to its circumvention.

The oxazaphosphorines cyclophosphamide, ifosfamide and trofosfamide remain a clinically useful class of anticancer drugs with substantial antitumour activity against a variety of solid tumors and hematological malignancies. A major limitation to their use is tumour resistance, which is due to multiple mechanisms that include increased DNA repair, increased cellular thiol levels, glutathione S-transferase and aldehyde dehydrogenase activities, and altered cell-death response to DNA damage. These mechanisms have been recently re-examined with the aid of sensitive analytical techniques, high-throughput proteomic and genomic approaches, and powerful pharmacogenetic tools. Oxazaphosphorine resistance, together with dose-limiting toxicity (mainly neutropenia and neurotoxicity), significantly hinders chemotherapy in patients, and hence, there is compelling need to find ways to overcome it. Four major approaches are currently being explored in preclinical models, some also in patients: combination with agents that modulate cellular response and disposition of oxazaphosphorines; antisense oligonucleotides directed against specific target genes; introduction of an activating gene (CYP3A4) into tumor tissue; and modification of dosing regimens. Of these approaches, antisense oligonucleotides and gene therapy are perhaps more speculative, requiring detailed safety and efficacy studies in preclinical models and in patients. A fifth approach is the design of novel oxazaphosphorines that have favourable pharmacokinetic and pharmacodynamic properties and are less vulnerable to resistance. Oxazaphosphorines not requiring hepatic CYP-mediated activation (for example, NSC 613060 and mafosfamide) or having additional targets (for example, glufosfamide that also targets glucose transport) have been synthesized and are being evaluated for safety and efficacy. Characterization of the molecular targets associated with oxazaphosphorine resistance may lead to a deeper understanding of the factors critical to the optimal use of these agents in chemotherapy and may allow the development of strategies to overcome resistance.

Human multidrug resistance associated protein 4 confers resistance to camptothecins.

PURPOSE: The multidrug resistance associated protein (MRP) 4 is a member of the adenosine triphosphate (ATP)-binding cassette transporter family. Camptothecins (CPTs) have shown substantial anticancer activity against a broad spectrum of tumors by inhibiting DNA topoisomerase I, but tumor resistance is one of the major reasons for therapeutic failure. P-glycoprotein, breast cancer resistance protein, MRP1, and MRP2 have been implicated in resistance to various CPTs including CPT-11 (irinotecan), SN-38 (the active metabolite of CPT-11), and topotecan. In this study, we explored the resistance profiles and intracellular accumulation of a panel of CPTs including CPT, CPT-11, SN-38, rubitecan, and 10-hydroxy-CPT (10-OH-CPT) in HepG2 cells with stably overexpressed human MRP4. Other anticancer agents such as paclitaxel, cyclophosphamide, and carboplatin were also included. METHODS: HepG2 cells were transfected with an empty vehicle plasmid (V/HepG2) or human MRP4 (MRP4/HepG2). The resistance profiles of test drugs in exponentially growing V/HepG2 and MRP4/HepG2 cells were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay with 4 or 48 h exposure time of the test drug in the absence or presence of various MRP4 inhibitors. The accumulation of CPT-11, SN-38, and paclitaxel by V/HepG2 and MRP4/HepG2 cells was determined by validated high-performance liquid chromatography methods. RESULTS: Based on the resistance folds from the MTT assay with 48 h exposure time of the test drug, MRP4 conferred resistance to CPTs tested in the order 10-OH-CPT (14.21) > SN-38 carboxylate (9.70) > rubitecan (9.06) > SN-38 lactone (8.91) > CPT lactone (7.33) > CPT-11 lactone (5.64) > CPT carboxylate (4.30) > CPT-11 carboxylate (2.68). Overall, overexpression of MRP4 increased the IC50 values 1.78- to 14.21-fold for various CPTs in lactone or carboxylate form. The resistance of MRP4 to various CPTs tested was significantly reversed in the presence of dl-buthionine-(S,R)-sulfoximine (BSO, a gamma-glutamylcysteine synthetase inhibitor), MK571, celecoxib, or diclofenac (all MRP4 inhibitors). In addition, the accumulation of CPT-11 and SN-38 over 120 min in MRP4/HepG2 cells was significantly reduced compared to V/HepG2 cells, whereas the addition of celecoxib, MK571, or BSO significantly increased their accumulation in MRP4/HepG2 cells. There was no significant difference in the intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells, indicating that P-glycoprotein was not involved in the observed resistance to CPTs in this study. MRP4 also conferred resistance to cyclophosphamide and this was partially reversed by BSO. However, MRP4 did not increase resistance to paclitaxel, carboplatin, etoposide (VP-16), 5-fluorouracil, and cyclosporine. CONCLUSIONS: Human MRP4 rendered significant resistance to cyclophosphamide, CPT, CPT-11, SN-38, rubitecan, and 10-OH-CPT. CPT-11 and SN-38 are substrates for MRP4. Further studies are needed to explore the role of MRP4 in resistance, toxicity, and pharmacokinetics of CPTs and cyclophosphamide.