Abdul Sami Shaikh

Non-proton ligand-sensing domain of acid-sensing ion channel 3 is required for itch sensation.

Itch, the unpleasant sensation that evokes a desire to scratch, accompanies numerous skin and nervous system disorders. However, the molecular mechanisms of itch are unclear. Acid‐sensing ion channel 3 (ASIC3) is a sensor of acidic and primary inflammatory pain. The whole‐cell patch clamp technique was used to determine the effect of chloroquine (CQ) on ASICs currents in primary sensory neurons or the Chinese hamster ovary cells transfected with rat ASIC1a or ASIC3. Site‐directed mutagenesis of plasmid was performed. Scratching behavior was evaluated by measuring the number of bouts during 30 min after injection. CQ, an anti‐malarial drug defined as a histamine‐independent pruritogen, selectively enhanced the sustained phase of ASIC3 current in a concentration‐dependent manner either in ASIC3‐transfected Chinese hamster ovary cells or in primary cultured rat dorsal root ganglion neurons. Further studies revealed that the effect of CQ on ASIC3 channels depends on the newly identified non‐proton ligand‐sensing domain. Importantly, CQ‐evoked scratching behavior was largely alleviated by APETx2, a selective ASIC3 channel blocker. Like CQ, other compounds such as amiloride, 2‐guanidine‐4‐methylquinazoline and neuropeptide FF, which have been previously reported to be non‐proton ligands that activate ASIC3, undoubtedly evoked the scratching response. In conclusion, ASIC3, a proton‐gated ion channel critical for pain sensation, also functions as an essential component of itch transduction.

Targeting autophagy augments the activity of DHA-E3 to overcome p-gp mediated multi-drug resistance

Multidrug resistance (MDR) is a major obstacle for successful chemotherapy treatment. Searching for effective MDR modulators and combining them with anticancer drug therapies has been a promising strategy against clinical MDR. In our previous study, we have found that DHA-E3, a synthetic derivative of DHA, has the ability to modulate the function of P-glycoprotein (P-gp) and reverse MDR in cancer cells. In this study, we further evaluated the reversal effect of DHA-E3 on MDR and explored its mechanism of action in vitro. Our findings showed that DHA-E3 significantly potentiated the cytotoxicity of vincristine(VCR) and adriamycin(ADR) in the P-gp over-expressing KB/VCR and A02 cells. The mechanistic study found that DHA-E3 increased the intracellular accumulation of ADR and rhodamine-123 by directly inhibiting the drug-transport activity of P-gp. In the present study, we found that DHA-E3 not only reversed MDR, but also induced autophagy in MDR cancer cells. To determine whether DHA-E3-induced autophagy is an adaptive survival response or contributes to cell death, we manipulated autophagic activity using autophagy inhibitor 3-MA or siRNA targeting Beclin1. We found that the reversal activity of DHA-E3 was significantly exacerbated in the presence of 3-MA or blocking the expression of Beclin1. These results suggest that DHA-E3 is capable of reversing MDR, induction of autophagy represents a defense mechanism and inhibiting this process may be an effective strategy to augment the reversal activity of reversal agents.

Therapeutic Drug Monitoring of Phenytoin by Simple, Rapid, Accurate, Highly Sensitive and Novel Method and Its Clinical Applications

BACKGROUND Phenytoin has very challenging pharmacokinetic properties. To prevent its toxicity and ensure efficacy, continuous therapeutic monitoring is required. It is hard to get a simple, accurate, rapid, easily available, economical and highly sensitive assay in one method for therapeutic monitoring of phenytoin. OBJECTIVE The present study is directed towards establishing and validating a simpler, rapid, an accurate, highly sensitive, novel and environment friendly liquid chromatography/mass spectrometry (LC/MS) method for offering rapid and reliable TDM results of phenytoin in epileptic patients to physicians and clinicians for making immediate and rational decision. METHODS 27 epileptics patients with uncontrolled seizures or suspected of non-compliance or toxicity of phenytoin were selected and advised for TDM of phenytoin by neurologists of Qilu Hospital Jinan, China. The LC/MS assay was used for performing of therapeutic monitoring of phenytoin. The Agilent 1100 LC/MS system was used for TDM. The mixture of Ammonium acetate 5mM: Methanol at (35: 65 v/v) was used for the composition of mobile phase. The Diamonsil C18 (150mm×4.6mm, 5μm) column was used for the extraction of analytes in plasma. The samples were prepared with one step simple protein precipitation method. The technique was validated with the guidelines of International Conference on Harmonisation (ICH). RESULTS The calibration curve demonstrated decent linearity within (0.2-20 µg/mL) concentration range with linearity equation, y= 0.0667855 x +0.00241785 and correlation coefficient (R2) of 0.99928. The specificity, recovery, linearity, accuracy, precision and stability results were within the accepted limits. The concentration of 0.2 µg/mL was observed as lower limit of quantitation (LLOQ), which is 12.5 times lower than the currently available enzyme-multiplied immunoassay technique (EMIT) for measurement of phenytoin in epilepsy patients. CONCLUSION A rapid, simple, economical, precise, highly sensitive and novel LC/MS assay has been established, validated and applied successfully in TDM of 27 epileptics patients. It was alarmingly found that TDM results of all these patients were out of safe range except two patients. However, it needs further evaluation. Besides TDM, the stated method can also be applied in bioequivalence, pharmacokinetics, toxicokinetics and pharmacovigilance studies.

Bioequivalence Study of Warfarin in Healthy Chinese Volunteers With a Validated High‐Performance Liquid Chromatography‐Mass Spectrometry Method

This study was designed to investigate the pharmacokinetics of an innovative film‐coated warfarin sodium tablet and to compare it with the marketed sugar‐coated warfarin sodium tablet in humans. A single‐dose, open‐label, randomized, two‐way crossover study was performed in 24 healthy Chinese male volunteers. They were administered 2.5 mg of innovative film‐coated warfarin sodium tablets or the marketed sugar‐coated warfarin sodium tablets. Blood samples were collected at different time points after dosing for investigation of the pharmacokinetics of warfarin in human plasma. A sensitive liquid chromatography mass spectrometry method was established to determine warfarin in plasma. Drug and Statistics 2.1.1 was applied to calculate the pharmacokinetics parameters. The main pharmacokinetic parameters for film‐coated and sugar‐coated warfarin were the following: t½, 103.5 ± 18.8 and 105.8 ± 21.3 hours; Tmax, 0.7 ± 0.5 and 1.3 ± 0.8 hours; Cmax, 347.8 ± 74.8 and 322.9 ± 75.7 ng/mL; AUC0∼360, 16,024.2 ± 3713.9 and 15,586.6 ± 3477.0 ng·mL−1·h; AUC0∼∞, 17,335.7 ± 4089.1 and 16,912.0 ± 3911.2 ng·mL−1·h, respectively. The human pharmacokinetics of film‐coated and sugar‐coated warfarin were slightly different. The oral absorption and bioavailability of innovative film‐coated warfarin were slightly higher than those of the sugar‐coated warfarin. This study is vital to clinical usage of warfarin not only because of the pharmacokinetic parameters of the 2 pharmaceutical dosage forms of warfarin but also to obtain data on the prevalence of CYP2C9 and VKORC1 genes and their influence on the concentration of warfarin.

Histone Acetyltransferase Mof Affects the Progression of DSS-Induced Colitis.

Background/Aims: Histone acetylation has been demonstrated to be associated with inflammation response. Histone acetyltransferase (HAT) Mof, specifically acetylating lysine 16 of histone H4 (H4K16), has been reported to regulate T cell differentiation. In addition, it has been suggested that acetylation of H4K16 is associated with the inflammatory response. We evaluated the role and potential mechanism of Mof in the development of experimental colitis. Methods: We used Mof conditional knockout mice to study the role of Mof in dextran sulfate sodium (DSS)-induced colitis and detected the differential expression of genes due to Mof deficiency involved in the inflammatory response, particularly the Th17 signaling pathway, by western blotting, quantitative PCR and RNA sequencing (RNA-seq). Results: A significant elevation of Mof was observed in colonic tissues of mice with DSS-induced colitis. Mof deficiency alleviated the severity of DSS- induced colitis in mice. We found that Th17 signaling pathway associated genes, including Il17a, Il22, RORγt, RORα, Stat3, TGF-β 1, and Il6, were downregulated in colon tissues with Mof deficiency. RNA-seq data analysis suggested that 68 genes were related to inflammatory response processing and 47 genes were downregulated in Mof defective colon tissues. Conclusion: Our study demonstrated that HAT Mof is involved in the development of colitis, and the lack of Mof ameliorates DSS-induced colitis in mice.

The histone acetyltranseferase KAT8 regulates cell differentiation by suppression of MN1 in AML

K(lysine) acetyltransferase 8 (KAT8, also known as MOF or MYST1) was originally identified as an essential component of the dosage compensation in Drosophila, causing a twofold increase in the expression of X-linked genes in male flies (Rea et al, 2007). The corresponding acetylation of histone H4 at lysine 16 (H4K16ac) is closely related to KAT8 expression level. The global reduction of H4K16ac and depletion of KAT8 in mammal cells can result in abnormal gene transcription, especially causing abnormal expression of certain tumours. Despite all the improvements in the study and treatment of acute myeloid leukaemia (AML), there is little information about KAT8 expression in leukaemic cells from AML patients. Bone marrow (BM) samples were obtained from 50 patients with AML upon diagnosis (see Table SI for Patient characteristics). U937, HL-60 and K562 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). U937 cells were treated with 100 nM

A comparison of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-multiplied immunoassay technique (EMIT) for the determination of the cyclosporin A concentration in whole blood from Chinese patients

Cyclosporin A (CyA) is an immunosuppressive agent widely used in clinical therapy. In the therapeutic process, the blood concentration of CyA should be monitored to avoid or prevent rejection and toxicity. The objectives of this study were to compare the correlation of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-multiplied immunoassay technique (EMIT) for the determination of the CyA concentration in human blood and to provide evidence for the rational usage of EMIT in clinical practice. Blood samples collected from 132 patients undergoing a liver or kidney transplant or patients with aplastic anemia at Qilu Hospital of Shandong University were tested using the two methods. The calibration curve was linear from 25-500 ng·mL-1 for LC-MS/MS and from 50-450 ng·mL-1 for EMIT. The inter- and intra-day RSDs were less than 15%. The CyA blood concentration according to EMIT was 3.5 ng·mL-1 more than that according to LC-MS/MS. The 95% confidence interval was -10.0~16.9 ng·mL-1. The CyA blood concentration according to the two methods did not differ significantly (p > 0.05). LC-MS/MS and EMIT were suitable methods for determining the CyA blood concentration. The two methods were closely correlated (r2 = 0.969), but the CyA blood concentration according to EMIT was slightly higher than that according to LC-MS/MS. The clinical significance of this finding needs to be further evaluated.