Formulation Development of Mirtazapine Liquisolid Compacts: Optimization Using Central Composite Design.

Mirtazapine is a tetracyclic anti-depressant with poor water solubility. The aim of this study was to improve the dissolution rate of mirtazapine by delivering the drug as a liquisolid compact. Central composite design (CCD) was employed for the preparation of mirtazapine liquisolid compacts. In this, the impacts of two independent factors, i.e., excipient ratio (carrier:coating) and different drug concentration on the response of liquisolid system were optimized. Liquisolid compacts were prepared using propylene glycol as a solvent, microcrystalline cellulose as a carrier, and silicon dioxide (Aerosil) as the coating material. The crystallinity of the formulated drug and the interactions between the excipients were examined using X-ray powder diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR), respectively. The dissolution study for the liquisolid compact was carried out as per FDA guidelines. The results showed loss of crystallinity of the mirtazapine in the formulation and was completely solubilized in non-volatile solvent and equally dispersed throughout the powder system. Moreover, drug dissolution was found to be higher in liquisolid compacts than the direct compressed conventional tablets (of mirtazapine). The liquisolid technique appears to be a promising approach for improving the dissolution of poorly soluble drugs like mirtazapine.

Assessment of Tocilizumab (Humanized Monoclonal Antibody) for Therapeutic Efficacy and Clinical Safety in Patients with Coronavirus Disease (COVID-19).

Background and objectives: COVID-19 patients exhibit a broad range of manifestations, presenting with a flu-like respiratory tract infection that can advance to a systemic and severe disease characterized by pneumonia, pulmonary edema, severe damage to the airways, and acute respiratory distress syndrome (ARDS, causing fatality in 70% of COVID-19 cases). A 'cytokine storm' profile is found in most severely influenced COVID-19 patients. The treatment protocol of the disease also includes tocilizumab, which is a humanized monoclonal antibody used to treat autoimmune and inflammatory conditions. This study was designed (1) to assess the role of tocilizumab in COVID-19 patients regarding therapeutic efficacy through evaluation of cytokine release syndrome (CRS) resolution and anticoagulant effect, analyzing clinical safety via monitoring of associated adverse effects profile; and (2) to compare the clinical safety and therapeutic efficacy of institutional treatment regimen (alone) versus tocilizumab added to an institutional treatment module in COVID-19 patients. Materials and Methods: In this study, the endpoints parametric assessment of severely diseased patients of COVID-19 was performed (total n = 172, control group (institutional protocol treatment provided), n = 101 and test group (tocilizumab provided), n = 71) at the Khyber Teaching Institution, MTI, Peshawar. The assessments were compared using non-parametric analyses at baseline and after a follow-up of 12−18 days until the patient discharged or expired. Results: Results of the study revealed an insignificant difference among the control vs. test group in resolving inflammatory parameters (C-reactive protein (CRP) 21.30 vs. 50.07; p = 0.470, ferritin 482.9 vs. 211.5; p = 0.612, lactate dehydrogenase (LDH) 29.12 vs.18.8; p = 0.0863, and D-dimer 464 vs.164.4; p = 0.131). However, a statistically significant difference was found between the control group and test group regarding coagulation parameters (international normalized ratio (INR) 0.12 vs. −0.07; p ≤ 0.001; activated partial thromboplastin time (aPTT) 0.42 vs. −1.16; p ≤ 0.001; prothrombin time (PT) 0.31 vs. −0.96; p ≤ 0.001; platelet count −12.34 vs. −1.47; p = 0.012) and clinical survival rate (89.10 vs. 90.14; p < 0.001). Furthermore, there was significantly higher infection rates and raised alanine aminotransferase (ALT) and alkaline phosphatase (ALP) associated with the tocilizumab group as compared to those receiving institutional treatment (bacterial infections: 0.99% vs. 15.49%; p ≤ 0.01, ALT: 3.96% vs. 28.16%; p ≤ 0.01, ALP: 1.98% vs. 22.53%; p ≤ 0.01). Conclusions: From this study, it was concluded that tocilizumab can be a better drug of choice in terms of efficacy, particularly in resolving coagulopathy in severe COVID-19 patients.

Evaluation of in-vitro and in-vivo antimicrobial potential of Typha elephantina leaves extracts using Cyprinus carpio as a research model.

Present study investigate the in-vitro antibacterial and antifungal potential of Typha elephantina leaves aqueous extract (T. Eaq), ethanolic extract (T. Eeth) and methanolic extract (T. Emth) at different dosages against selected bacteria and fungi using dis diffusion method and Potato Dextrose Agar method. The study was also proceeded in- vivo against one strain of fungi (Aspergillus niger) using aqueous (T. Eaq) extract only. In-vitro study showed that Citrobacter freundii was highly sensitive while Salmonella typhimurium was the least among all. The antifungal activity was dose dependent and differs according to the fungal strain. Aspergillus niger was highly sensitive in order of aqueous extract (T. Eaq), ethanolic extract (T. Eeth) and methanolic extract (T.Emth), followed by Alterneria solani, Candida albicans and Aspergillus ustus. The in-vivo antifungal study was carried using Cyprinus carpio which were first infected with Aspergillus niger and then treated with (T. Eaq) at different doses. During in-vivo study various hematobiochemicl parameters and bio-accumulative stress of some heavy metals were assessed. Highly significant (P<0.05) remedial effects were observed at day 21st of treatment with extract at 100mg/ kg body weight. Differential accumulation was found i.e in skin the accumulation was highest followed by intestine gills and muscles tissues. Liver showed least accumulation.

Association of Non-Hodgkin lymphoma risk with CYP1A1, GSTM1 and GSTT1 gene variants, in tobacco addicted patients of Pashtun ethnicity of Khyber Pakhtunkhwa, Pakistan.

The current study determines the possible antitumor and immunomodulatory effects of thymosin against the in vivo and in vitro growth of tumor-derived cell line in mice. Peritoneal phagocytes count, Ehrlich ascites tumor (EAT) cells, T- lymphocytes, and B- lymphocytes activities were determined. In addition, serum level of interleukin 2 (IL-2) and liver functions were measured. In animal testing, thymosin at doses of 0.50 and 1mg activated the phagocytic function of macrophages, as well as T- and B- cell function. Thymosin caused a marked shortage in the proliferation of EAT cells in the peritoneal fluid with dose 0.50g as compared with that of the corresponding control group. Furthermore, treatment with thymosin caused effectively elevate in serum level of IL-2, on the contrary reduce in serum levels of ALT, AST and total proteins. The size of solid Ehrlich tumor was significantly decreased, as measured morphologically with the doses 0.50 and 1 mg (P<0.01). These results confirmed that many biological activities attributed to thymosin and is as an adjuvant for immune enhancement.

Association of nasopharyngeal cancer risk with genetic polymorphisms of drug-metabolizing enzyme genes GSTM1, GSTT1 and CYP1A1 (rs4646903 variant), in tobacco addicted patients of Pashtun ethnicity of Khyber Pakhtunkhwa Province of Pakistan.

Associations of GSTM1, GSTT1 and CYP1A1 gene variants with risk of developing nasopharyngeal cancer were evaluated in this case-control study, in which 130 cases along with 151 population-based healthy controls of Pashtun ethnicity from Khyber Pakhtunkhwa province of Pakistan were recruited. Socio-demographic data were obtained and blood samples were collected with informed consent for analysis. Specific RT-PCR and conventional PCR methods were used to detect CYP1A1 and GSTs, respectively, and results analyzed through SPSS version 20. Study showed that CYP1A1 homozygous (C/C) had an almost 4-fold increased risk for nasopharyngeal cancer; while heterozygous (T/C) had an almost 2 times increased risk. Overall the C allele is significantly associated with nasopharyngeal cancer as compared to T allele. Null genotypes of GSTM1 were having 3-fold increased risk; whereas null genotype of GSTT1 was having 2 times increased risk. Similarly, GSTM/GSTT both null genotype was having more than 5 times increased association. Presence of all three gene variants showed strong and significant association. Findings of the study suggest that presence of GSTM1 and/or GSTT1 null genotypes along with variant alleles of CYP1A1 are significant risk factors for nasopharyngeal cancer susceptibility in Pashtun population.

Rosuvastatin pharmacokinetics in Pakistani healthy volunteers in comparison with other population.

In current study, the pharmacokinetics (PK) of rosuvastatin were evaluated in Pakistani healthy volunteers and compared with those reported in other population. This was a randomized and open labeled clinical trial in which a single oral dose of 40 mg rosuvastatin was administered to the overnight fasted healthy volunteers. Plasma concentrations of rosuvastatin were quantified by a validated liquid chromatography-tandem mass spectrometry method. The PK parameters of rosuvastatin and its metabolite N-desmethyl-rosuvastatin were determined by PK specific software i.e., PK-Summit® (PK-Solutions). A total of 20 healthy volunteers having BMI in the normal ranges were included in this study. All PK parameters were represented as mean ± SD and 95% confidence intervals of the means have been calculated. The Cmax (29.07 ± 6.88 ng/mL), [AUC]xo (206.65 ± 55.27 ng/hr/mL) and CL/F (3275.26 ± 1072.87 mL/hr) were slightly higher in our study, whereas the values of Vd (19377.23 ± 9114.29 mL) and tmax (3.0 ± 0.46 hr) were comparatively smaller. Overall, the PK parameters of rosuvastatin determined in our study were in compliance with other reported. Therefore, no adjustments in the dosing schedule or dose are warranted.

Mini-Review: Drug-food interactions of commonly available juices of Pakistan.

Medicines are often consumed concurrently with food; sometimes to improve its absorption and efficacy. However, certain foods may modify the function of drug metabolizing enzymes or transport mechanisms that are crucial determinants of systemic drug availability. Extensive work has been reported on certain juices like grapefruit that affects the bioavailability of more than 60 medications. However, relatively less work has been reported on certain other commonly used fruit juices, especially in Pakistan, such as mango, strawberry, apple, banana, pomegranate and grape etc. Present review has taken an account of the current work done in this area.

Comparative pharmacokinetics of Omeprazole and its metabolites in poor and extensive metabolizer Pakistani healthy volunteers and a review of different studies.

This study was designed to evaluate a comparative single dose (40mg) pharmacokinetics (PK) of Omeprazole (OMP) and its two metabolites, 5-hydroxy Omeprazole (5-OH-OMP) and Omeprazole sulphone (OMP-S) in poor (PM) and extensive (EM) metabolizer Pakistani healthy adult volunteers. The frequency of CYP2C19 and CYP3A4 varies widely in different populations. The present study was conducted to evaluate the PK of OMP and its two metabolites in Pakistani population and to review different studies conducted after administration of single dose of OMP. Twenty two subjects were enrolled in this study and divided into two groups. The CYP2C19 phenotyping was evaluated by the metabolic ratio of OMP to 5-OH-OMP. It was a single dose, open label study and the blood samples from subjects were collected at different time intervals until 24 hours. The PK parameters were calculated using the PK-summit software. The metabolic ratio of area under the plasma concentration-time curve AUCOMP/5-OH-OMP was 1.86 ± 0.572 and13.84 ± 2.504 for EM and PM, respectively; maximum plasma concentration (Cmax) of OMP was increased by two folds for PM while the AUC∞ was increased by 3 folds; the Cmax and AUC∞ of 5-OH-OMP decreased for PM by 2 folds while there was 3 fold increase observed in the Cmax and AUC∞ of OMP-S. The PK of OMP and its metabolites in different populations were also discussed, and issues regarding CYP2C19 and CYP3A4 genotyping were also extensively reviewed. In EM of CYP2C19 the concentration of 5-OH-OMP is higher while that of OMP-S is lower. This study as well as reported studies reveals that in PM of CYP2C19 more drugs are available for CYP3A4 to be metabolized. A correlation between CYP2C19 EM and PM activity with CYP3A4 needs to be established.

Effect of Omeprazole on the Pharmacokinetics of Rosuvastatin in Healthy Male Volunteers.

The current study aimed at the evaluation of, in vivo, the effect of omeprazole on the pharmacokinetics of rosuvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. Omeprazole is an acid suppressant and CYP2C9, CYP3A4, and CYP2C19 substrate and inhibitor, as well as inhibitor of transporters (like P-gp). This was a randomized, open-label, 2-period, crossover study. Healthy male volunteers (N = 20), divided into 2 groups, were given single oral doses of rosuvastatin 40 mg either alone (treatment period I) or concomitantly with omeprazole 40-mg capsule (treatment period II). Plasma concentrations of rosuvastatin (rosuva) and its metabolite N-desmethyl rosuvastatin (NDM-rosuva) were quantified by a validated liquid chromatography-tandem mass spectrometry method developed in our laboratory. An insignificant decrease (P > 0.05) has been observed in the values of maximum plasma concentrations, clearance, and half-life of rosuva, whereas an insignificant increase (P > 0.05) has been observed in the area under the plasma concentration-time curves from zero time to the last measurable concentration(Equation is included in full-text article.), that extrapolated to infinity (Equation is included in full-text article.), and mean residence time values after concomitant administration with omeprazole. Although omeprazole concomitant administration altered the pharmacokinetics of NDM-rosuva metabolite significantly, rosuva's very little metabolism (10%) suggests that these changes are of no clinical significance. Concomitant administration of omeprazole with rosuva did not alter the pharmacokinetics of rosuva in healthy volunteers. These data are consistent with other reported studies, indicating that rosuva is not a good candidate for metabolism-based drug-drug interactions. Therefore, rosuva can be administered safely along with omeprazole.

Comparison of toxic heavy metals concentration in medicinal plants and their respective branded herbal formulations commonly available in Khyber Pakhtunkhwa.

The present study was conducted on fifteen medicinal plants and their respective branded formulations, commonly used in Khyber Pakhtunkhwa, for the evaluation of toxic heavy metals. The purpose of the study was to assess the toxic profile of the crude medicinal plants with respect to the worldwide permissible limits of metal concentrations and to correlate it with their respective herbal formulations available on the market. Chromium (Cr), Copper (Cu), Lead (Pb), Manganese (Mn) and Nickel (Ni) content were evaluated using wet digestion and Atomic Absorption Spectrophotometry technique. The results exhibited that in 100% of the analyzed medicinal plants Cr and Ni are present in excess of the maximum limits, Cu and Pb in 73% and 60% respectively, while Mn is in the normal range. Likewise in the respective branded formulations Cr and Ni exceed the normal limit in 100% of the products, Cu and Pb in 27% and 20% of the products respectively, while Mn is in the normal range. It indicates that majority of people in Pakistan who frequently use herbal drugs in various forms are exposed to the hazardous elements, which may pose serious health effects. Regulatory measures should therefore be taken to protect the general public from their hazardous health effects.

Report: Antibacterial and antifungal activities of leaf extract of Achyranthes aspera (Amaranthaceae) from Pakistan.

Alcoholic extract and various fractions of Achyranthes aspera leaves, traditionally used in Pakistan for treatment of infectious diseases was screened for in vitro antibacterial and antifungal activity. The chloroform and butanol fractions were found to be the most active among the fractions, showing considerable antibacterial activity against Shigella flexneri and Escherichia coli. The highest activity was found in the ethylacetate fraction (17 mm zone of inhibition) against gram-negative (Salmonella typhi) bacteria, with MIC value as 0.29 mg/mL. In antifungal screening, moderate activity was shown by the chloroform fraction (50 % inhibition) against Microsporum canis, with MIC value as 0.25mg/mL. Considerable level of antifungal activity was depicted by crude extract, hexane and butanol fractions against Aspergillus flavus and Microsporum canis. The ability of various extracts of Achyranthes aspera to inhibit different strains of fungi and bacteria indicates its potential use for the treatment of microbial infections.

Development and evaluation of pluronic- and methylcellulose-based thermoreversible drug delivery system for insulin.

The objective of the current work was to develop and evaluate thermoreversible subcutaneous drug delivery system for Insulin. Thermoreversible in-situ gel system was developed and evaluated both in-vitro and in-vivo comprising of pluronic F-127 alone or in combination with methylcellulose in different ratios. The drug release kinetics and mechanism was predicted by applying various mathematical models to the in-vitro dissolution data. Rabbits were used as animal model following subcutaneous injection to predict various pharmacokinetic parameters by applying Pk-Summit® software. The in-vitro and in-vivo data revealed that the formulation IPM 15/3 consisting of the pluronic F-127 (15% w/v) and methylcellulose (3% w/v) was the most robust and capable formulation for extending the drug release and maintaining basal plasma insulin level between 10 and 40 µU/ml for 240 h (10 d).

Report: screening of selected medicinal plants for their enzyme inhibitory potential - a validation of their ethnopharmacological uses.

In present study four medicinal plants namely Valeriana wallichii, Xanthium strumarium, Achyranthes aspera and Duchesnea indica belonging to different families were collected in Khyber Pakhtunkhwa province and crude extract and subsequent fractions were analyzed for their inhibitory potential against acetylcholinesterase, butyrylcholinesterase and α-glucosidase enzymes. Valeriana wallichii, Xanthium strumarium and Achyranthes aspera were significantly active against cholinesterases. Chloroform and ethylacetate fractions of Valeriana wallichii exhibited significant activity against acetylcholinesterase (IC50: 61µg/ml) and butyrylcholinesterase enzymes (IC50: 58µg/ml), respectively. Similarly ethylacetate fraction of Achyranthes aspera showed significant activity against acetylcholinesterase (IC50: 61 µg/ml) and butyrylcholinesterase enzymes (IC50: 61 µg/ml), respectively. In case of α-glucosidase enzyme, the chloroform fraction of Xanthium strumarium exhibited significant inhibitory activity (IC50: 72 µg/ml) as compared to the standard compound acarbose (IC50: 483 µg/ml). Duchesnea indica showed no such activities.

Evaluation of anti-inflammatory activity of selected medicinal plants of Khyber Pakhtunkhwa, Pakistan.

In present study, the anti-inflammatory potential of three medicinal plants, Xanthium strumarium, Achyranthes aspera and Duchesnea indica were evaluated, using both in vitro and in vivo assays. Carrageenan induced hind paw edema model was used to carry out the in vivo anti-inflammatory activity, while for in vitro screening lipoxygenase inhibition assay was used. Crude extract of all the selected plants depicted significant (plt;0.001) anti-inflammatory activity, at late phase of inflammation. Achyranthes aspera also showed considerable anti-inflammatory activity (47%) at relatively lower concentration (200 mg/ml), at the initial phase of inflammation. Similarly the ethyl acetate fraction of all the selected plants showed significant lipoxygenase inhibition activity when compared with the standard drug (Baicalein). The results obtained from both in vitro and in vivo anti-inflammatory activity suggest that the ethyl acetate fraction of the crude extract of all the selected plants can be used for the isolation of new lead compounds with better anti-inflammatory activity.

Anti-inflammatory activity of the topical preparation of Valeriana wallichii and Achyranthes aspera leaves.

In vivo and in vitro screening of anti inflammatory activity of Valeriana wallichii and Achyranthes aspera leaves crude extract was performed, using standardized procedures. Methanolic crude extract topical formulation (cream) of Valeriana wallichii and Achyranthes aspera leaves (Family Valerianaceae and Amaranthaceae respectively), were screened for their anti-inflammatory activity, through "Carrageenan induced hind paw edema" test, for their effect on the acute and chronic phase inflammation models in male Wistar rats. Methanolic extract and its fractions were also evaluated for their in vitro anti-inflammatory activity using lipoxygenase inhibition assay. Leaves of Valeriana wallichii showed significant (P<0.001), dose dependant anti inflammatory activity, comparable with that of the standard, in animal model. The ethyl acetate fraction of Valeriana wallichii also showed considerable (IC 50=73 ± 0.36) in vitro anti-inflammatory activity as compared to standard (6.11 ± 0.02). Similarly Achyranthes aspera leaves showed relatively weak (p>0.05) in vivo anti-inflammatory activity. However, its activity was comparable with that of standard at 10% concentration after 5 hrs of carrageenan injection. This activity was present in ethyl acetate fraction during in vitro screening (IC 50=76 ± 0.14) as compared to that of standard (IC 50=6.11 ± 0.02). The combined in vitro and in vivo Anti-inflammatory screening shows that the ethyl acetate fraction of the crude extract of Valeriana wallichii and Achyranthes aspera can be used for the isolation of new Anti-inflammatory lead compounds.

Validation of Genome-Wide Association Studies (GWAS)-Identified Type 2 Diabetes Mellitus Risk Variants in Pakistani Pashtun Population.

Objective: Recent GWAS largely conducted in European populations have successfully identified multiple genetic risk variants associated with Type 2 Diabetes Mellitus (T2DM). However, the effects conferred by these variants in the Pakistani population have not yet been fully elucidated. The objective of this study was to examine European GWAS-identified T2DM risk variants in the Pakistani Pashtun population to better understand the shared genetic basis of T2DM in the European and Pakistani cohorts. Methodology: A total of 100 T2DM patients and 100 healthy volunteers of Pashtun ethnicity were enrolled in this study. Both groups were genotyped for 8 selected single nucleotide polymorphisms (SNPs) using the Sequenom MassARRAY® platform. The association between selected SNPs and T2DM was determined by using appropriate statistical tests. Results: Of the 8 studied SNPs, 5 SNPs, SLC30A8/ rs13266634 (p=0.031, OR=2.13), IGF2BP2/ rs4402960 (p=0.001, OR=3.01), KCNJ11/ rs5219 (p=0.042, OR=1.78), PPARG/ rs1801282 (p=0.042, OR=2.81) and TCF7L2/ rs7903146 (p=0.00006, 3.41) had a significant association with T2DM. SNP GLIS3/ rs7041847 (p=0.051, OR=2.01) showed no sufficient evidence of association. SNPs KCNQ1/ rs2237892 (p=0.140, OR=1.61) and HHEX/IDE/ s1111875 (p=0.112, OR=1.31) showed opposite allelic effects and were not validated for T2DM risk in the study population. Among the studied SNPs, TCF7L2/ rs7903146 showed the most significant association. Conclusion: Our study finding indicates that selected genome-wide significant T2DM risk variants previously identified in European descent also increase the risk of developing T2DM in the Pakistani Pashtun population.

Estimation of dihydroartemisinin in human plasma using a highly sensitive LTQ Orbitrap mass spectrometer with Xcalibur software.

Background: Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is considered a first-line antimalarial agent. Artemether is extensively metabolized in vivo to its active metabolite DHA, and therefore its determination offers considerable difficulties. In the present study, DHA identification and estimation were accurately performed by the mass spectrometric analysis, using a high-resolution liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) LTQ Orbitrap hybrid mass spectrometer. Methods: The plasma samples were taken from healthy volunteers, and the spiked plasma was extracted by adding 1 mL of a mixture of dichloromethane and tert.-methyl butyl ether (8:2 v/v) to 0.5 mL of plasma. The internal standard solution (artemisinin 500 ng/mL) was added to the plasma samples. After vertexing and centrifugation, the organic layer was separated and transferred into another tube and dried under nitrogen. The residue was reconstituted in 100 µL of acetonitrile and was injected onto the LC-MS system for analysis. Measurement of standards and samples was carried out isocratically on a Surveyor HPLC system combined with an LTQ Orbitrap mass spectrometer using an ACE 5 C18-PFP column. Mobile phase A consisted of 0.1% v/v formic acid in water, Mobile phase B consisted of acetonitrile only, and isocratic elution was carried out with A:B 20:80, v/v. The flow rate was 500 µL/min. The ESI interface was operated in a positive ion mode with a spray voltage of 4.5 kV. Results: Artemether is not a very biologically stable compound and is immediately metabolized to its active metabolite dihydroartemisinin, so no clear peak was observed for artemether. Both artemether and DHA after ionization undergo neutral losses of methanol and water, respectively, in the source of the mass spectrometer. The ions observed were (MH-H2O) m/z 267.15 for DHA and (MH-m/z 283.15 for internal standard artemisinin. The method was validated according to international guidelines. Discussion: The validated method was applied successfully for the determination and quantification of DHA in plasma samples. This method works well for the extraction of drugs, and the Orbitrap system with the help of Xcalibur software accurately and precisely determines the concentration of DHA in spiked as well as volunteer's plasma.

Decoding type 2 diabetes mellitus genetic risk variants in Pakistani Pashtun ethnic population using the nascent whole exome sequencing and MassARRAY genotyping: A case-control association study.

Genome-wide association studies have greatly increased the number of T2DM associated risk variants but most of them have focused on populations of European origin. There is scarcity of such studies in developing countries including Pakistan. High prevalence of T2DM in Pakistani population prompted us to design this study. We have devised a two stage (the discovery stage and validation stage) case-control study in Pashtun ethnic population in which 500 T2DM cases and controls each have been recruited to investigate T2DM genetic risk variants. In discovery stage Whole Exome Sequencing (WES) was used to identify and suggest T2DM pathogenic SNPs, based on SIFT and Polyphen scores; whereas in validation stage the selected variants were confirmed for T2DM association using MassARRAY genotyping and appropriate statistical tests. Results of the study showed the target positive association of rs1801282/PPARG (OR = 1.24, 95%Cl = 1.20-1.46, P = 0.010), rs745975/HNF4A (OR = 1.30, 95%Cl = 1.06-1.38, P = 0.004), rs806052/GLIS3 (OR = 1.32, 95%Cl = 1.07-1.66, P = 0.016), rs8192552/MTNR1B (OR = 1.53, 95%Cl = 0.56-1.95, P = 0.012) and rs1805097/IRS-2 (OR = 1.27, 95%Cl = 1.36-1.92, P = 0.045), with T2DM; whereas rs6415788/GLIS3, rs61788900/NOTCH2, rs61788901/NOTCH2 and rs11810554/NOTCH2 (P>0.05) showed no significant association. Identification of genetic risk factors/variants can be used in defining high risk subjects assessment, and disease prevention.

Development and Evaluation of Self-Emulsifying Drug-Delivery System-Based Tablets for Simvastatin, a BCS Class II Drug.

Background: Self-emulsifying drug-delivery systems (SEDDSs) are designed to improve the oral bioavailability of poorly water-soluble drugs. This study aimed at formulating and characterization of SEDDS-based tablets for simvastatin using castor and olive oils as solvents and Tween 60 as surfactant. Methods: The liquids were adsorbed on microcrystalline cellulose, and all developed formulations were compressed using 10.5 mm shallow concave round punches. Results: The resulting tablets were evaluated for different quality-control parameters at pre- and postcompression levels. Simvastatin showed better solubility in a mixture of oils and Tween 60 (10:1). All the developed formulations showed lower self-emulsification time (˂200 seconds) and higher cloud point (˃60°C). They were free of physical defects and had drug content within the acceptable range (98.5%-101%). The crushing strength of all formulations was in the range of 58-96 N, and the results of the friability test were within the range of USP (≤1). Disintegration time was within the official limits (NMT 15 min), and complete drug release was achieved within 30 min. Conclusion: Using commonly available excipients and machinery, SEDDS-based tablets with better dissolution profile and bioavailability can be prepared by direct compression. These S-SEDDSs could be a better alternative to conventional tablets of simvastatin.

Association of APOE (rs429358 and rs7412) and PON1 (Q192R and L55M) Variants with Myocardial Infarction in the Pashtun Ethnic Population of Khyber Pakhtunkhwa, Pakistan.

Coronary Artery Diseases (CAD) remains the top among Non-communicable Diseases (NCDs). Variations in Apolipoprotein E (APOE) and Paroxonase 1 (PON1) have been associated with Myocardial Infarction (MI) in several populations. However, despite the high prevalence of CAD, no such study has been reported in the Pashtun ethnic population of Pakistan. We have conducted a two-stage (i.e., screening and validation) case-control study in which 200 cases and 100 control subjects have been recruited. In the first stage, Whole Exome Sequencing (WES) was used to screen for pathogenic variants of Myocardial Infarction (MI). In the second stage, selected variants of both APOE and PON1 genes (rs7412, rs429358, rs854560, and rs662) were analyzed through MassARRAY genotyping. Risk Allele Frequencies (RAFs) distribution and association of the selected SNPs with MI were determined using the Chi-square test and logistic regression analysis. WES identified a total of 12 sequence variants in APOE and 16 in PON1. Genotyping results revealed that APOE variant rs429358 (ɛ4 allele and ɛ3/ɛ4 genotype) showed significant association in MI patients (OR = 2.11, p value = 0.03; 95% CI = 1.25-2.43); whereas no significant difference (p˃ 0.05) was observed for rs7412. Similarly, the R allele of PON1 Q192R (rs662) was significantly associated with cases (OR = 1.353, p value = 0.048; 95% CI = 0.959-1.91), with particular mention of RR genotype (OR = 1.523, p value = 0.006; 95% CI = 1.087-2.132). Multiple logistic regression analysis showed that rs429358 (C allele) and rs662 (R allele) have a significantly higher risk of MI after adjustment for the conventional risk factors. Our study findings suggested that the rs429358 variant of APOE and PON1 Q192R are associated with MI susceptibility in the Pashtun ethnic population of Pakistan.