RP-HPLC method to assay ethinylestradiol and drospirenone with 3D chromatograph model in fixed dose in rat plasma for pharmacokinetic study analysis.

For the simultaneous measurement of Ethinylestradiol (EE) and Drospirenone (DP) in fixed-dose combination hormones tablets, a reverse-phase high-performance liquid chromatographic (RP-HPLC) method was developed. A specific, precise and accurate RP-HPLC method was developed and validated to analyse the drugs in rat plasma. The fluorescence detection for EE was made at λ= 200-310 nm and Ultraviolet-visible (UV/Vis) detection for DP was made at 270 nm. The typical EE and DP retention times were 4.19 and 5.30 minutes, respectively. The limit of detection (LOD) and limit of detection (LOQ) for EE were 0.121 and 0.282µg/mL and LOD and LOQ for DP were 2.23 and 7.697µg/mL respectively. The regression coefficient (r2) of EE and DP were 0.9937 and 0.9913 respectively. Precision's relative standard deviation (RSD) was less than 5%. The analyte recoveries of both drugs stayed within 95% of each other. All other validation parameters adhered to ICH standards. Throughout the analytical process, the analyte was stable. The advantages of the method developed include stability under different conditions and a low limit of quantification that was in micrograms. Its applicability was confirmed by the analysis of EE and DP levels in plasma samples in a designed pharmacokinetic study in rats after oral administration.

Control synthesis of mesoporous silica microparticles: Optimization and in vitro cytotoxicity studies.

The current paper explains how to make mesoporous silica microparticles (MSM) by mixing water and dichloromethane. Several dichloromethane-water ratios were used to adjust the reaction mixture for the first time to easily synthesize mesoporous silica micro particles with regulated particle size. By carefully modifying the concentrations of water and dichloromethane, a higher level of consistency was achieved in the production of micro particles, i.e. to a 2:1 v/v ratio. It was discovered that variations in the dichloromethane-to-water ratios significantly affect the surface roughness and morphologies of mesoporous silica particles along with size. This is most likely because the solvent affects how quickly tetraethyl orthosilicate (TEOS) and how quickly inorganic species polymerize. In all experiments, conditions were maintained the same at 25oC temperature and 1000 rpm. Scanner electron microscopy (SEM), Fourier transform infrared (FTIR) and X-ray powder diffraction (XRD) methods were used to identify the structure of MSM. The in vitro cytotoxicity assays showed that the produced particles, which had a diameter of 1.0 m, were safe for usage in the cellular system.

Genetic Investigation of Consanguineous Pakistani Families Segregating Rare Spinocerebellar Disorders.

Spinocerebellar disorders are a vast group of rare neurogenetic conditions, generally characterized by overlapping clinical symptoms including progressive cerebellar ataxia, spastic paraparesis, cognitive deficiencies, skeletal/muscular and ocular abnormalities. The objective of the present study is to identify the underlying genetic causes of the rare spinocerebellar disorders in the Pakistani population. Herein, nine consanguineous families presenting different spinocerebellar phenotypes have been investigated using whole exome sequencing. Sanger sequencing was performed for segregation analysis in all the available individuals of each family. The molecular analysis of these families identified six novel pathogenic/likely pathogenic variants; ZFYVE26: c.1093del, SACS: c.1201C>T, BICD2: c.2156A>T, ALS2: c.2171-3T>G, ALS2: c.3145T>A, and B4GALNT1: c.334_335dup, and three already reported pathogenic variants; FA2H: c.159_176del, APTX: c.689T>G, and SETX: c.5308_5311del. The clinical features of all patients in each family are concurrent with the already reported cases. Hence, the current study expands the mutation spectrum of rare spinocerebellar disorders and implies the usefulness of next-generation sequencing in combination with clinical investigation for better diagnosis of these overlapping phenotypes.

A homozygous founder variant in PDE2A causes paroxysmal dyskinesia with intellectual disability.

Intellectual developmental disorder with paroxysmal dyskinesia or seizures (IDDPADS, OMIM#619150) is an ultra-rare childhood-onset autosomal recessive movement disorder manifesting paroxysmal dyskinesia, global developmental delay, impaired cognition, progressive psychomotor deterioration and/or drug-refractory seizures. We investigated three consanguineous Pakistani families with six affected individuals presenting overlapping phenotypes partially consistent with the reported characteristics of IDDPADS. Whole exome sequencing identified a novel missense variant in Phosphodiesterase 2A (PDE2A): NM_002599.4: c.1514T > C p.(Phe505Ser) that segregated with the disease status of individuals in these families. Retrospectively, we performed haplotype analysis that revealed a 3.16 Mb shared haplotype at 11q13.4 among three families suggesting a founder effect in this region. Moreover, we also observed abnormal mitochondrial morphology in patient fibroblasts compared to controls. Belonging to diverse age groups (13 years-60 years), patients presented paroxysmal dyskinesia, developmental delay, cognitive abnormalities, speech impairment, and drug-refractory seizures with variable onset of disease (as early as 3 months of age to 7 years). Together with the previous reports, we observed that intellectual disability, progressive psychomotor deterioration, and drug-refractory seizures are consistent outcomes of the disease. However, permanent choreodystonia showed variability. We also noticed that the later onset of paroxysmal dyskinesia manifests severe attacks in terms of duration. Being the first report from Pakistan, we add to the clinical and mutation spectrum of PDE2A-related recessive disease raising the total number of patients from six to 12 and variants from five to six. Together, with our findings, the role of PDE2A is strengthened in critical physio-neurological processes.

A Novel Nonsense Variant in GRM1 Causes Autosomal Recessive Spinocerebellar Ataxia 13 in a Consanguineous Pakistani Family.

Background and objectives: Autosomal recessive spinocerebellar ataxia-13 (SCAR13) is an ultra-rare disorder characterized by slowly progressive cerebellar ataxia, cognitive deficiencies, and skeletal and oculomotor abnormalities. The objective of this case report is to expand the clinical and molecular spectrum of SCAR13. Methods: We investigated a consanguineous Pakistani family with four patients partially presenting with clinical features of SCAR13 using whole exome sequencing. Segregation analysis was performed by Sanger sequencing in all the available individuals of the family. Results: Patients presented with quadrupedal gait, delayed developmental milestones, non-progressive peripheral neuropathy, and cognitive impairment. Whole exome sequencing identified a novel pathogenic nonsense homozygous variant, Gly240*, in the gene GRM1 as a cause of SCAR13 that segregates with the recessive disease. Discussion: We report a novel homozygous nonsense variant in the GRM1 gene in four Pakistani patients presenting with clinical features that partially overlap with the already reported phenotype of SCAR13. In addition, the family presented quadrupedal gait and non-progressive symptoms, manifestations which have not been recognized previously. So far, only four variants in GRM1 have been reported, in families of Roma, Iranian, and Tunisian origins. The current study adds to the mutation spectrum of GRM1 and provides a rare presentation of SCAR13, the first from the Pakistani population.

Preparation of smart PVP/HPMC based IPN hydrogel, its characterization and toxicity evaluation.

In this study, the interpenetrating polymeric network (IPN) were fabricated via free radical polymerization using polymers hydroxypropyl methylcellulose (HPMC), Polyvinylpyrrolidone (PVP) and monomer Methacrylic acid (MAA) and also investigated their influence by changing their concentrations. The developed polymeric network is crosslinked via N' N' -methylene bis-acrylamide (MBA). Different characterizations have been performed to analyze fabricated interpenetrating polymeric network structure i.e., Scanning Electron Microscopy (SEM), X-ray Powder Diffraction (XRD), Fourier-Transform Infrared Spectroscopy (FT-IR), Thermo-gravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). Letrozole (LTZ) was loaded as a model drug in the developed system. Swelling dynamics as well as drug release behavior were thoroughly examined. FTIR studies corroborated the formation of interpenetrating polymeric network. SEM uncovered porous structure while TGA depicted enhanced thermal stability of polymeric network. PXRD depicted amorphous dispersion of LTZ. Swelling dynamics as well as LTZ release behavior from developed interpenetrating polymeric network hydrogels were dependent upon pH of the medium and concentration of pure reactants employed. Higuchi model was best fit to regression coefficient which indicated diffusion controlled mechanism of drug release. Acute oral toxicity study depicted no mortality or any signs relating to acute toxicity throughout the whole observed period. Hence, the designed interpenetrating polymeric network might turn out to be a safe and a potential carrier system for the delivery of LTZ in the treatment of breast cancer (BC).

Modifier Genes in Microcephaly: A Report on WDR62, CEP63, RAD50 and PCNT Variants Exacerbating Disease Caused by Biallelic Mutations of ASPM and CENPJ.

Congenital microcephaly is the clinical presentation of significantly reduced head circumference at birth. It manifests as both non-syndromic-microcephaly primary hereditary (MCPH)-and syndromic forms and shows considerable inter- and intrafamilial variability. It has been hypothesized that additional genetic variants may be responsible for this variability, but data are sparse. We have conducted deep phenotyping and genotyping of five Pakistani multiplex families with either MCPH (n = 3) or Seckel syndrome (n = 2). In addition to homozygous causal variants in ASPM or CENPJ, we discovered additional heterozygous modifier variants in WDR62, CEP63, RAD50 and PCNT-genes already known to be associated with neurological disorders. MCPH patients carrying an additional heterozygous modifier variant showed more severe phenotypic features. Likewise, the phenotype of Seckel syndrome caused by a novel CENPJ variant was aggravated to microcephalic osteodysplastic primordial dwarfism type II (MOPDII) in conjunction with an additional PCNT variant. We show that the CENPJ missense variant impairs splicing and decreases protein expression. We also observed centrosome amplification errors in patient cells, which were twofold higher in MOPDII as compared to Seckel cells. Taken together, these observations advocate for consideration of additional variants in related genes for their role in modifying the expressivity of the phenotype and need to be considered in genetic counseling and risk assessment.

Antiviral activity of green silver nanoparticles produced using aqueous buds extract of Syzygium aromaticum.

In the present study, we synthesized silver (Ag) nanoparticles using aqueous extracts of clove (Syzygium aromaticum) (SAE). This synthesis of green silver nanoparticles (AgNP) was a novel and effectual tool against the Newcastle Viral Disease (NDV). Syzygium aromaticum extract was used as reducing and stabilizing agent for synthesis of silver nanoparticles. AgNP were characterized using diversity of biophysical methods inclusive of Fourier transform infrared spectroscopy (FTIR), UV-VIS spectroscopy and Transmission electron microscopy (TEM) for morphology and size. Furthermore, XRD analysis confirmed the crystalline nature of the particles. In current investigations, the antiviral activity of clove buds silver nanoparticles was inspected in-vitro and in-ovo. Embryonated chicken eggs were used to perform the cytotoxicity assay of the clove extract silver nanoparticles (CESN). CESN showed in vitro antiviral activity against NDV in embryonated eggs.

Formulation and in vitro evaluation of colchicines and naproxen sodium sustain release tablets in combination for treatment of gout.

Present investigation concern with combination of two drugs for the treatment of gout. One of these drug (naproxen sodium) is pain killer which is sustain their action within the body for 12 hours and the other drug (colchicine) is anti-gout, which release as conventional dosage. After oral administration naproxen will act as sustain release dosage and increase patient compliance about six batches of tablet were developed and evaluate .For the sustain release action polymers Methocel K4M and HPMCK15were used. These polymers were used in combination used with other inactive ingredients. Two methods were used for proration of final tablets. In 1st method only naproxen sodium granules were prepared which are sustained released. In second method these granules were mixed with colchicines powder and other all inactive ingredients. This method is easy and cost effective characterization of pallets and final tablets were performed. Final tablets were evaluated for all tests like appearance, friability, dissolution, hardness, assay, weight variation and in-vitro release study performed. The results obtained were satisfactory and complies with USP specification. Formulation containing combination of Methocel K4M and HPMC K15 showed good sustain release profile for 12 hours.