Development and assessment of immediate-release tablets containing clopidogrel bisulphate & aspirin-strategy for optimizing the combination formulation.

The main goal of the study was to improve the compliance and convenience of patients by designing and development of an immediate release (IR) fixed-dose combination (Clopidogrel bisulphate and Aspirin) tablets. The proposed combination product utilizes Clopidogrel to protect the moisture-sensitive aspirin component, enhancing its stability against atmospheric conditions. Response-surface approach (Design Expert vs. 13) was used to generate this IR tablet by calculating the right composition of independent variables such as Microcrystalline cellulose 102, pregelatinized starch and Hydroxypropyl cellulose. 32 factorial design was used to estimate the effects of these independent variables on the responses of dependent variables (disintegration & friability) and constructed a total of nine (9) formulations. Pre and Post formulation, quality control parameters were investigated as per pharmacopeia. A systematic approach was used for the optimization process and a prototype checkpoint batch (CPB) based on the better contrast of independent variables was prepared. In vitro analysis of formulations was carried out to estimate the responses. Friability was found in the range of 0.088-1.076%w/w, except F1 = 1.076 all are within limits (NMT 1.0%). Disintegration time was recorded 7.3 ± 1.20 as lower and 24.5 ± 1.63 min was the highest. The release of drugs from their dosage form was fast and rapid, for clopidogrel after 15min was 70.42-96.82% with SD ± 8.71 and aspirin was 69.88-91.49% in 15 min with SD ± 6.41, all the tablets were released more than 80% in 20 min. The stability outcomes of CPB tablets after 15 days of stress study (60 ± 2°C and 75 ± 5%) indicated good compatibility and stability of APIs with excipients. It was concluded that the direct compression method can be preferred to prepare a combination product with cost-effectiveness. It was also concluded that the proposed methodology could increase Aspirin's stability and allow for an aqueous coating system to finish the product with a film coating. By using Design Expert software, the best composition of the formulation can be selected and optimized in a short period of time with minimum trial and errors. The results also demonstrated that the use of a fixed-dose combination tablet instead of the individual is expected to be more convenient to patients and thus improves patient compliance and decreases the occurrence of adverse effects and side effects.

Sequence- and structure-specific RNA oligonucleotide binding attenuates heterogeneous nuclear ribonucleoprotein A1 dysfunction.

The RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (A1) regulates RNA metabolism, which is crucial to maintaining cellular homeostasis. A1 dysfunction mechanistically contributes to reduced cell viability and loss, but molecular mechanisms of how A1 dysfunction affects cell viability and loss, and methodologies to attenuate its dysfunction, are lacking. Utilizing in silico molecular modeling and an in vitro optogenetic system, this study examined the consequences of RNA oligonucleotide (RNAO) treatment on attenuating A1 dysfunction and its downstream cellular effects. In silico and thermal shift experiments revealed that binding of RNAOs to the RNA Recognition Motif 1 of A1 is stabilized by sequence- and structure-specific RNAO-A1 interactions. Using optogenetics to model A1 cellular dysfunction, we show that sequence- and structure-specific RNAOs significantly attenuated abnormal cytoplasmic A1 self-association kinetics and A1 cytoplasmic clustering. Downstream of A1 dysfunction, we demonstrate that A1 clustering affects the formation of stress granules, activates cell stress, and inhibits protein translation. With RNAO treatment, we show that stress granule formation is attenuated, cell stress is inhibited, and protein translation is restored. This study provides evidence that sequence- and structure-specific RNAO treatment attenuates A1 dysfunction and its downstream effects, thus allowing for the development of A1-specific therapies that attenuate A1 dysfunction and restore cellular homeostasis.

Evaluation of Hepatoprotective effects of different doses of Lyophilized Beetroot powder in albino rabbits.

Beta vulgaris L. is a vegetable most commonly consumed in salads and has been shown to possess multiple benefits. This research was carried out to observe the effects of Beta vulgaris powder at different doses orally in albino rabbits on liver biochemical parameters and coagulation. The study was carried out on albino rabbits which were divided into three groups designated as Group I (administered distilled water) Group II and III (administered beetroot powder at 500mg/kg and 1000mg/kg dose respectively) orally for 2 month duration. The sample was withdrawn at day 0, 30th and 60th day through cardiac puncture. The results showed that both doses of Beta vulgaris were considered safe for use as all the liver parameters were significantly decreased compared to control. Among both doses 500mg/kg dose was considered safer as it reduced the parameters significantly compared to 1000mg/kg dose. Blood coagulation factors at both the doses showed significant increase which was in reference range. Beta vulgaris is a highly beneficial dietary product with ample amount of flavonoids and anti-oxidant agents which might help in improving the liver function and also play a role in coagulation by increasing both fibrinogen levels and prothrombin time.

Determination of anxiolytic and antidepressant like activity of hydro alcoholic fruit extract of Pyrus communis (L.) and its impact on the memory after chronic dosing in animal models.

The purpose of this study was to evaluate the anxiolytic and antidepressant activity of ethanolic fruit extract of Pyrus communis (pear), in comparison with escitalopram in rodents (rats and mice). Thirty Wistar rats of about 200-250gm and albino mice of 25-30gm, male gender were divided into three groups each comprising of (n=10) animal respectively. Control group received distilled water, positive control received 10mg escitalopram & treated group received 200mg/kg/day of Pyrus communis ethanolic fruit extract orally for 30 days. They were evaluated by using the open field test, forced swim test (FST), plus maze test, light and dark test, hole poking test, stationary rod test, water maze test & cage crossing activity. Results were expressed as mean ± SD. Data was analyzed by using SPSS software (VERSION 21) one way ANOVA followed by Tukey test was used for post hoc analysis. Our result showed that fruit extract had significant antidepressant-like behavior in FST (p<0.001), open field (p<0.05), cage crossing (p<0.001) , significant anxiolytic activity in light and dark box test, plus-maze activity and significantly enhanced learning in water maze and stationary rod test when compared with control. The Pyrus communis fruit extract showed the anxiolytic and antidepressant-like profile in rats and mice. However, further studies need to be carried out in clinical trials for its use in different neuropsychological disorders.

A herbal treatment approach for the management of clinical, hormonal and ultrasound parameters in reproductive age group women with polycystic ovarian syndrome: A randomized clinical trial.

Around fifteen percent women of reproductive age have been effected by Polycystic Ovarian Syndrome (PCOS); a complicated disorder; and apparently there is no standard therapy available. Considering this lack, we design present work; for the assessment of a herbal medicine (Femitex-SP4) in managing PCOs. During 2016-17; this study was carried out at Abbasi Shaheed hospital, Karachi, Pakistan. A total of 150 patients aged between 18-44 years were included as per Rotterdam criteria. Patients received 500 mg of powdered herbs in capsule form twice daily. The primary outcomes were regular menstruation and ovulation plus change in fasting blood sugar levels. Changes in free testosterone levels and ovarian morphology was secondary outcome measures. Continuous outcomes before and after treatment were compared by Student's t-test (one tailed, independent). P = 0.05 was considered as significant. Women menstrual cycle was considerably improved. Fasting blood sugar levels did not change (p=0.103392). Progesterone levels were same at the starting point and after treatment (P=0.318322). With complete recovery in 6 patients; a notable change was found in ovarian size. Free testosterone levels were also dropped significantly (p<0.00001). Our main success was drastic improvement in normalizing menstrual cycle during therapy. Herbal treatment is proven to be clinically effective in most of the patients; particularly PCOs patients with menstrual irregularities. Hence, Femitex-SP4 can be taken as a better treatment for PCOs.

Higher incidence of prescribing rates and utilization of broad spectrum cephalosporin in young patients: A retrospective observational study in a tertiary care hospital.

Frequent use of antibiotics has been developed resistance and the use of broad spectrum cephalosporin must be limited in children. The study evaluated the association of prescribing patterns of third generation cephalosporin with diagnosis, age, availability of cultural sensitivity report and gender. It is an observational study that was carried out in the duration of six months in a low socio-economic tertiary care hospital. The data of six hundred and eighty-five (685) patients were collected from the medical records of the tertiary hospital. The cephalosporin are the most prescribed antibiotics in children 118/217 (54.3%) followed by adults 119/403 (29.5%) and teenagers 18/65 (27.6%). Whereas, 75/217 (34.5%), 126/403 (31.2%) and 22/65 (33.8%) were prescribed cephalosporin with combination in patients respectively. The culture sensitivity was performed only in 25% of patients i.e., 173/685, Of 173 culture reports 70 and 91 cases from children and adults respectively. Blood is mostly examined specimen in children and urine in adults. Escherichia coli was highly recovered pathogen in culture sensitivity report. The study concluded broad spectrum cephalosporin antibiotics were highly prescribed in children. The culture sensitivity was performed in limited number of patients. Antibiotics stewardship programme will be implemented to reduce the prescribing of broad spectrum cephalosporin in young patients.

In-vitro bioassays of aqueous and ethanol extracts of Aseel dates.

Aseel is amongst the most vital date variety of Pakistan. Beside its nutritional value, it also got remedial uses therefore for the first time different in-vitro bioassays were performed to assess its medicinal value. Aqueous (DFAE) and ethanol (DFEE) extracts of fresh Aseel dates were used for the purpose. Microplate alamar blue assay was done for antibacterial activity, Brine shrimp lethality test for cytotoxicity and MTT assays with different cancer cell lines were used for anti-cancer activity. Antioxidant and anti-inflammatory activity were also evaluated by free radical scavenging bioassay and chemiluminescence technique. Alamar blue assay of both extracts exhibited weak antibacterial activity against E.coli, S, flexenari and S. aureus. Brine shrimp lethality revealed absence of cytotoxicity at 1000µ/mL concentration. DFEE 50 µ/mL was effective against MCF-7,MDA-MB-231, PC3, 3T3 and Hela cancer cell lines showing 17.59%, 20.90%, 37.60%, 22.35% and 36.70% inhibition whereas DFAE exhibits 20.46%, 30.86%, 15.21%, 29.70% and 16.40 % inhibition respectively. Similarly both extracts also showed varying degree of anti-oxidant and anti-inflammatory activity against standard drug. The results are suggestive of weak bioactivity of Aseel date extracts might because of reduced potency however further studies are required for better understanding of observed results and separation of active ingredients from Aseel dates.

Investigation of anti-inflammatory and analgesic activities of camel milk in animal models.

Opioids and non-opioids have long been used as analgesic, anti-inflammatory and antipyretic. Long-term use of these drugs may lead to severe toxicities. Therefore natural remedies are now being explored to avoid risk of adverse effects associated with the use of these conventional medicines. Bioactive components from milk of different species have been identified as nutraceuticals, but no experimental or clinical study is conducted so far to explore the analgesic and anti-inflammatory potential of camel milk. In this study we evaluated camel milk for its possible analgesic and antiinflammatory activity. The anti-inflammatory effects of camel milk was studied in rats using paw edema method (induced by acetic acid) while tail-flick method was used to evaluate its analgesic effect in mice. Significantly increased tail-flick latency was shown after camel milk (33ml/kg) treatment when compared with acetylsalicylic acid at all time intervals. Anti-inflammatory activity of camel milk was significant (p<0.001) at 4th hour of treatment as shown by maximum percentage inhibition in edema volume (46.84%) in comparison to control. Results of our present study suggested possible use of camel milk as adjuvant therapy in treating various chronic pain and inflammatory ailments. Camel milk could further be investigated in future for recognition of biochemical constituents responsible for its antiinflammatory and pain relieving activities.

Community onset of CTX-M extended spectrum ß-lactamases among uropathogenic E. coli and K. pneumoniae from Karachi, Pakistan.

Urinary tract infections (UTIs) are major health issue in developing countries like Pakistan, become more complicated with extended spectrum ß-lactamase (ESBL) expression in Escherichia coli and Klebsiella pneumoniae. The ground of this present study was to evaluate the incidence of cefotaxime (CTX-M) gene in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. The clinical isolates from various specimens were collected for one-year duration from January till December 2015. After initial screening (n=352) isolates were examined for phenotypic expression of ESBLs by double disc synergy test. Furthermore, eight-four isolates were analyzed by polymerase chain reaction for identification of Cefotaxime (CTX-M), Temoneira (TEM) and Sulfhdryl variable (SHV) genes. Among eighty-four clinical isolates CTX-M was dominant and found positive in 50 isolates (59.5%) followed by TEM in 35 (41.6%) and SHV in 11 (13%). In uropathogenic E. coli and K. pneumoniae, ESBLs gene was found in 50 and 6 isolates out of 57 and 7 respectively. Among uropathogens CTX-M was most prevalent 78% (39/50) in E. coli followed by K. pneumoniae. In uropathogenic E. coli, CTX-M was found dominant in females. The study concluded that ESBL related uropathogenic E. coli were CTX-M dominant, showed community onsets of UTIs that can be preventive and controlled with modified hygienic practices.

Phytochemical standardization of Vasicinein cough syrup by high performance liquid chromatography-densitometry.

The highly oriented modern detection techniques provide a precise and definite tool for investigation in natural medicines. Current study directed the standardization of eminent biomarker Vasicine in a natural cough syrup. A highly accurate and precise method of High-performance thin layer chromatography (HPTLC) has been developed to certify the quantity of vasicine inside the syrup. Ethyl acetate, chloroform, ethanol and ammonia (6:3:1: 1 v/v) were mobile phase for the study. The TLC plate silica gel G60F254 was used with CAMAG Scanner III and CAMAG Linomate 5. The detected Rf value was 0.51 in both sample and reference standard at 254 nm. International conference of Harmonization (ICH) guidelines were followed for the validation of the developed method. Linearity was achieved in the range of 200µg to 1600µg with co-efficient correlation r2=0.9995. Accuracy was found in between 98.9 to 101.4% however precision was good at both inter and intra-day. As per the standardization of ICH, the developed method was found to be reproducible and showed sharp similar peak with high resolution.

Prevalence of mecA: Genotyping screening of community acquired-MRSA isolates in Karachi, Pakistan.

Among resistant nosocomial and community pathogens, MRSA has become the most serious pathogen, causing life threatening infections worldwide. In S.aureus, quick and exact recognition of methicillin (cefoxitin) resistance has become essential. The benchmark for MRSA identification among S.aureus is the detection of the mecA gene that causes the expression of protein (PBP2a) culpable for classic ß-lactam resistance. However, the utter reliance on amplification of mecA gene as a hallmark in confirmation of methicillin (cefoxitin) resistant S. aureus is the matter of distrust by some investigators. The current investigation designed to analyse the prevalence of mecA gene among phenotypically positive MRSA isolates using molecular method and to correlate its prevalence to conventional techniques. Furthermore, antimicrobial sensitivity of mecA positive staphylococci was determined by Kirby Baeuer method. For this purpose, 201 clinical staphylococcal specimens were recovered from various diagnostic laboratories in Karachi City, Pakistan. Phenotypic existence of methicillin resistance in S. aureus was observed to be 51.7%. In contrast, when organisms were subjected for amplification of mecA gene by PCR, mecA positive isolates were 36/104 (35%) MRSA isolates. Current work raise question towards the usefulness of molecular identification of mecA gene in confirmation of methicillin resistance without correlating with conventional methods. Therefore, it is essential to consider the other possible resistance mechanisms for ß-lactams that may interact with mecA gene in the development of methicillin resistance mechanism in Staphylococcus.

Incidence of multidrug resistance and extended-spectrum beta-lactamase expression in community-acquired urinary tract infection among different age groups of patients.

OBJECTIVES: Urinary tract infection (UTI) is a frequent disorder and depends on age and gender. Ineffective empiric treatment of UTI is common when associated with extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae. The aim of the study was to investigate the prevalence of Gram-negative uropathogens of E. coli and K. pneumoniae in different age groups along with the identification of ESBL-producing uropathogens and antimicrobial susceptibility profiles. MATERIALS AND METHODS: A total of 247 uropathogens of E. coli and K. pneumoniae were collected over a period of 1 year (January-December 2015) from various diagnostic centers of Karachi city (Pakistan). Antimicrobial susceptibility analysis was performed by disc diffusion method, and identification of ESBL was performed by double disc synergy test. Categorical data of ESBL and non-ESBL uropathogens were analyzed by Pearson's Chi-square test. RESULTS: The study of 247 patients with community-acquired UTI comprised 72% females and 28% males, illustrating an increased prevalence of UTIs among females. It was also revealed that 90% belonged to the age group of 16-30 years whereas 78% related to the age group of 46-60 years. ESBL was found positive in 33.5% (63/188) of E. coli and 15.25% (9/59) in K. pneumonia, with a significant association i.e., (p =0.007). Amikacin, fosfomycin, imipenem, and tazobactam/piperacillin were found to be the effective treatment options. A significant association was found between ESBL-producing uropathogens against ciprofloxacin, enoxacin, and amoxicillin/clavulanic acid resistance (P < 0.05). CONCLUSIONS: It was concluded that for effective treatment of UTIs, appropriate screening of ESBL and culture sensitivity must be employed instead of empiric treatment.

Phenotypic expression and prevalence of multi drug resistant extended spectrum beta-lactamase producing Escherichia coli and Klebsiella pneumoniae in Karachi, Pakistan.

The aim of the present study was to evaluate the prevalence rate of ESBL producing Gram negative isolates of E. coli, K. pneumoniae and P. mirabilis, to determine the association of various factors with ESBL production and therapeutic options for the treatment. Total 352 isolates were subjected for identification of ESBL by double disc synergy test. Antimicrobial susceptibility was performed using CLSI guidelines and statistical association between ESBL/Non ESBL producers were determined by chi square at significant level of 0.05. A total of 96 isolates were ESBL positive (27%), females were 67% whereas males were 33%. E. coli was most prevalent pathogen (82%) followed by Klebsiella pneumoniae (17%). Furthermore 75% of ESBL associated infections were urinary tract infections. 95% of ESBL producing isolates were multidrug resistant and tazobactam/piperacillin combination and imipenem are good choices with 100% and 97% susceptibility respectively. E coli (OR 2.83, 95% CI 1.585-5.072, RR 2.22, p 0.0004) and K. pneumoniae (OR 0.52, 95% CI 0.285-0.952, RR 0.609, p 0.032) were significantly associated with ESBL production. The spread of ESBL producing multidrug resistant E. coli and K. pneumoniae has increased and proper screening for ESBL identification is needed because of limited therapeutic antibiotic choices.

MRSA: Prevalence and susceptibility pattern in health care setups of Karachi.

This assessment aims to determine the prevalence of methicillin resistance and multidrug resistance (MDR) among the clinical isolates of Staphylococcus aureus and antimicrobial susceptibility profile of methicillin resistant Staphylococcus aureus (MRSA) to the frequently prescribed antibiotics in Karachi. Isolates of MRSA, recovered from various clinical samples were included in this prospective, cross-sectional study from Jan 2015 to June 2017. Agar diffusion method was employed according to the protocols of Clinical Laboratory Standards Institute. Out of total 346 S. aureus strains, the frequency rate of MRSA was 52% (n = 180). MRSA infection was found higher among the age group 21-30 years i.e. 30% (n=54), followed by 20% (n=36) in 31-40 years. Frequency of MRSA percentage in male and female was and 70% and 30% respectively. MRSA was more frequently observed in blood 20% (n=36). MRSA showed high resistance (100%) to Oxacillin and Cefoxitin while 25% Vancomycin resistant S. aureus (VRSA) isolates and 25% Teicoplanin resistance were also reported. MRSA exhibited 16% resistance to Minocycline. It was concluded that MRSA pose a challenging threat to public health in Karachi. In addition, MDR should be periodically checked to avoid treatment failure.

Evaluation of anti-inflammatory and anti-oxidant potential of seed extracts of Vernonia anthelmintica.

Seeds of Vernonia anthelmintica in the form of Ethanol seed extract of Vernonia anthelmintica (EEVA), Hexane extract of Vernonia anthelmintica (HEVA) and water decoction of Vernonia anthelmintica (WDVA) were evaluated for their in-vivo anti-Inflammatory potential in carrageenan induced rat paw model. The results were compared to anti-inflammatory activity of standard drug (ibuprofen) and untreated groups. In-vitro evaluation of antioxidant potential of EEVA and HEVA were also conducted by "DPPH scavenging assay". The results of present study depicts that HEVA and EEVA in higher dose possess a strong anti-inflammatory potential as compared to standard anti-inflammatory drugs, whereas WDVA showed milder anti-inflammatory potential. DPPH assay has revealed strong anti-oxidant potential of EEVC with the percentage Radical Scavenging activity (%RSA) of 89.709 at concentrations of 500 ul as compared to standard drugs gallic acid (23.436±0.43) and acetyl salicylic acid (111.44±0.7) at concentrations of 95.95 µM. The other extract HEVC has shown to have insignificant %RSA at the concentration of 500µl. Hence the present study revealed that selected extracts of Vernonia anthelmintica exhibited significant in-vitro antioxidant and in-vivo anti-inflammatory potential.