Cytochrome P-450 CYP2C19 genetic polymorphism and its relation with clopidogrel resistance.

Objectives: To find out the prevalence of CYP2C19*2 genetic polymorphism in ischaemic heart disease patients, and to determine its relation with clopidogrel resistance in different genotype groups. METHODS: The cross-sectional study was conducted from August 2015 to December 2019 at the Army Medical College, National University of Medical Sciences, Rawalpindi, Pakistan, and comprised ischaemic heart disease patients of either gender who were on clopidogrel therapy. CYP2C19*2 genotyping of all the patients was carried out through polymerase chain reaction-restriction fragment length polymorphism. Platelet aggregation analysis was done using a light transmission aggregometer. Data was analysed using SPSS 23. RESULTS: Of the 390 patients, 232(59.5%) were males and 158(40.5%) were females. The overall age range was 16-82 years. Clinical indications of clopidogrel were angina 198(50.8%), myocardial infarction 146(37.4%) and acute coronary syndrome 46(11.8%). CYP2C19*2 genotyping showed that 196(50.24%) patients were homozygous wild type carriers (GG or *1/*1), 159(40.8%) were heterozygous carriers (GA or *1/*2), and 35(9%) were homozygous polymorphic allele carriers (AA or *2/*2). Platelet aggregation studies showed that there were 157(80.1%) clopidogrel responders and 39(19.9%) clopidogrel-resistant patients among GG carriers, 118(74.2%) clopidogrel responders and 41(25.8%) clopidogrel-resistant among GA carriers, and 18(51.4%) clopidogrel responders and 17(48.6%) clopidogrel-resistant among AA carriers (p=0.001). Intergroup mean platelet aggregation was significantly different (p=0.025). Allelic frequency of dominant allele *1 and polymorphic variant allele *2 was 0.706(70.6%) and 0.294(29.4), respectively. CONCLUSIONS: Homozygous and heterozygous carriers of CYP2C19 allele *2 was found to have higher prevalence of clopidogrel resistance in the studied population.

Effects of body mass index on propofol-induced cardiovascular depression in the Pakistani population.

Objective: To determine the relationship between the patient's Body Mass Index (BMI) and the cardiovascular effects produced by propofol at a dose of 1.5 mg/kg in the Pakistani population. Methods: This descriptive cross-sectional study was conducted in the Holy Family Hospital Rawalpindi from August 2021 to January 2022. According to their BMI, one hundred twenty Pakistani individuals 18 to 60 years of age were equally divided into three groups. Group N (n = 40) with a BMI of 18 to 24.9, group OW (n=40) with a BMI of 25 to 29.5, and group O (n=40) with a BMI of 30 to 34.9 were randomized to receive propofol injections at a 1.5 mg/kg dose for induction of anesthesia. We measured mean blood pressure before the propofol and then at one, three, and ten minutes after the injection. Data were analyzed by using SPSS 22. Results: Mean blood pressure decreases significantly in all groups, as shown by p-values of <0.001 for the first two readings. In group N, blood pressure returned to near normal within ten minutes (p-value 0.061), but in groups, OW and O, mean blood pressure was significantly lower even after ten minutes (p-values 0.005 and 0.001, respectively). Individual variations in propofol response were also observed. Conclusion: In the Pakistani population, propofol at an induction dose of 1.5 mg/kg to patients with different body weights produces cardiovascular effects with marked standard deviations in each group, which indicate different individual responses. Clinical Trial Number: NCT05383534 https://register.clinicaltrials.gov/.

Unraveling Catechol-O-Methyltransferase rs4680 SNP's Role in Patients' Response to Tramadol and Its Adverse Effects: A Pharmacogenetics Insight into Postoperative Pain Management.

Effective postoperative pain management is essential for patient well-being and an efficient healthcare system. Variations in the Catechol O-Methyltransferase (COMT) gene, specifically rs4680, play a crucial role in pain perception and opioid response. This study seeks to elucidate the impact of rs4680 polymorphism on tramadol efficacy and adverse reactions in post-surgical patients. We performed an uncontrolled cohort pharmacogenetics study in which participants underwent postoperative tramadol administration. The frequencies of rs4680 alleles were determined and the association between rs4680 genotypes and the efficacy of tramadol analgesic as pain relief, measured by the Numeric Rating Scale (NRS), was analyzed. Secondary outcomes included tramadol-induced sedation levels, opioid-induced nausea and vomiting, and other adverse effects of tramadol. Data analysis, using IBM SPSS Statistics 23, focused on pain and side effect differences across genotypes, with statistical significance set to p ≤ 0.05. The COMT (rs4680) genotype distribution exhibited a 'G' allele frequency of 41.5% and an 'A' allele frequency of 58.5%, with the AA genotype present in 44% of individuals, adhering to the Hardy-Weinberg equilibrium (p = 0.788). Patients with the AA genotype reported lower pain scores post-tramadol administration across all times examined (p < 0.001), but also experienced statistically significant (p < 0.001) higher incidences of tramadol-induced nausea, vomiting, and sedation. However, GG genotype individuals experienced poor pain relief from tramadol, requiring more supplemental analgesia. These significant findings underscore the critical role of COMT rs4680 polymorphism in response to tramadol and the necessity of a personalized approach to postoperative pain management.

Reno Protective Role Of N Acetyl Cysteine And Aqueous Extract Of Berberis Lycium Royale Root Bark On Rats.

BACKGROUND: N acetyl cysteine and Berberis lycium Royale root bark have been used to treat kidney diseases. Objectives of the study were to evaluate the individual and combined effect of N acetyl cysteine and aqueous extract of Berberis lycium Royale root bark in Gentamicin induced nephrotoxicity in rats. This randomized control trial conducted at Islamic International Medical College, Rawalpindi in collaboration with NIH, Islamabad in 1 month from Sep to Oct 2020. METHODS: Fifty Wister albino rats of 10-12 weeks old were divided into five groups with 10 in each group. Group 1 was normal control given food and water only and remaining 40 were in treatment groups. Nephrotoxicity was induced by intraperitoneal injection of Gentamicin (80mg/kg) for 6 days in group 2, 3, 4 and 5. After induction of nephrotoxicity, Group 3 was administered N acetyl cysteine 140mg/kg per oral, Group 4 was given aqueous extract of Berberis lycium Royale root bark 400 mg/kg per oral and Group 5 was given both N acetyl cysteine 140mg/kg per oral and aqueous extract of Berberis lycium Royale root bark 400 mg/kg per oral for 21 days. Serum uric acid was measured in all groups after 30 days to observe the reversal of renal injury. RESULTS: The results of this study indicate that Group 3, Group 4 and Group 5 showed a decrease in serum uric acid level as compared to Disease Control Group (Group 2). However, Group 5 significantly reduced uric acid (p-0.05). CONCLUSIONS: Combined effect of N acetyl cysteine and aqueous extract of Berberis lycium Royale root bark showed improvement in uric acid level in Gentamicin induced nephrotoxicity in rats.

Effect of vitamin D supplementation on various parameters in non-alcoholic fatty liver disease patients.

There are still no FDA approved drugs for NAFLD so far. Vitamin D may be a good therapeutic option for NAFLD patients due to its insulin sensitizing and anti-inflammatory properties. The purpose is to investigate the effect of oral vitamin D supplementation on various parameters in NAFLD patients. In this double blind randomized placebo controlled trial, 109 patients of NAFLD diagnosed by abdominal ultrasound and liver enzymes were divided into two groups for treatment with oral capsule of vitamin D3 50,000 IU and capsule placebo weekly for a period of 12 weeks. Anthropometric, chemical, metabolic and inflammatory parameters were assessed pre and post treatment by using SPSS 16. After 12 weeks oral treatment with vitamin D , its level increased significantly in vitamin D group from 12.5±4.2 to 24.5±3.8 ng/ml p =0.003 vs placebo group. This rise was further accompanied by decrease in HOMA-IR (4.56±1.6 to 3.26± 1.8 p=0.003) liver enzymes (i.e. ALT: 72.±17.6 to 54.5±14.5 IU/L p=0.04; AST: 68±14.5 to 46.± 10.5 p =0.002) serum CRP 3.25±0.68 to 2.28±0.44 mg/L p =0.06 and increase in serum adiponectin 8.56 ±1.12 to 10.44±2.35 mg/L p =0.03 as compared to placebo group. However non significant changes were observed in both groups in terms of body weight, BMI, and serum lipid profiles. Vitamin D supplementation not only improved its own status but also caused a significant amelioration in metabolic, chemical and inflammatory parameters in NAFLD patients. So it should be consider as an adjunctive therapy in NAFLD patients.

Zinc and iron complexes of oleanolic acid, (OA) attenuate allergic airway inflammation in rats.

Oleanolic acid (OA) is a hydroxyl pentacyclic triterpene acid (HTAs) used in various ailments. Inflammatory diseases may be profoundly influenced by iron (Fe) and zinc (Zn) status. We studied the anti-asthmatic effects of two metal complexes (Fe and Zn) of OA in the ovalbumin (OVA)-induced rat model. Delayed type hypersensitivity (DTH) was measured. Total and differential leucocyte count was done in blood as well as bronchoalveolar lavage fluid (BALF). The mRNA expression levels of pro-inflammatory cytokines were measured in lung tissue by reverse transcription polymerase chain reaction. The levels of cyclooxygenase-2 (COX-2), immunoglobulin E (IgE) and 5-lipoxygenase (5-LOX) were estimated by enzyme linked immunosorbent assay. Splenocyte proliferation was performed through BrdU uptake method and nitric oxide levels were measured by colorimetric assay kit. The acute toxicity study was also done for the complexes. The asthmatic group developed allergic airway inflammation shown by increased DTH and inflammatory markers in blood and BALF. OA + Fe and OA + Zn displayed significant decrease in DTH, NO, expression of IL-4, 5, 13, 17, toll-like receptor-2, nuclear factor-kappa B and tumor necrosis factor-α; serum IgE, COX-2, and 5-LOX. The metal complexes also attenuated OVA-stimulated splenocyte proliferation. While no hepatotoxic or nephrotoxic potential was shown by OA + Fe and OA + Zn. Our findings indicate that both OA + Fe and OA + Zn possess significant anti-asthmatic effect which may be ascribed to its immunomodulatory and anti-inflammatory features.

Potentiating effect of rifampicin on methimazole induced hepatotoxicity in mice.

Methimazole (MMI) is a widely used drug for hyperthyroidism. However, its clinical use is associated with hepatotoxicity. Though the precise mechanism of hepatic damage is still far from clear, role of metabolic activation and reactive metabolites have been implicated. The present study was designed to investigate the role of enzyme induction in bioactivation based hepatotoxicity of methimazole in mice. Thirty male mice were randomly divided into five groups. Hepatotoxicity was induced by single intraperitoneal injection of methimazole (1000mg/kg). Pretreatment with rifampicin which is a potent enzyme inducer was carried out for 6 days prior to administration of methimazole. The extent of hepatic damage was determined by measuring serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) along with histopathological grading of liver samples. The elevated levels of biochemical markers by methimazole were potentiated by pretreatment with rifampicin. This potentiation of hepatic injury was also observed in liver histopathological examination. These findings suggest induction of microsomal enzymes as a potentiating factor of methimazole induced hepatotoxicity.

Association of anti-emetic efficacy of Ondansetron with 18792A>G polymorphism in a drug target gene 5-HT3B in Pakistani population.

OBJECTIVE: To evaluate the association of anti-emetic efficacy of ondansetron with 18792A>G polymorphism in the target gene of 5-hydroxytryptamine type 3 subtype B. METHODS: The prospective clinical study was conducted at Combined Military Hospital, Rawalpindi and the genetic analysis was carried out at Institute of Biomedical and Genetic Engineering, Islamabad from August 2012 to September 2013. The subjects enrolled were undergoing elective laparoscopic cholecystectomy under general anaesthesia. All the patients were given anti-emetic ondansetron (4mg) intravenously 30 minutes before the end of surgery. Within the first two hours after surgery the response to ondansetron was noted down. Patients with the complaints of vomiting and those who had no vomiting were analysed for 18792A>G polymorphism using polymerase chain reaction- restriction fragment length polymorphism method. RESULTS: Of the 350 patients, 183(52%) had complaints of vomiting and 167(48%) had no such complaints. Overall, 195(56%) patients had 18792AA genotype, 130(37%) had genotype AG, and 25(7%) had GG genotype. No significant association was found between the incidence of vomiting and the 18792A>G genotypes at 2 hours after surgery (p>0.05). CONCLUSIONS: No association of anti-emetic efficacy of ondansetron with 18792A>G polymorphism in the target gene of 5-hydroxytryptamine type 3 subtype B was found.

Alpha-tocopherol ameliorates nephrotoxicity associated with the use of colistin in rabbits.

The alarming rise in the rate of multi drug resistant, life threatening gram negative infections has brought renaissance in the use of Colistin for last two decades. The major constraint in its utilization is its nephrotoxicity. Therefore it is being underused which is favoring the development of resistance. This study assesses the prevention of nephrotoxicity associated with high and low toxic doses of Colistin by alpha-tocopherol. Thirty rabbits were randomly divided into five groups. Baseline serum urea, creatinine and electrolytes were estimated. A loading dose of colistin was given in the form of infusion followed by I.M injections for six days. In the preventive groups α-tocopherol was additionally given orally for two weeks. Rabbits were sacrificed 24 hours after the last dose. The kidney slides graded and statistically analyzed using "chi square". The results of serum analysis were compared using one way analysis of variance followed by post hoc tukey test. There was marked nephrotoxicity in high toxic group where as in low toxic group mild nephrotoxicity was evident. Alpha-tocopherol attenuated the renal insult in both the toxic groups. As damage induced by colistin is oxidative in nature, thus it was concluded that the protection offered by α- tocopherol is due to its antioxidant activity.

Mini Review - Analysis of Artemether and Dihydroartemisinin by high performance high liquid chromatography in biological fluids-issues and solutions.

Artemether-Lumefantrine is the most widely recommended antimalarial combination used to treat millions of patients suffering from malaria. Artemether undergoes rapid metabolism and gets converted to its active metabolite dihydroartemisisn. Drug analysis is a vital aspect to evaluate drugs in research. There are a number of methods available for the determination of artemether in biological fluids. These methods include HPLC based UV detection, GS-MS, HPLC-ECD and HPLC-MS/MS. This article reviews different methods for the determination of artemether in the biological fluids. Among the available methods HPLC-MS/MS proves to be the most accurate and reliable one for analysis. This has the advantage of improved sensitivity and selectivity with smaller sample volume.

Frequency of CYP2D6*10 genotypes in Pakistani breast cancer patients taking adjuvant tamoxifen.

OBJECTIVE: To investigate the frequency of cytochrome P2D6*10 in breast cancer patients. METHODS: This retrospective study was conducted at the Nuclear Medicine, Oncology and Radiotherapy Institute, Islamabad, and the Combined Military Hospital, Rawalpindi, Pakistan, and comprised medical records of breast cancer patients from January 2000 to September 2013. Pre- and post-menopausal women with diagnosed breast cancer who were advised 20mg/day of tamoxifen as adjuvant therapy were included. The frequency of the cytochrome was determined using polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: Of the 232 participants, 25(10.8%) had stage I disease, 127(54.7%) had stage II and 80(34.5%) had stage III disease. The overall mean age was 46.9±9.9 years. The allele frequency of cytochrome CYP2D6*1 was 431(93%) and that of CYP2D6*10 was 33(7 %). Pakistanis differed significantly from the Asian populations and other ethnic groups in the distribution of the allele cytochrome, but its frequency was comparable to South Indians. CONCLUSIONS: The frequency of CYP2D6*10 allele in Pakistani breast cancer women was comparable to the South Indians, but was significantly lower than other populations.

Influence of genetic variations in ABCB1 on the clinical efficacy of ondansetron - A pharmacogenetic analysis of Pakistani population.

OBJECTIVE: To study the association of C1236T single-nucleotide polymorphisms of ABCB1 gene with non-responsiveness to antiemetic treatment in post-operative patients. METHODS: The prospective, clinical trial was conducted at Combined Military Hospital, Rawalpindi, and the Institute of Biomedical and Genetic Engineering, Islamabad, in 2012-13, and comprised patients undergoing elective laparoscopic cholecystectomy. All patients were given 0.1 mg/kg ondansetron intravenously 30 minutes before the end of surgery and Deoxyribonucleic acid samples were obtained. The frequencies of genotypes of Single Nucleotide Polymorphism were determined by polymerase chain reaction followed by restriction fragment length polymorphism. RESULTS: Of the 426 patients, 201(47%) were responders having no nausea or vomiting, and 225(52.8%) were non-responders having nausea or vomiting. The incidence of post-operative nausea and vomiting during the first 2 hours after surgery was significantly lower in patients with 1236TT genotype than other 1236 genotypes (p<0.001). It was significantly higher in patients with CC genotype at 2 hours than other 1236 genotypes (p<0.001). CONCLUSIONS: Polymorphism of ABCB1 gene may be a good guide for predicting responsiveness for ondansetron.

Polymorphisms of the ABCB1 Gene in the Pakistani Population.

OBJECTIVE: To investigate the frequency of the single nucleotide polymorphism C1236T in exon 12 of the ABCB1 gene in Pakistani population and to compare it with published data on Asian and Caucasian populations. STUDY DESIGN: Across-sectional observational study. PLACE AND DURATION OF STUDY: Combined Military Hospital, Rawalpindi and Institute of Biomedical and Genetic Engineering (IBGE), Islamabad, from August 2012 to May 2013. METHODOLOGY: C1236T polymorphism was investigated in 426 Pakistani subjects. The frequency was compared with the published data on other Asian and Caucasian populations. RESULTS: The frequencies of ABCB1 C1236T were 16.4% for CC, 44.1% for CT and 39.4% for TT. Pakistanis differed significantly from all the European populations compared in the distribution of the TT genotype of C1236TABCB1 (p < 0.05). The Pakistani population also differed significantly from some of the European populations in the distribution of CC and CT genotype (p < 0.05). CONCLUSION: There was significant difference in the genotype frequency of the ABCB1 gene compared to other populations. This study has provided a framework for future pharmacogenetic and pharmacokinetic studies on this polymorphic variant of ABCB1 gene in the Pakistani population.

Association of Anti-Emetic Efficacy of Ondansetron with G2677T Polymorphism in a Drug Transporter Gene ABCB1 in Pakistani Population.

OBJECTIVE: To determine the association of ABCB1 polymorphism G2677T with anti-emetic efficacy in patients treated with ondansetron for preventing postoperative nausea and vomiting. STUDY DESIGN: A clinical trial. PLACE AND DURATION OF STUDY: Combined Military Hospital, Rawalpindi and Institute of Biomedical and Genetic Engineering, Islamabad, from 2012 to 2013. METHODOLOGY: Four mg ondansetron was administered intravenously 30 minutes before the end of surgery. A total of 246 patients with the complaints of nausea and vomiting and 244 patients without nausea and vomiting were analyzed for G2677T polymorphism using PCR-RFLP method. Results were described as frequency percentages and chi-square test with significance at p < 0.05. RESULTS: The patients with TT genotype had significantly lower incidence of postoperative nausea and vomiting during the first 2 hours (p < 0.001) and between 2 - 24 hours after surgery as compared to other genotypes (p < 0.001). The patients with GG genotypes had significantly higher incidence of this complaint (p=0.014). CONCLUSION: Polymorphism of ABCB1 has an association with responsiveness for ondansetron. There is a role for genetics in the management of PONV.

Protective effect of metformin against gentamicin induced nephrotoxicity in rabbits.

Gentamicin is used against gram negative infections, but major problem encountered is nephrotoxicity that occurs in 10-20% of therapeutic regimes. Gentamicin induced oxidative stress plays an important role in this nephrotoxicity. Recent data has shown metformin to possess antioxidant activity. Purpose of study was to evaluate potential role of metformin in protecting kidney from nephrotoxicant insult. Thirty-six rabbits were randomly divided into six groups (n=6). G-1 received 1 ml isotonic saline intraperitoneally (IP) daily for 13 days. G-2 received gentamicin (150 mg/kg/day) IP for last 6 days of 13 days. G-3 received gentamicin (40 mg/kg/day) IP for 13 days. G-4 received metformin salt (100 mg/kg/day) dissolved in drinking water via feeding tube for 13 days. G-5 received metformin salt (100 mg/kg/day) via feeding tube for 13 days plus gentamicin (150mg/kg/day) IP for last 6 days of 13 days. G-6 received gentamicin (40mg/kg/day) IP plus metformin salt (100mg/kg/day) via feeding tube for 13 days. Blood was collected on days 0 and 14 for serum urea & creatinine estimation. All animals were sacrificed and kidneys were removed for renal histological examination. Metformin showed nephroprotective effect. It blunted nephrotoxic insult at 150mg/kg/day of gentamicin, whereas showed complete nephroprotection at 40mg/kg/day of gentamicin. Metformin offers complete nephroprotection at low toxic dose ranges of gentamicin. This could offer an efficacious and cheaper treatment alternative in those diabetics who also suffer from gram-negative infections.

Effects of rocuronium pretreatment on muscle enzyme levels following succinylcholine.

Succinylcholine revolutionized anaesthetic practice by providing intense neuromuscular blockade of very rapid onset and ultrashort duration, thereby greatly easing the maneuver of tracheal intubation. However the worth of succinylcholine is limited by the frequent occurrence of muscular side effects which manifest biochemically in the form of rise in serum creatine kinase (CK). The administration of small doses of nondepolarizing muscle relaxants before the administration of succinylcholine has been shown to decrease the incidence and severity of muscular side effects experienced by the patients. This study was aimed at evaluating the efficacy of technique in reducing the muscular side effects of succinylcholine, biochemically manifested as rise in CK. Sixty healthy adults were enrolled in the study who were scheduled for minor muscle cutting surgeries under general anaesthesia. They were assigned at random to two groups of thirty patients each. They randomly received succinylcholine for intubation and a precurarization dose of rocuronium followed by succinylcholine for intubation. Blood samples were drawn for estimation of serum creatinine kinase. There was a significantly raised CK in the succinylcholine group. In the precurarization group the rise in CK was prevented and the levels were significantly less as compared to the group which received succinylcholine alone. Present study concluded that precurarization with rocuronium was effective in reducing the succinylcholine-induced rise in creatinine kinase.

Analgesic and anti-inflammatory effects of Pistacia integerrima extracts in mice.

AIM OF STUDY: Extracts of Pistacia integerrima galls have been dispensed by Traditional Practitioners of Subcontinent for chest diseases as well as for aches and pains in the body. This study was planned to evaluate the possible analgesic and antiinflammatory effects of Pistacia integerrima extracts. MATERIALS AND METHODS: Analgesia was determined using acetic acid induced abdominal constriction and formalin induced paw licking in mice. Antinociceptive effect was observed by thermally induced algesia in mice. RESULTS: Pistacia integerrima leaves extracts showed significant response against chemically induced pain (P<0.001) whereas galls extracts had highly significant protection (P<0.0001) in a dose dependent manner. In thermally induced algesia, Pistacia integerrima galls extracts 200 mg/kg (p.o.), showed significant (P<0.05) response but less than pentazocine and diclofenac, positive references. The extracts of Pistacia integerrima 50-200 mg/kg (p.o.) had modest activity against hind paw acute and chronic inflammation induced by formalin (P<0.01). CONCLUSION: These results demonstrate that Pistacia integerrima extracts have antinociceptive and analgesic effects and no apparent acute toxicity on oral administration.

Clinical investigation of hypoglycemic effect of seeds of Azadirachta-inidca in type-2 (NIDDM) diabetes mellitus.

The present study was designed to investigate clinically the hypoglycemic effect of seeds of Azadirachta indica in Type-2 diabetes mellitus. After assaying fasting plasma and urinary glucose, 10 patients of type-2 diabetes mellitus with no previous medication, 10 patients of type-2 diabetes mellitus taking oral hypoglycemic agents with history of inadequate control and six control subjects were given low (0.5 g tid) and high (2 g tid) doses of powdered part, aqueous extract and alcoholic extract of Azadirachta indica for 14 days. On 15th day blood and urine samples for glucose were taken. Based on results obtained it was found that Azadirachta indica has significant hypoglycemic activity in high dose and can be successfully combined with oral hypoglycemic agents in type-2 diabetic patients whose diabetes is not controlled by these agents.