Assessment of active pharmaceutical ingredients in drug registration procedures in Pakistan : implications for the future

Introduction: There are concerns over the quality of generic medicines in Pakistan. This is due to perceived non-compliance with good manufacturing practice (GMP), whereby the quality of the raw materials is not being assessed. If not addressed, this will impact on the potential for generics exports from Pakistan, as well as on patient care. Consequently, there is a need to assess the current assessment and regulatory situation in Pakistan and to recommend a way forward that ensures the future quality of products. Objective: To assess the quality of the raw materials that are either imported to, or manufactured in Pakistan, that are then used to produce a leading analgesic (ibuprofen). As part of the assessment, the presence and levels of impurities will be determined. Subsequently, the findings will be used to recommend potential regulatory changes to improve patient care. Methods: The quality of 27 sourced raw materials and the reference product were assessed using a variety of quality assessment methodologies, including assay tests, and infrared spectroscopy and UV-spectrophotometry. The calculated values were then compared to values documented in the Certificate of Analysis (CoA), and high performance liquid chromatography (HPLC). Results: All but one sample passed the spectroscopy identification tests. However, 81.5% of samples failed to comply with pharmacopoeia assay limits. There were also concerns with the use of HPLC methods used to assess the quality of raw materials, and over the fact that assay values obtained were not the same as those listed in the CoA. In addition, where manufacturers had concerns over the amount of ibuprofen in the raw material, rather than rejecting the product, they typically used higher quantities to make up any shortfall. Conclusions: The results of this study demonstrate that there is an urgent need to improve the registration process for generic products in Pakistan. This should include implementing Common Technical Documents (CTDs) based on international standards. In this way, we will see generics being produced that result in improved patient care and that have potential for export from Pakistan

Adolescent Afghan refugees display a high prevalence of hyperhomocysteinemia and associated micronutrients deficiencies indicating an enhanced risk of cardiovascular disease in later life

A growing body of research evidence suggests that elevated homocysteine level (hyperhomocysteinemia) is an independent risk factor for cardiovascular diseases. The current study aimed to investigate the prevalence and associated risk factors for hyperhomocysteinemia among adolescent Afghan refugees aged 10−19 years. In total, 206 healthy adolescent boys and girls were randomly recruited from a refugee village in Peshawar, Pakistan, in 2020. Socio-demographic data, anthropometric assessment, and blood sample collection were performed following standard methods. Serum homocysteine was assessed using a chemiluminescent microparticle immunoassay, with hyperhomocysteinemia defined as levels ≥ 15 µmol/L. The overall prevalence of hyperhomocysteinemia was 25%, with mean homocysteine levels significantly (p = 0.004) higher among boys (14.1 µmol/L) than girls (11.8 µmol/L). Multivariate logistic regression analysis revealed a significant association between hyperhomocysteineimia and serum levels of vitamin B12 (OR 0.29; 95% CI of 0.14 to 0.62; p 0.01) and folate (OR 0.1; 95% CI of 0.03 to 0.27; p 0.001). Overall, our study findings indicate high prevalence of hyperhomocysteinemia among adolescent Afghan refugees who are potentially at high risk of developing cardiovascular diseases in future. There is a dire need to develop and implement nutritional and public health strategies to control hyperhomocysteinemia, protect against related diseases and complications in future, and ensure healthy lives and well-being among these vulnerable populations

Effect of Drought on Trichome Density and Length in Cotton (Gossypium Hirsutum)

Cotton is a major cash crop and backbone of the textile industry in Pakistan which is badly affected by sucking insects. Drought is an important abiotic factor in trichome development. The objective of the study was to determine the effects of drought on trichome density and length. Trichome density was measured in two ways, one through the scaling method and the other through counting the trichome density manually. The scaling method is qualitative grading while quantitative grading includes trichomecount in a card of optimized length. Three scales were finalized to classify leaves on the basis of trichomes which were counted in a specific area (0.25cm2) on abaxial side of the leaf. In drought stress, trichomes density and length were measured and compared to that in normal conditions. Trichome density varies from 12 to 56 in 0.25cm2 under drought stress. On the basis of correlation of trichome density with stomatal conductance, photosynthetic rate, PAR and transpiration ratio under drought and normal conditions, it was concluded that trichome density increased as a result of drought stress

FREQUENCY OF BONE FRACTURES DETECTED BY PLAIN RADIOGRAPHY AND KEEPING CT AS GOLD STANDARD

Background: The main cause of bone fracture is traffic accidents are (72.2%), falls (11.6%), blunt injuries are (7.7%) and others (5.8%)). About 84 patients were included in our studies. Out of which 21 (25.0%) were females and 63(75.0%) were males. Objective: The objective of this study is to evaluate the frequency of bone fractures detected by plain radiography and keeping CT as gold standard. Methodology: In our cross-sectional study all the patients with fracture undergoing CT and X-Ray were included. Patients with bone surgery were excluded as well as patients who declined to give consent and those who were uncooperative were all excluded. X-ray performed for all the fractures and the complex one sent to CT. The consent form was obtained by patients in this study. We reviewed our data of patients who underwent CT and X-ray. CT (Toshiba 64) was performed from the exact area of fracture and images reconstructed at 3mm and for reporting 5mm for filming. X-ray performed according to the appropriate range of kVp and mAs. All findings of CT were considered by the advice of consultant radiologist. Data was represented with means of standard deviation of frequency and percentage where appropriate. Chi square testing was used to compare CT and X-ray qualitative data. Data was entered into IBM SPSS Statistics 24.0. P-value <0.05 considered the significant. Results: In our study 84 patients were included. Out of which 21 (25.0%) were females and 63 (75.0%) were males and the standard deviation was 0.436. There causes of fracture were 18(21.4%) injury, 8(9.5%) were compression fractures, 54(64.3%) RTA, 2(2.4%) pathologic fractures and 1(1.2%) was sports injury fracture and the standard deviation was .997. Conclusion: Our study concluded that the bone fractures are more common in males than females. The most common cause of bone fractures is RTA (Road Traffic Accidents). Study also concludes that most common type of bone fracture is Transverse bone fracture. CT was more efficient in detecting bone fractures than plain radiography because more fractures were detected on CT than plain radiography. Keywords: Non-enhanced CT, X- ray, Bone fracture, Road Traffic Accidents DOI: 10.7176/JHMN/93-09 Publication date:September 30th 202

Micronutrient Status and Dietary Diversity of Women of Reproductive Age in Rural Pakistan

Consuming a diverse diet is essential to ensure an adequate intake of micronutrients. The aim of this study was to assess the nutritional status and dietary diversity of women of reproductive age (WRA) living in a marginalized community in rural Pakistan. Forty-seven WRA (35 ± 7 years old) who were not pregnant or lactating at enrollment, were recruited to participate in the study. Twenty-four-hour dietary recall interviews were conducted by the study nutritionist, and the data collected were used to create a minimum dietary diversity for women score (MDD-W) on five occasions during the monsoon and winter seasons (October to February). Nutritional status was assessed using anthropometry and biochemical markers of micronutrient status. Height and weight were used to determine body mass index (BMI), and mid-upper-arm circumference was measured. Plasma zinc, iron, and selenium concentrations were measured using inductively coupled mass spectrometry, and iron status was assessed using serum ferritin and blood hemoglobin concentrations. The mean (±SD) food group diversity score was 4 ± 1 with between 26% and 41% of participants achieving an MDD-W of 5. BMI was 27.2 ± 5.5 kg/m2 with 28% obese, 34% overweight, and 6% underweight. The prevalence of zinc deficiency, based on plasma zinc concentration, was 29.8%; 17% of the participants had low plasma selenium levels; 8.5% were iron deficient; and 2% were suffering from iron deficiency anemia. The findings indicate that the women living in this community consume a diet that has a low diversity, consistent with a diet low in micronutrients, and that zinc deficiency is prevalent. Public health interventions aimed at increasing the dietary diversity of WRA are needed to improve the micronutrient intake, particularly of zinc, in this population

Examining the effectiveness of consuming flour made from agronomically biofortified wheat (Zincol-2016/NR-421) for improving Zn status in women in a low resource setting Pakistan: Study protocol for a randomised, double blind, controlled cross over trial (BiZiFED)

Introduction: Dietary zinc (Zn) deficiency is a global problem, particularly in low and middle-income countries where access to rich, animal-source foods of Zn is limited due to poverty. In Pakistan, Zn deficiency affects over 40% of the adult female population, resulting in sub-optimal immune status and increased likelihood of complications during pregnancy. Methods and analysis: We are conducting a double blind, randomised controlled feeding study with crossover design, in a low resource setting in Pakistan. Households were provided with flour milled from genetically and agronomically biofortified grain (Zincol-2016/NR-421) or control grain (Galaxy-2013. Fifty households were recruited. Each household included a female aged 16-49 years who is neither pregnant nor breastfeeding, and not currently consuming nutritional supplements. These women were the primary study participants. All households were provided with control flour for an initial 2-week baseline period, followed by an 8-week intervention period where 25 households receive biofortified flour (Group A) and 25 households receive control flour (Group B). After this eight-week period, Group A and B crossed over, receiving control and biofortified flour respectively for eight weeks. Tissue (blood, hair and nails) have been collected from the women at five time points: baseline, mid and end of period 1, mid and end of period 2. Ethics and dissemination: Ethical approval was granted from the lead University (reference number: STEMH 697 FR) and the collaborating institution in Pakistan. The final study methods (including any modifications) will be published in peer reviewed journals, alongside the study outcomes on completion of the data analysis. In addition, findings will be disseminated to the scientific community via conference presentations and abstracts and communicated to the study participants through the village elders at an appropriate community forum

The Impact of Consuming Zinc-Biofortified Wheat Flour on Haematological Indices of Zinc and Iron Status in Adolescent Girls in Rural Pakistan: A Cluster-Randomised, Double-Blind, Controlled Effectiveness Trial

Biofortification of wheat is potentially a sustainable strategy to improve zinc intake; however, evidence of its effectiveness is needed. A household-based, double-blind, cluster-randomized controlled trial (RCT) was conducted in rural Pakistan. The primary objective was to examine the effects of consuming zinc-biofortified wheat flour on the zinc status of adolescent girls aged 10–16 years (n = 517). Households received either zinc-biofortified flour or control flour for 25 weeks; blood samples and 24-h dietary recalls were collected for mineral status and zinc intake assessment. Plasma concentrations of zinc (PZC), selenium and copper were measured via inductively coupled plasma mass spectrometry and serum ferritin (SF), transferrin receptor, alpha 1-acid glycoprotein and C-reactive protein by immunoassay. Consumption of the zinc-biofortified flour resulted in a moderate increase in intakes of zinc (1.5 mg/day) and iron (1.2 mg/day). This had no significant effect on PZC (control 641.6 ± 95.3 µg/L vs. intervention 643.8 ± 106.2 µg/L; p = 0.455), however there was an overall reduction in the rate of storage iron deficiency (SF < 15 µg/L; control 11.8% vs. 1.0% intervention). Consumption of zinc-biofortified flour increased zinc intake (21%) but was not associated with an increase in PZC. Establishing a sensitive biomarker of zinc status is an ongoing priority

Biofortified Wheat Increases Dietary Zinc Intake: A Randomised Controlled Efficacy Study of Zincol-2016 in Rural Pakistan

A new variety of zinc biofortified wheat (Zincol-2016) was released in Pakistan in 2016. The primary aim of this study was to examine the effects of consuming Zincol-2016 wheat flour on biochemical and functional markers of zinc status in a population with widespread zinc deficiency. An individually-randomised, double-blind, placebo-controlled cross over design was used. Fifty households were recruited to participate in the study, with each household included at least one woman of reproductive age (16–49 years) who was neither pregnant nor breast feeding or currently taking nutritional supplements. All households were provided with control flour for an initial 2-week baseline period, followed by the intervention period where households were randomly allocated in a 1:1 ratio to receive biofortified flour (group A; n = 25) and control flour (group B; n = 25) for 8-weeks, then switched to the alternate flour for 8-weeks. The trial has been registered with the ISRCTN (https://www.isrctn.com), ID ISRCTN83678069. The primary outcome measure was plasma zinc concentration, and the secondary outcome measures were plasma selenium and copper concentrations, plasma copper:zinc ratio and fatty acid desaturase and elongase activity indices. Nutrient intake was assessed using 24-h dietary recall interviews. Mineral concentrations in plasma were measured using inductively coupled plasma mass spectrometry and free fatty acids and sphingolipids by mass spectrometry. Linear Mixed Model regression and General Linear Model with repeated measures were used to analyse the outcomes. Based on an average flour consumption of 224 g/day, Zincol-2016 flour provided an additional daily zinc intake of between 3.0 and 6.0 mg for white and whole grain flour, respectively. No serious adverse events were reported. This resulted in significant, increase in plasma zinc concentration after 4 weeks [mean difference 41.5 μg/L, 95% CI (6.9–76.1), p = 0.02]. This was not present after 8 weeks (p = 0.6). There were no consistent significant effects of the intervention on fatty acid desaturase and elongase activity indices. Regular consumption of Zincol-2016 flour increased the daily zinc intake of women of reproductive age by 30–60%, however this was not associated with a sustained improvement in indices of zinc status

Estimation of heritability and genetic advance for various metric traits in seven F2 populations of bread wheat (Triticum aestivum L.)

Heritability estimates provide information about the traits that are transmitted from parents to their offsprings and it has a crucial role in selection criteria to improve grain yield. The purpose of the present study was to compute heritability and genetic advance of seven F2 populations derived through cross combinations of five parental varieties/lines of bread wheat Iqbal-2000, Lasani- 2008, 9444, 9432 and 9436. Data were recorded for seven metric traits: plant height, days to heading, flag leaf area, peduncle length, number of kernels per spike, kernel weight per spike and grain yield per plant. The estimates of heritability were low to high for all the traits studied except for kernel weight per spike which had a moderate to high value of heritability. Results depict that all the mentioned traits can be enhanced easily due to high values of heritability and phenotypic variations. The cross combinations 9436 × Iqbal-2000 and 9436 × Lasani-2008 had high heritability for most of the traits. Therefore, selection could be practiced among wheat crosses with special emphasis on crosses, namely 9436 × Iqbal-2000, to improve grain yield per plant

Effect of Drought on Trichome Density and Length in Cotton (Gossypium Hirsutum)

Cotton is a major cash crop and backbone of the textile industry in Pakistan which is badly affected by sucking insects. Drought is an important abiotic factor in trichome development. The objective of the study was to determine the effects of drought on trichome density and length. Trichome density was measured in two ways, one through the scaling method and the other through counting the trichome density manually. The scaling method is qualitative grading while quantitative grading includes trichomecount in a card of optimized length. Three scales were finalized to classify leaves on the basis of trichomes which were counted in a specific area (0.25cm2) on abaxial side of the leaf. In drought stress, trichomes density and length were measured and compared to that in normal conditions. Trichome density varies from 12 to 56 in 0.25cm2 under drought stress. On the basis of correlation of trichome density with stomatal conductance, photosynthetic rate, PAR and transpiration ratio under drought and normal conditions, it was concluded that trichome density increased as a result of drought stress