A novel home-based method for preparing suspensions of anti-TB drugs.

BACKGROUND: Tablets are the most widely available dosage form for the treatment of TB; however, adult tablets fail to meet the needs of young children who cannot swallow these tablets or require dose titration. We tested a new, simple device (XTEMP-R®) and the methodology for converting tablets of TB drugs into a homogeneous suspension for home use by children and caregivers.METHODS: XTEMP-R is a new device used for converting tablets into liquid preparations. Four TB drugs - pretomanid, delamanid, clofazimine and bedaquiline - were dispersed in the device utilizing water and simple syrup. The reproducibility of accurately delivering aliquots from the suspension upon preparation and upon redispersion after storing for 2 days was studied.RESULTS: Suspensions of each of the drugs tested were easily prepared in about 10 min and were visually uniform in consistency. Dosages in 2 and 5 mL were assessed in suspension, and those in 5 mL tested upon redispersion after 2 days. The observed range for these dosages spanned from 94.6% to 101.1% of the theoretical concentration for the suspensions under examination. The cleaned device had no detectable residual drug.CONCLUSION: XTEMP-R can be used at home by caregivers to prepare doses of suspensions accurately for children and patients who cannot swallow tablets.

Stable, compounded bedaquiline suspensions to support practical implementation of pediatric dosing in the field.

BACKGROUND: Bedaquiline (BDQ) tablets are indicated as part of a combination regimen for the treatment of multidrug-resistant TB in adults, adolescents and children. A dispersible tablet formulation is now approved but is not currently available in all settings. The aim of this study was to develop stable extemporaneous liquid formulations of BDQ that can be stored at room temperature or 30°C for several weeks, to support pragmatic pediatric dosing in the field and reduce wastage.METHODS: BDQ tablets were suspended in simple syrup and a sugar-free vehicle. Each 20 mg/mL formulation was stored at room temperature or 30°C for 30 days in amber dispensing bottles. Appearance, BDQ potency, pH and microbial counts were determined on Days 0, 15 and 30.RESULTS: The BDQ potency in both formulations remained at 98-101% of the theoretical concentration for 30 days. The appearance, pH and microbial count of sugar-free formulation did not change during the 30-day storage. The simple syrup formulation was stable for 15 days as microbial growth was observed on Day 30.CONCLUSIONS: BDQ may be prepared in syrup or sugar-free suspensions: syrup suspensions can be stored for 15 days at room temperature and 30C, whereas sugar-free suspensions can be stored for 30 days at room temperature and 30C. This information will support practical BDQ dosing for children in the field.

Extemporaneously compounded liquid formulations of clofazimine.

BACKGROUND: Clofazimine (CFZ) is routinely used worldwide for the treatment of leprosy and TB. However, no liquid or dispersible tablet formulations of CFZ are currently available commercially for patients with challenges ingesting soft gelatin capsules or solid formulations. The aim of this research was to develop stable extemporaneous liquid formulations of CFZ that can be stored at room temperature for several weeks to enable practical dosing in the field. METHODS: Two formulations were prepared in syrup and sugar-free vehicles with CFZ tablets using a simple method that can be used in a routine pharmacy. Suspensions were stored at room temperature and at 30°C for 30 days. Formulation aliquots were tested on Days 0, 15 and 30 for appearance, pH, potency and microbial counts. RESULTS: Appearance remained unchanged during storage. The pH of both formulations was between 4.0 and 6.0. Potency was between 90% and 110% for 30 days in the syrup formulation and for 15 days in the sugar-free formulation. Microbial counts met United States Pharmacopeia 1111 limits for oral aqueous liquids and specific organisms were absent. CONCLUSIONS: A simple field-friendly method was successfully developed for the preparation of CFZ liquid formulations using commonly available ingredients. This will permit practical dosing and titration for children and other patients with swallowing challenges.

Comparison of pharmacokinetics and bioavailability of bedaquiline fumarate, benzoate and maleate in dogs.

BACKGROUND: Bedaquiline (BDQ) as a fumarate salt is indicated as part of a regimen to treat multidrug-resistant TB (MDR-TB). BDQ benzoate and maleate have been identified as promising alternatives that will encourage generic pharmaceutical houses to manufacture this drug. Our study compared the pharmacokinetics (PK) of BDQ fumarate vs. the maleate and benzoate salts in dogs.METHODS: The PK of BDQ and its active N-desmethyl metabolite M2 following intravenous administration of 1 mg/kg BDQ (as fumarate) and oral administration of 10 mg/kg BDQ as fumarate, benzoate, or maleate salts in suspension to fasted male beagle dogs was evaluated in a parallel-group and crossover study with N = 4 per group. BDQ and M2 plasma concentrations were determined up to 168 h post-dose. T-tests were conducted to compare the area under the curve, AUC0-t among groups.RESULTS: Orally administered fumarate, benzoate, and maleate salts, in parallel-group design, resulted in mean BDQ AUC0-t of 9,267 ± SD 10,182, 19,258 ± SD 11,803, and 15,396 ± SD 9,170 ng.h/ml, respectively; and in a crossover design of 9,267 ± SD 10,182, 17,441 ± SD 24,049, and 18,087 ± SD 19,758 ng.h/ml, respectively. P values were >0.05.CONCLUSION: There was no statistically significant difference in BDQ and M2 AUC0-t following oral administration of fumarate, benzoate and maleate salts in dogs.

Sugar and sugar-free liquid formulations of delamanid for patients with rifampicin-resistant TB.

BACKGROUND: Delamanid (DLM) tablets are recommended for the treatment of rifampicin-resistant TB. However, no liquid or dispersible tablet formulation of DLM is currently commercially available for patients with challenges ingesting these tablets. The aim of this study was to develop stable extemporaneous liquid formulations of DLM that can be stored at room temperature for several weeks.METHODS: DLM tablets were suspended in 1) simple syrup and 2) a specially formulated sugar-free vehicle. These suspensions containing DLM 5 mg/mL were stored in plastic prescription bottles at room temperature or 30°C for 30 days. These suspensions were evaluated for appearance, potency, pH, and microbial counts at Days 0, 15, and 30.RESULTS: The potency of DLM in each formulation remained at 98-104% of the theoretical concentration for 30 days. The appearance, pH, and microbial count did not change for the sugar-free formulation during the 30-day storage period. Microbial growth, however, was observed in the simple syrup formulation on Day 30 but not on Day 15.CONCLUSION: DLM can be formulated in sugar or sugar-free suspensions and stored at room temperature or 30°C for at least 15 and 30 days, respectively.

Stable sugar and sugar-free suspensions of pretomanid.

BACKGROUND: Pretomanid (PMD) tablets are indicated as part of a combination regimen for the treatment of adults with pulmonary extensively drug-resistant, treatment-intolerant or non-responsive multidrug-resistant TB. No commercial liquid formulation is currently available for patients unable to swallow these tablets.OBJECTIVE: To develop stable extemporaneous liquid formulations of PMD that can be stored at room temperature or 30°C for at least 4 weeks.METHODS: Crushed PMD tablets were formulated into 20 mg/mL suspensions in a simple syrup and sugar-free formulation. The PMD formulations were stored at room temperature and at 30°C for 30 days in dispensing bottles. Appearance, pH, potency and microbial counts of the suspensions were determined on Days 0, 15 and 30.RESULTS: The potency of PMD remained at 99.7-103.4% of the theoretical concentration in each formulation. The appearance, pH and microbial count did not change during the 30-day storage period. Simple syrup formulations did not require preservatives for microbial stability.CONCLUSIONS: PMD oral suspension formulations in simple syrup or in sugar-free vehicle were easily prepared by utilising commonly available equipment and ingredients and were stable for 30 days. These formulations are appropriate alternatives for patients with swallowing difficulties.

Gut Microbiome Composition Is Predictive of Incident Type 2 Diabetes in a Population Cohort of 5,572 Finnish Adults.

OBJECTIVE: To examine the previously unknown long-term association between gut microbiome composition and incident type 2 diabetes in a representative population cohort. RESEARCH DESIGN AND METHODS: We collected fecal samples from 5,572 Finns (mean age 48.7 years; 54.1% women) in 2002 who were followed up for incident type 2 diabetes until 31 December 2017. The samples were sequenced using shotgun metagenomics. We examined associations between gut microbiome composition and incident diabetes using multivariable-adjusted Cox regression models. We first used the eastern Finland subpopulation to obtain initial findings and validated these in the western Finland subpopulation. RESULTS: Altogether, 432 cases of incident diabetes occurred over the median follow-up of 15.8 years. We detected four species and two clusters consistently associated with incident diabetes in the validation models. These four species were Clostridium citroniae (hazard ratio [HR] 1.21; 95% CI 1.04-1.42), C. bolteae (HR 1.20; 95% CI 1.04-1.39), Tyzzerella nexilis (HR 1.17; 95% CI 1.01-1.36), and Ruminococcus gnavus (HR 1.17; 95% CI 1.01-1.36). The positively associated clusters, cluster 1 (HR 1.18; 95% CI 1.02-1.38) and cluster 5 (HR 1.18; 95% CI 1.02-1.36), mostly consisted of these same species. CONCLUSIONS: We observed robust species-level taxonomic features predictive of incident type 2 diabetes over long-term follow-up. These findings build on and extend previous mainly cross-sectional evidence and further support links between dietary habits, metabolic diseases, and type 2 diabetes that are modulated by the gut microbiome. The gut microbiome can potentially be used to improve disease prediction and uncover novel therapeutic targets for diabetes.

Diagnosis and Management of Pancreatic Adenocarcinoma in the Background of Chronic Pancreatitis: Core Issues.

BACKGROUND: The incidence of pancreatic adenocarcinoma (PDAC) in patients with chronic pancreatitis (CP) is as high as 5%. It is a commonly encountered diagnostic challenge in patients with CP on long-term follow-up. SUMMARY: This review consolidates the existing literature on assessment of PDAC in background of CP, its evaluation through the available investigations, surgical management, and prognostication. Recent change in symptomatology of an otherwise stable CP should raise a suspicion of malignancy. Endoscopic ultrasound (EUS) is more specific and sensitive in establishing the diagnosis of PDAC compared to cross-sectional imaging (computed tomography/magnetic resonance imaging). Intraoperative assessment with careful palpation coupled with careful clinical judgment helps in differentiating between an inflammatory mass and pancreatic cancer. Confirmation can be obtained with either preoperative EUS-guided fine needle cytology/core biopsy or intraoperative core biopsy under ultrasound guidance. However, despite complete evaluation with above options, 1-6% patients often show malignancy on final histopathological examination. Key Messages: Diagnosis of PDAC in CP needs a high index of suspicion. Cross-sectional imaging has poor negative predictive value. CA 19-9 with MUC5AC combination may become an ideal investigation. EUS with core biopsy/frozen section has a good sensitivity and specificity and low false negative results. Complete radical resection should be the aim to get long-term survival.

An ethnobotanical study of medicinal plants used in sacred groves of Kumaon Himalaya, Uttarakhand, India.

ETHNOPHARMACOLOGICAL RELEVANCE: International organizations recognize the importance of sacred groves and place them into the context of sustainable development and also emphasize to conserve biodiversity through protection of sacred groves and sties. The significance of medicinal plants from Himalayan region is well known to the world. Therefore, present study was conducted in identified sacred groves of Kumaon Himalaya to investigate and document the utilization of medicinal plants by various local communities and tribal people. MATERIALS AND METHODS: The study was conducted during 2008-2011 in four seasons of the year. Information was collected from 70 locals from different sacred groves by using free listing interviews with randomly selected informants and semi-structured questionnaires; plant specimens were collected, identified and deposited at the CSIR-NBRI herbarium (LWG), Lucknow, India. RESULTS: Seven sacred groves viz., Dhwaj, Haat Kali, Hokra, Malay Nath, Nakuleshwar, Narayan Swami Ashram and Patal Bhuvneshwar were identified from the Pithoragarh district of Kumaon Himalaya. 89 medicinal plants belonging to 52 families and 77 genera of which, 2 are lichens, 4 are pteridophytes, 3 are gymnosperms and remaining 80 plant species are angiosperms. 6 plant species are reported with new therapeutic uses for the first time in this paper. Highest informant׳s consensus factor value was found in liver disorder (0.55) and least by body pains (0.23). 55 ethnomedicinal plants are showing 100% fidelity level against various diseases. CONCLUSION: Sacred groves in Kumaon region of Indian Himalaya are rich sources and best repository of ethno-medicinally important plants with many rare, endangered and threatened species. It is an excellent example of unique traditional way of in situ conservation of different plant species.

Next-generation protein-rich potato expressing the seed protein gene AmA1 is a result of proteome rebalancing in transgenic tuber.

Protein deficiency is the most crucial factor that affects physical growth and development and that increases morbidity and mortality especially in developing countries. Efforts have been made to improve protein quality and quantity in crop plants but with limited success. Here, we report the development of transgenic potatoes with enhanced nutritive value by tuber-specific expression of a seed protein, AmA1 (Amaranth Albumin 1), in seven genotypic backgrounds suitable for cultivation in different agro-climatic regions. Analyses of the transgenic tubers revealed up to 60% increase in total protein content. In addition, the concentrations of several essential amino acids were increased significantly in transgenic tubers, which are otherwise limited in potato. Moreover, the transgenics also exhibited enhanced photosynthetic activity with a concomitant increase in total biomass. These results are striking because this genetic manipulation also resulted in a moderate increase in tuber yield. The comparative protein profiling suggests that the proteome rebalancing might cause increased protein content in transgenic tubers. Furthermore, the data on field performance and safety evaluation indicate that the transgenic potatoes are suitable for commercial cultivation. In vitro and in vivo studies on experimental animals demonstrate that the transgenic tubers are also safe for human consumption. Altogether, these results emphasize that the expression of AmA1 is a potential strategy for the nutritional improvement of food crops.

Analysis of Fluorescence Lifetime Imaging Microscopy (FLIM) data. Based on a fully automated Laguerre deconvolution method.

OBJECTIVES: A novel Fluorescence Lifetime Imaging Microscopy (FLIM) deconvolution method based on the linear expansion of fluorescence decays on a set of orthonormal Laguerre functions was recently proposed. The Laguerre deconvolution method applies linear least-square estimation to estimate the expansion coefficients of all pixel decays simultaneously, performing at least two orders of magnitude faster than the other algorithms. In the original Laguerre FLIM deconvolution implementation, however, the Laguerre parameter α is selected using a heuristic approach, making it unsuitable for online applications. METHODS: In this study, we present a fully automated implementation of the Laguerre FLIM deconvolution, whereby the Laguerre parameter α is treated as a free parameter within a nonlinear least-squares optimization scheme. RESULTS: The performance of this method has been successfully validated on simulated data, and experimental FLIM images of standard fluorescent dyes and endogenous tissue fluorescence. CONCLUSIONS: The main advantage of the proposed method is that it does not require any user intervention for tuning up the deconvolution process. Thus, we believe this method will facilitate the translation of FLIM to online applications, including real-time clinical diagnosis.

Comparative biomonitoring of leachates from hazardous solid waste of two industries using Allium test.

Hazardous industrial wastes are inevitable source of environmental pollution. Leachates from these wastes might contaminate the origins of potable water and affect human health. The study was carried out to determine the possible genotoxic effects of leachates from solid waste of a metal and dye industry using the Allium cepa chromosome aberrations assay. The 10% leachates were prepared from solid wastes obtained from both the industries and examined for the presence of heavy metal content and genotoxicity. To simulate the field and laboratory conditions, A. cepa bulbs were exposed through soil and aqueous medium for 48 h to 2.5-10% leachates. The results revealed that both metal waste leachate (MWL) and dye waste leachate (DWL) contained high concentrations of chromium, nickel and iron that significantly induced cytogenetic alterations. Significant inhibition of mitotic index (MI), inductions of chromosomal/mitotic aberrations (CA/MA) and micronuclei (MN) formation were found in all experimental groups exposed to MWL and DWL. The effects observed were concentration dependent and the frequency of aberrations was higher with treatment of MWL than DWL. The MI was severely inhibited at 10% aqueous exposure it was 4.59+/-0.69 (P<0.001) in MWL and almost half to that induced by DWL that was 8.62+/-0.69 (P<0.05). Significant frequency of CA/MA and MN induced by MWL was 14.21 (P<0.001) and 0.33 (P<0.001) whereas CA/MA and MN induced by DWL was 7.81 (P<0.001) and 0.13 (P<0.05) in the aqueous medium. The investigations inferred that abnormalities caused by MWL were higher than DWL both in soil and aqueous media. These toxic responses may have relied on raised heavy metal concentrations of metal-based than dye industrial wastes.

Cytogenetic effects of leachates from tannery solid waste on the somatic cells of Vicia faba.

The contamination of surface- and groundwater by the leaching of solid wastes generated by industrial activities as a result of water runoff and rainfall is a matter of great concern. The leachates from tannery solid waste (TSW), a major environmental pollutant, were examined for their possible genotoxic effects on the somatic cells of Vicia faba. Leachates were prepared from solid wastes procured from leather-tanning industrial sites, and V. faba seedlings were exposed to three test concentrations, 2.5%, 5%, and 10%, through soil and aqueous media for 5 days. The root tips examined for cytogenetic damage revealed that leachate of TSW significantly inhibited the mitotic index and induced significantly frequent chromosomal and mitotic aberrations (CA/MA) in a dose-dependent manner. The chemical analysis of TSW samples revealed that the chief constituents were chromium and nickel, which may cause genetic abnormalities. The frequency of aberrations was found to be higher in the root meristematic cells of Vicia faba exposed through the aqueous medium than those exposed through the soil medium. The results of the present study indicated that contamination of potable water bodies by leachates of TSW may cause genotoxicity. For the biomonitoring of complex mixtures of toxicants with the V. faba bioassay, the use of the aqueous medium seems to be a more promising method than the use of the soil medium.

Prognostic factors in betel and tobacco related oral cancer.

Paucity of well-defined prognostic molecular markers severely hampers prediction of the clinical course of squamous cell carcinoma (SCC) of oral cavity. The aim of the study was to evaluate the prognostic significance of impairments in the expression of proteins involved in cell cycle regulation and locoregional spread in oral SCC of habitual betel and tobacco chewers. A prospective study was performed in 105 betel and tobacco consumers with oral SCCs during the period 1988-1999, to evaluate the prognostic relevance of impairments in the expression of proteins involved in cell cycle regulation and locoregional spread. Alterations in the expression of p53, pRb, p16, MDM2, p21, and Ets-1 proteins were determined by immunohistochemical analysis in formalin fixed, paraffin embedded tissue sections from oral SCCs. Analysis of multiple molecular biological factors showed overexpression of p53 in 69/105 (66%) cases, MDM2 in 72/105 (69%) cases, p21 in 57/105 (54%) cases and Ets-1 in 64/105 (61%) cases. Loss of pRb was observed in 58/105 (55%) cases and p16 loss was observed in 72/105 (69%) cases. Interestingly, multivariate analysis revealed loss of pRb as the most significant predictor of advanced tumour stage [P=0.001; Odd's Ratio (OR)=3.5] and overexpression of Ets-1 protein was an independent risk factor for lymph node metastasis (OR: 10.1; P<10(-6)). Multiple logistic regression models showed that pRb loss [Hazard's Ratio (HR): 3.93] and p53 overexpression (HR: 2.97) may serve as adverse prognosticators for disease free survival of the patients. The data demonstrate multiple impairments in p53/MDM2/p21/Ets-1 and p16/pRb pathways in betel and tobacco related oral tumourigenesis.

Coexpression of Ets-1 and p53 in oral carcinomas is associated with P-glycoprotein expression and poor prognosis.

PURPOSE: To determine the association between the expression of P-gp with Ets-1 and p53 proteins in oral squamous cell carcinomas (SCCs). MATERIALS AND METHODS: Immunohistochemical analysis of Ets-1, P-glycoprotein (P-gp), and p53 proteins was carried out in 40 formalin-fixed, paraffin-embedded tissue sections from oral SCCs using specific antibodies for these proteins. RESULTS: Expression of Ets-1 protein was observed in 27/40 (68%) cases, P-gp was overexpressed in 27/40 (68%) cases, and p53 accumulation was observed in 26/40 (65%) cases. Twenty-two of 27 (82%) SCCs showed concomitant overexpression of Ets-1 and P-gp underpinning an association between the expression of these two proteins ( P=0.007). Twenty-one of 27 (78%) Ets-1 overexpressing oral SCCs showed accumulation of p53 protein ( P=0.015). Nineteen of the 27 (70%) P-gp expressing tumours showed p53 accumulation. Concomitant Ets-1 and P-gp overexpression was significantly associated with poor prognosis ( P=0.002). In multivariate analysis using Cox's proportional hazards model, P-glycoprotein emerged as the most significant adverse predictor of disease-free survival (HR=6.2, P=0.003). The hallmark of the study was the significant association between the expression of Ets-1, P-gp, and p53 proteins in oral SCCs and their association with poor prognosis. Oral cancer patients showing concomitant expression of Ets-1, P-gp, and p53 proteins had shorter disease-free survival (median time of no recurrence=18 months) and worst prognosis ( P=0.001) as compared to the cases overexpressing any of these proteins. CONCLUSION: Concomitant expression of Ets-1, P-gp, and p53 proteins adversely affects the clinical outcome in oral SCCs.

Prognostic impact of Ets-1 overexpression in betel and tobacco related oral cancer.

In oral squamous cell carcinoma (SCC), the presence of lymph node metastasis is one of the most important prognostic factors, correlating locoregional spread, recurrence, distant metastasis and survival. However, the biological factors implicated and the mechanisms underlying these events are not completely elucidated. We reported Ets-1 expression in oral SCC and proposed that it may serve as a plausible marker of invasive potential and lymph node metastasis. Herein, we investigate the prognostic significance of Ets-1 expression in oral cancer. One hundred oral SCCs assessed for Ets-1 expression showed significant association with tumor stage (P = .027), lymph node involvement (P < 10(-6)) and distant organ metastasis (P = .007). The transcription factor Ets-1 regulates the expression of several genes involved in extracellular matrix remodeling that may account for its association with lymph node and distant organ metastasis. Kaplan-Meier survival analysis entailed significant association of Ets-1 expression in oral SCCs with reduced disease free survival (P = .0041), suggesting its utility as a prognosticator for oral cancer. In conclusion, these findings underscore the role of Ets-1 in oral tumor invasion and metastasis and may thus account for its association with diminished disease free survival.

Sequence-specific delivery of a quinone methide intermediate to the major groove of DNA.

Silyl-protected phenol derivatives serve as convenient precursors for generating highly electrophilic quinone methide intermediates under biological conditions. Reaction is initiated by addition of fluoride and has previously exhibited proficiency in DNA alkylation and cross-linking. This approach has now been extended to the modification of duplex DNA through triplex recognition and fluoride-dependent quinone methide induction. Both oligonucleotides of a model duplex were alkylated in a sequence specific manner by an oligonucleotide conjugate that is consistent with triplex association. Optimum reaction required the presence of the two complementary target sequences and a pH of below 6.5. In addition, one guanine in each strand adjacent to the triplex region was the predominant site of alkylation. The yield of modification varied from approximately 20% for the purine-rich strand to only 4% for the pyrimidine-rich strand. This surprising difference indicates that the linker between the recognition and reactive elements may limit productive interaction between the quinone methide and the reactive nucleophiles of DNA. Restricted orientation of this intermediate may also be responsible for the lack of target cross-linking at detectable levels.

Plumbism--a mimicker of common childhood symptoms.

Lead poisoning is a preventable entity that can affect almost every system of the body. Its toxic effects range from subtle common childhood symptoms to even death. In a prospective study forty six children with at least one symptom compatible with plumbism were enrolled after screening patients attending medical services at AIIMS. The work-up included a detailed clinical history and examination including intelligence quotient (IQ) and behaviour. Blood lead levels were estimated in all with due precautions. Their ages ranged from 2.5 to 18 years and M:F ratio was 2:1. The common symptoms included unexplained recurrent colic (39), anorexia (21), sporadic vomiting and constipation (13), pigmented gum lines (11), growth failure (11), history of pica (9) etc. None of the children had acceptable blood lead levels (< 10 micrograms/dl). Ninety three per cent children had levels > 20 micrograms/dl and 47.8% had > 45 micrograms/dl. Statistically significant correlation with blood lead levels was seen with most symptoms. Plumbism can mimic common childhood illnesses and should be investigated in children with a clinically compatible profile. Community awareness with regard to pica reduction may be an important preventive measure. This silent epidemic needs to be aggressively tackled by a multipronged approach.