Drug loaded essential oil microemulsions enhance photostability and evaluation of in vitro efficacy.

Loss of stability of pharmaceutical APIs (Active Pharmaceutical Ingredient) due to photolytic activity has been a major concern in the pharmaceutical industry as it leads to loss of activity of API and excipients, the formation of toxic by-products, change in color and flavor. Itraconazole (ITZ) bulk drug was exposed to UVC (254 nm) irradiation in an environmental chamber (37 °C, 75 %RH) and its photoprotection by cinnamon, clove, eugenol, and oregano based microemulsion was analyzed. No significant change in the spectra was observed at various time points, confirming the photo-protective activity of microemulsions, unlike the bulk drug. FTIR spectra illustrate the fundamental peaks of the functional groups of ITZ and ITZ loaded MEs. The overlaid spectra showed that there was a minor change in peaks of UV exposed ITZ bulk drug but the ITZ loaded microemulsions were able to protect all the major functional groups. The in vitro anti-microbial assay against C. albicans demonstrated no significant change in the activity of ITZ loaded microemulsion between untreated, 7th day and 15th day while the activity of bulk drug was reduced drastically in the UVC exposed sample. It was concluded that microemulsions can be used as an effective photo-protective drug delivery vehicle for light-sensitive compounds.

Molecular Characterization of a Novel Germline VHL Mutation by Extensive In Silico Analysis in an Indian Family with Von Hippel-Lindau Disease.

Von Hippel-Lindau [VHL] disease, an autosomal dominant hereditary cancer syndrome, is well known for its complex genotype-phenotype correlations. We looked for germline mutations in the VHL gene in an affected multiplex family with Type 1 VHL disease. Real-Time quantitative PCR for deletions and Sanger sequencing of coding regions along with flanking intronic regions were performed in two affected individuals and one related individual. Direct sequencing identified a novel heterozygous single nucleotide base substitution in both the affected members tested, segregating with VHL phenotype in this family. This variant in exon 3, c.473T>A, results in substitution of leucine, a highly conserved acid, to glutamine at position 158 [p.L158Q] and has not been reported thus far as a variant associated with disease causation. Further, this variant was not observed in 50 age and ethnicity matched healthy individuals. Extensive in silico prediction analysis along with molecular dynamics simulation revealed significant deleterious nature of the substitution L158Q on pVHL. The results of this study when collated support the view that the missense variation p.L158Q in the Elongin C binding domain of pVHL may be disease causing.

Usefulness of Succinate dehydrogenase B (SDHB) immunohistochemistry in guiding mutational screening among patients with pheochromocytoma-paraganglioma syndromes.

Genetic testing of pheochromocytomas (PCC) and paragangliomas (PGL), although expensive, is gradually becoming a part of the routine laboratory investigation for patients with PCC-PGL syndrome. Recently, Succinate dehydrogenase B (SDHB) immunochemistry has been shown to be an excellent indicator of germline mutations in the SDH genes and could help significantly reduce cost. This study assesses the utility of SDHB immunohistochemical analysis when used to guide genetic analysis, with emphasis on cost benefits it could provide in a resource-limited setting. Forty-four cases of PCC/PGL characterized by genetic analysis were included to determine their SDHB expression pattern by immunohistochemistry. SDHB antibody expression was negative among three cases each, with SDHB and SDHD mutations. Immunohistochemistry results were positive for all three cases of RET, a single case of neurofibromatosis and for two cases with Von Hippel-Lindau (VHL) mutations while the remaining two cases with VHL mutations showed a diffuse 'cytoplasmic blush'. Thirty of the remaining 31 samples demonstrated positive staining and were negative for mutations, while a lone sample that was negative for staining and mutation was not included in the final analysis as the internal control for the sample was not adequately stained. Cost analysis in our settings showed that triaging with SDHB immunohistochemistry could potentially reduce costs by USD 320-500 per patient. SDHB immunohistochemistry, when used as a guide to genetic testing, can significantly reduce the effort, time and costs of testing among patients with PCC-PGL, a huge benefit in resource limited settings.

P.Arg82Leu von Hippel-Lindau (VHL) gene mutation among three members of a family with familial bilateral pheochromocytoma in India: molecular analysis and in silico characterization.

Various missense mutations in the VHL gene have been reported among patients with familial bilateral pheochromocytoma. However, the p.Arg82Leu mutation in the VHL gene described here among patients with familial bilateral pheochromocytoma, has never been reported previously in a germline configuration. Interestingly, long-term follow-up of these patients indicated that the mutation might have had little impact on the normal function of the VHL gene, since all of them have remained asymptomatic. We further attempted to correlate this information with the results obtained by in silico analysis of this mutation using SIFT, PhD-SNP SVM profile, MutPred, PolyPhen2, and SNPs&GO prediction tools. To gain, new mechanistic insight into the structural effect, we mapped the mutation on to 3D structure (PDB ID 1LM8). Further, we analyzed the structural level changes in time scale level with respect to native and mutant protein complexes by using 12 ns molecular dynamics simulation method. Though these methods predict the mutation to have a pathogenic potential, it remains to be seen if these patients will eventually develop symptomatic disease.

Detection of large deletions in the VHL gene using a Real-Time PCR with SYBR Green.

Mutation in VHL gene causes the von Hippel-Lindau (VHL) disease, a dominantly inherited familial cancer syndrome. The VHL mutation pattern includes point mutations, small deletions and large deletions. While most mutations can be identified during sequencing, large deletions often remain unnoticed in initial mutational screening. We evaluated the utility of a previously described real-time quantitative PCR (RQ-PCR) using SYBR Green for detection of larger deletions in the VHL gene and normalized the data using two reference genes with a normal copy number i.e., ZNF80 (3q13.31) and GPR15 (3q12.1). DNA sequencing was also done on all cases included in the study. SJNB-6 cell line demonstrating distal 3p loss was used as a positive control for deletion. Out of 21 individual cases included of VHL disease, 2 cases were found with partial deletion by RQ-PCR, with an exon 1 deletion, while PCR-sequencing identified 5 cases with base pair substitution and 1 with splice site variant which were not picked up by RQ-PCR. RQ-PCR proved to be fast, accurate and sensitive for identifying large deletions and can be incorporated into the routine work-up for detection of large deletions in VHL disease.

Familial carotid body tumors in patients with SDHD mutations: a case series.

OBJECTIVE: To describe a family with hereditary paraganglioma due to a disease-causing mutation in the SDHD gene. METHODS: We present the clinical findings, diagnostic test results, treatment, and genetic test results in a family with hereditary paraganglioma. RESULTS: Three siblings with bilateral carotid body tumors presented at different time points and with varied clinical presentations. While the proband, a 20-year-old man, was not hypertensive and had normal urinary metanephrine and normetanephrine levels, his sister and brother had a more severe clinical picture, with hypertension in both and elevated normetanephrine levels in his brother (his brother had pheochromocytoma and 2 intra-abdominal paragangliomas). Mean age at presentation was 24 years. A 4-base pair frameshift mutation, c.337-340delGACT, was detected in exon 4 of the SDHD gene in all 3 patients. CONCLUSION: This is the first report of the c.337-340delGACT mutation being associated with hereditary paraganglioma; this report emphasizes the need to screen all at-risk first-degree relatives for the disease-causing SDHD mutation once it has been identified in an affected family member.

Role of cortical sparing adrenalectomy and novel variant of mutation in patient with von Hippel-Lindau disease.

Neurofibromatosis type 1 is the most common phakomatoses and is inherited in autosomal dominant fashion with complete penetrance. Secondary hypertension is common in these patients due to various causes including adrenal tumors. Pheochromocytoma is a rare catecholamine producing tumor seen in 0.5% to 5% of patients with neurofibromatosis. The combination of pheochromocytoma with neurofibromatosis is rarely reported in the literature. We recently encountered an elderly lady with this combination who successfully underwent adrenalectomy. We report the case for the uncommon occurrence and to highlight the relevant literature review about pheochromocytoma in neurofibromatosis.