Insulin-Loaded Barium Salt Particles Facilitate Oral Delivery of Insulin in Diabetic Rats.

Oral delivery is considered as the most preferred and yet most challenging mode of drug administration; especially a fragile and sensitive peptide like insulin that shows extremely low bioavailability through the gastro-intestinal (GIT) route. To address this problem, we have designed a novel drug delivery system (DDS) using precipitation-induced Barium (Ba) salt particles. The DDS can load insulin molecules and transport them through the GIT route. There were several in vitro simulation tests carried out to prove the efficiency of Ba salt particles as oral delivery candidates. All three Ba salt particles (BaSO4, BaSO3, and BaCO3) showed very good loading of insulin (>70% in all formulations) and a degree of resistance throughout a wide range of pHs from basic to acidic conditions when assessed by spectrophotometry. Particles and insulin-associated particles were morphologically assessed and characterized using FE-SEM and FT-IR. A set of tests were designed and carried out with mucin to predict whether the particles are potentially capable of overcoming one of the barriers for crossing intestinal epithelium. The mucin binding experiment demonstrated 60-100% of mucin adhesion to the three different particles. FT-IR identifies the characteristic peaks for mucin protein, particles, and particle-mucin complex re-confirming mucin adhesion to the particles. Finally, the effectiveness of nano-insulin was tested on streptozotocin (STZ) induced diabetic rats. A short acting human insulin analog, insulin aspart, was loaded into Ba salt particles at a dose of 100 IU/Kg prior to oral administration. Among the three formulations, insulin aspart-loaded BaSO4 and BaCO3 particles dramatically reduced the existing hyperglycemia. BaSO4 with loaded Insulin showed an onset of glucose-lowering action within 1 hr, with blood glucose level measured significantly lower compared to the 2nd and 3rd h (p < 0.05). Insulin-loaded BaCO3 particles showed a significant decrease in blood glucose level at 1-2 h, although the glucose level started to show a slight rise at 3rd h and by 4th h, it was back to baseline level. However, although BaSO3 particles with loaded insulin showed a trend of reduction in blood glucose level, the reduction was not found to be significant (p < 0.05) at any point in time. Therefore, oral formulations of insulin/BaSO4 and insulin/BaCO3 particles were observed as effective as native insulin aspart subcutaneous formulation in terms of onset and duration of action. Further investigation will be needed to reveal bioavailability and mechanism of action of this novel Nano-Insulin formulations.

Current strategies in extending half-lives of therapeutic proteins.

Macromolecular protein and peptide therapeutics have been proven to be effective in treating critical human diseases precisely. Thanks to biotechnological advancement, a huge number of proteins and peptide therapeutics were made their way to pharmaceutical market in past few decades. However, one of the biggest challenges to be addressed for protein therapeutics during clinical application is their fast degradation in serum and quick elimination owing to enzymatic degradation, renal clearance, liver metabolism and immunogenicity, attributing to the short half-lives. Size and hydrophobicity of protein molecules make them prone to kidney filtration and liver metabolism. On the other hand, proteasomes responsible for protein destruction possess the capability of specifically recognizing almost all kinds of foreign proteins while avoiding any unwanted destruction of cellular components. At present almost all protein-based drug formulations available in market are administered intravenously (IV) or subcutaneously (SC) with high dosing at frequent interval, eventually creating dose-fluctuation-related complications and reducing patient compliance vastly. Therefore, artificially increasing the therapeutic half-life of a protein by attaching to it a molecule that increases the overall size (eg, PEG) or helps with receptor mediated recycling (eg, albumin), or manipulating amino acid chain in a way that makes it more prone towards aggregate formation, are some of the revolutionary approaches to avoid the fast degradation in vivo. Half-life extension technologies that are capable of dramatically enhancing half-lives of proteins in circulation (2-100 folds) and thus improving their overall pharmacokinetic (PK) parameters have been successfully applied on a wide range of protein therapeutics from hormones and enzymes, growth factor, clotting factor to interferon. The focus of the review is to assess the technological advancements made so far in enhancing circulatory half-lives and improving therapeutic potency of proteins.

A community-based prospective cohort study of dengue viral infection in Malaysia: the study protocol.

BACKGROUND: Globally, dengue infections constitute a significant public health burden. In recent decades, Malaysia has become a dengue hyper-endemic country with the co-circulation of the four dengue virus serotypes. The cyclical dominance of sub-types contributes to a pattern of major outbreaks. The consequences can be observed in the rising incidence of reported dengue cases and dengue related deaths. Understanding the complex interaction of the dengue virus, its human hosts and the mosquito vectors at the community level may help develop strategies for addressing the problem. METHODS: A prospective cohort study will be conducted in Segamat district of Johor State in Peninsular Malaysia. Researchers received approval from the Malaysian Medical Research Ethics Committee and Monash University Human Research Ethics Committee. The study will be conducted at a Malaysian based health and demographic surveillance site over a 1 year period in three different settings (urban, semi-urban and rural). The study will recruit healthy adults (male and female) aged 18 years and over, from three ethnic groups (Malay, Chinese and Indian). The sample size calculated using the Fleiss method with continuity correction is 333. Sero-surveillance of participants will be undertaken to identify asymptomatic, otherwise healthy cases; cases with dengue fever who are managed as out-patients; and cases with dengue fever admitted to a hospital. A genetic analysis of the participants will be undertaken to determine whether there is a relationship between genetic predisposition and disease severity. A detailed medical history, past history of dengue infection, vaccination history against other flaviviruses such as Japanese encephalitis and Yellow fever, and the family history of dengue infection will also be collected. In addition, a mosquito surveillance will be carried out simultaneously in recruitment areas to determine the molecular taxonomy of circulating vectors. DISCUSSION: The research findings will estimate the burden of asymptomatic and symptomatic dengue at the community level. It will also examine the relationship between virus serotypes and host genotypes, and the association of the clinical manifestation of the early phase with the entire course of illness.

Targeted CYP2E1 quantification and its correlation to currently acceptable clinical biochemical indices.

BACKGROUND: The Cytochrome P450 enzymes are commonly known for their major role in metabolism. Besides its metabolic role, CYP2E1 gene expression has been associated with the onset of diabetic nephropathy. CYP2E1 protein elevation has also been reported to be responsible for the production of reactive oxygen species. The aims of this study were (i) to optimize and validate a targeted proteomic approach for quantitating CYP2E1 and validating it as a suitable clinical test, (ii) to investigate the concurrency between ESI-LCMS-MS quantitated circulating CYP2E1 and gold standard indices in the context of outpatient point-of-care clinical settings involving various groups of diabetic patients and (iii) to investigate the concurrency profile of circulating CYP2E1 protein, CYP2E1 gene expression and reactive oxygen species (ROS). This is a cross sectional study involving three groups of subjects (n = 166): control, pre-diabetes, and diabetes. We optimized a targeted proteomic approach for absolute quantification of CYP2E1. "YPEIEEK" and "GTVVVPTLYDNQEFPDPEK" were the representative peptides of CYP2E1 for our analytical method. Deuterated forms of "YPEIEEK" and "GTVVVPTLYDNQEFPDPEK" were used as internal standards. Lymphocytes were isolated from whole blood, microsomes were prepared, followed by in-solution digestion for production of tryptic peptides. Amounts of "YPEIEEK" and "GTVVVPTLYDNQEFPDPEK" from patients' samples were calculated from a calibration curve. RESULTS: "YPEIEEK" is a unique and reliable representative peptide for CYP2E1 quantification. "GTVVVPTLYDNQEFPDPEK" showed poor reproducibility and sensitivity. Incremental amounts of CYP2E1 protein in the peripheral circulation clearly showed concurrency with CYP2E1 gene expression and ROS levels in our study population. Elevations of CYP2E1 were observed even when gold standard clinical indicator for glycemic control (HbA1c) was within normal reference limits. Quantitated amounts of CYP2E1 protein in the pre-diabetes and diabetes groups showed significant difference relative to control group (p < 0.001). No significant differences were observed between the medians of pre-diabetes and diabetes groups (p = 0.870). CONCLUSIONS: CYP2E1 protein in peripheral blood can be reliably quantitated by the validated targeted proteomic approach method. Quantifiable amounts of CYP2E1 preceded abnormal HbA1C levels which indicates quantitation of CYP2E1 could be useful as an additional tool for early indication of diabetic risks and it complications.