Gagan Kaushal

Genetically Engineered Normal Flora for Oral Polypeptide Delivery: Dose–Absorption Response

Genetically modified Lactococcus lactis (L. lactis), a probiotic bacterium, able to secrete beta-lactamase (29 kDa), was used as a vector for the oral delivery of beta-lactamase to the rats. Three different doses of L. lactis were administered to the rats, and the resulted beta-lactamase oral bioavailability was studied, and compared to the solution form. The oral administration of 1.2 x 10(7), 3 x 10(7), and 8 x 10(7) colony-forming units of L. lactis led to 145, 209, and 364 mU of beta-lactamase absorbed, and the corresponding bioavailability was 8.7%, 15.5%, and 20.8% based on the in vitro production of beta-lactamase by L. lactis. The oral administration of 504 mU and 1008 mU beta-lactamase free solution resulted in 30 and 47 mU absorbed, a bioavailability of 5.9% and 4.7%, respectively. L. lactis significantly (p < 0.01) increased the oral bioavailability compared to the free solution form. A significant (p < 0.01) increase in the MAT value as compared to the solution, demonstrated that L. lactis can be used as a sustained delivery system. In conclusion, there is a linear relationship between L. lactis dose and these absorption PK parameters within L. lactis dose range of the current study.

A randomized placebo-controlled pilot study of the efficacy and safety of D-cycloserine in people with chronic back pain:

Background Few effective pharmacological treatment options exist for chronic back pain, the leading cause of disability in the US, and all are associated with significant adverse effects. Objective To determine the efficacy and safety of D-cycloserine, a partial agonist to the N-methyl-D-aspartate receptor, in the treatment of chronic low back pain. Methods A total of 41 participants with chronic back pain who met all inclusion and exclusion criteria were enrolled in a double-blind, placebo-controlled randomized pilot trial of D-cycloserine. Treatment was administered orally for six weeks at escalating daily doses of 100 mg, 200 mg, and 400 mg, each for two weeks. The primary outcome measure was back pain intensity using the Numeric Rating Scale (0–10). Secondary measures were back pain-related questionnaires: McGill Pain Questionnaire short form, painDETECT, PANAS, and BDI. The pre-specified analysis was a two-way repeated measures analysis of variance. Results A treatment difference was observed between groups treated with D-cycloserine and placebo at six weeks of 1.05 ± 3.1 units on the Numeric Rating Scale, with an effect size of 0.4 and p = 0.14. This trend of better chronic back pain relief with D-cycloserine was also observed in the secondary measures. No safety issues were seen. Conclusion The difference in mean pain between the D-cycloserine and placebo groups did not reach statistical significance. However, a clinically meaningful effect size in the magnitude of pain relief was observed with a consistent pattern across multiple outcome measures with good safety, supporting further research into the effectiveness of D-cycloserine for chronic back pain.

D-cycloserine nasal formulation development for anxiety disorders by using polymeric gels

D-cycloserine (DCS), a partial agonist at N-methyl-D-aspartate (NMDA) receptors, is used as an enhancer of exposure therapy for anxiety disorders. The purpose of the present study was to investigate the feasibility of using polymeric gels to increase the viscosity of the formulation and thereby increase the nasal residence time and sustained release of DCS in vitro. Hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), and methyl cellulose (MC) were prepared at concentrations of 0.5 to 5% w/v. Pluronic F-127 (PF-127) was prepared at concentrations of 15 to 35% w/v. pH, viscosity and in vitro DCS release behavior of the formulated gels were analyzed. All four gels that were tested, demonstrated sustained DCS release behavior over a 24-hour period, but with different rates. Based on the results of this study, HPMC, HPC, MC, and PF-127 are capable of increasing the viscosity of nasal gel formulations and of releasing DCS in sustained manner. Therefore, these polymeric gels can be suitable carriers for DCS nasal gel formulation.

Abstract LB-049: Differential effects of functionalized and non-functionalized short multi-wall carbon nanotubes on survival of pancreatic cancer cells

Pancreatic cancer is an aggressive type of cancer with poor prognosis and low five-year survival rate. Current treatments such as chemotherapy and radiation therapy are not effective. Thus, new therapeutic strategies are urgently needed to treat patients with this devastating disease and to improve their prognosis. Carbon nanotubes (CNTs) constitute a novel nanomaterial extensively studied to develop and exploit their potential in biomedical applications (e.g. drug delivery vehicles and diagnostic agents), including cancer nanobiomedicine. Modification of CNTs with functional groups may improve their aqueous solubility and selectively facilitate the attachment of drugs as well as macromolecules to CNTs, thereby creating new avenues for designing novel and highly targeted drugs. Even though the putative cytotoxicity of functionalized and non-functionalized CNTs is poorly understood, it can be gainfully exploited to enhance the efficacy of anti-cancer drugs. The aim is this study is to investigate the hypothesis that functionalized and non-functionalized CNTs exert differential cytotoxic effects on pancreatic cancer cells. We employed the MTT assay to determine the effects of functionalized and non-functionalized short multi-wall carbon nanotubes (SMWCNTs) on survival of PANC1 cells and Western-blot analysis to elucidate some of the cell survival/proliferation mechanisms (e.g., ERG) underlying the effects induced by SMWCNTs. We used the Seahorse XFp technology to examine the effects of the SMWCNTs on cellular energetics (especially glycolysis & mitochondrial metabolism) in pancreatic cancer cells. Additionally, we employed Annexin V/PI staining in combination with flow cytometry to determine the effects of SMWCNTs in inducing apoptosis in MiaPaca-2 cells. Dose-response studies on pancreatic cancer cells using the MTT assay indicated non-functionalized CNTs to be more cytotoxic than functionalized CNTs. Cell signaling studies indicate no alteration in AKT & phospho-AKT levels between functionalized & non-functionalized CNTs treatment. However, phospho-ERK levels were observed to consistently decrease with increasing concentrations of non-functionalized CNTs. Flow cytometric analysis showed increased apoptotic cell death with non-functionalized CNTs relative to that with functionalized CNTs. Overall, results from these studies indicate the differential effects of CNTs (functionalized versus non-functionalized) on pancreatic cancer cells and as such they may have implications in designing novel therapies for treatment of pancreatic cancer. Citation Format: Alok Bhushan, Rutika Kokate, Vilas Desai, Annie Chhun, Gagan Kaushal, Yao Zhang, James Lai. Differential effects of functionalized and non-functionalized short multi-wall carbon nanotubes on survival of pancreatic cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-049.

Stability of extemporaneously compounded diltiazem hydrochloride infusions stored in polyolefin bags

PURPOSE The stability of extemporaneously compounded diltiazem hydrochloride infusions stored in polyolefin bags was studied. METHODS Sterile preparations of diltiazem hydrochloride were compounded to a concentration of 1 mg/mL in 5% dextrose solutions in accordance with United States Pharmacopeia chapter 797. The infusions were stored at -20 °C, 2-6 °C, and 22-25 °C. Three samples from each temperature were withdrawn and assessed for stability immediately after preparation (day 0) and on days 7, 15, 21, and 30 using a high-performance liquid chromatography (HPLC) assay. The physical stability of diltiazem samples was assessed by visual examination. Infusions were evaluated against black and white backgrounds for evidence of visible particulate matter, cloudiness, and color changes. The concentration of diltiazem hydrochloride in all samples was examined using a stability-indicating HPLC method at each time point. Diltiazem was considered stable if the solution retained over 90% of the initial concentration. RESULTS No precipitation, cloudiness, or color change was observed at any of the temperatures studied. pH did not significantly increase or decrease among the samples, regardless of temperature, over the study period. The diltiazem hydrochloride infusions retained greater than 90% of the initial concentrations for at least 30 days. CONCLUSION Diltiazem hydrochloride diluted to 1 mg/mL in 5% dextrose injection was stable for 30 days when stored at -20 °C, 2-6 °C, and 22-25 °C.

STABILITY-INDICATING HPLC METHOD FOR THE DETERMINATION OF THE STABILITY OF EXTEMPORANEOUSLY PREPARED NOREPINEPHRINE PARENTERAL SOLUTIONS

A stability-indicating HPLC method was developed for analysis of norepinephrine in the presence of the degradation products. The stability of extemporaneously compounded norepinephrine syringes was investigated using this method. The sterile preparations of norepinephrine were compounded to the strength of 8 µg/mL in accordance with USP (United States Pharmacopeia) <797> standards. To carry out the stability testing of these products, these syringes were stored under three different temperature conditions of −20°C, 2–6°C, 22–25°C, and light-protected at 22–25°C. Three injections from each temperature were withdrawn and were assessed for stability on days 0, 7, 15, 21, and 30 as per the USP guidelines. The assay of norepinephrine was examined by this calibrated stability-indicating HPLC method. No precipitation, cloudiness, or color change was observed during this study at all temperatures. The assay content by HPLC revealed that norepinephrine syringes retain greater than at least 90% of the initial concentrations for 30 d. Norepinephrine syringes in the final concentration of 8 µg/mL and diluted in normal saline are stable for at least 30 d under all the conditions studied. The stability analysis results show that the shelf-life observed was far better than their recommended expiration dates.

Extended Stability of Magnesium Sulfate Infusions Prepared in Polyolefin Bags

Purpose To investigate the stability of extemporaneously compounded 50 g per 600 mL magnesium sulfate parenteral solution diluted in Lactated Ringers solution. Methods The sterile preparations of magnesium sulfate were compounded in accordance with USP <797> standards. To carry out the stability testing, these products were stored under 3 different temperature conditions of −20°C, 2°C to 6°C, and 22°C to 25°C. Under the stability studies, pH, particulate matter, and the active content were monitored for 30 days. Results Magnesium sulfate infusions are stable for 30 days when stored at −20°C or 2°C to 6 °C and stable for 30 days under all the temperature conditions studied. These data demonstrate that magnesium sulfate infusions have an extended physical and chemical stability after preparation. Conclusions The stability analysis results show that the shelf-life observed was far better than their recommended expiration dates. This will allow the hospitals to give longer dating to magnesium sulfate preparations, provided they are prepared in a sterile environment and are in compliance with USP <797> guidelines.

Extended Stability of Sodium Bicarbonate Infusions Prepared in Polyolefin Bags

Purpose To investigate the stability of 0.1 mEq/mL and 0.15 mEq/mL strengths of extemporaneously compounded sodium bicarbonate preparations. Methods Sterile preparations of sodium bicarbonate of 2 strengths (0.1 mEq/mL and 0.15 mEq/mL) were compounded in accordance with USP <797> standards. To carry out the stability testing, these products were stored under 3 temperature conditions: −20°C, 2°C-8°C, and 23°C-25°C. Under the stability studies, pH, particulate matter, and the active content were monitored for 30 days. Results Sodium bicarbonate infusions are stable for 21 days when stored at −20°C or 2°C −8°C and are stable for 7 days when stored at room temperature (23°C-25°C). These data demonstrate that sodium bicarbonate infusions have extended physical and chemical stability after preparation. Conclusions The stability analysis results show that the observed shelf life was far better than the recommended expiration dates. This will allow the hospitals to give longer dating to sodium bicarbonate preparations, provided they are prepared in a sterile environment and are in compliance with USP <797> guidelines.

STABILITY OF FAMOTIDINE IN POLYPROPYLENE SYRINGES USING A STABILITY-INDICATING HPLC ASSAY

Famotidine syringes (2 mg/mL) were prepared under sterile conditions in accordance with United States Pharmacopeia (USP). To test the stability, syringes were kept for 30 d under three different temperature conditions: room temperature (22–25°C), refrigerated (2–6°C), and frozen (−20°C). Three samples from each temperature were withdrawn and were assessed for stability on days 0, 7, 15, 22, and 30 as per the USP guidelines. Physical stability was assessed by visual examination and pH values. The assay of famotidine was examined by a stability-indicating HPLC method and a significant loss of stability was defined as 10% or greater decrease in famotidine content over time. Physical stability results illustrated that no precipitation, cloudiness, or color change was observed in famotidine syringes at all the temperatures studied. Results from the assay content by HPLC showed that famotidine injections retain greater than 90% of the initial concentrations for 30 d when stored at room temperature and frozen; and 22 d when refrigerated. Thus, we can conclude that when stored at room temperature or frozen, diluted famotidine injections (2 mg/mL in sterile water) in 12 mL polypropylene syringes are stable for at least 30 d and for 22 d when refrigerated.