Combating Alcohol Adduct Impurity in Immunosuppressant Drug Product Manufacturing: A Scientific Investigation for Enhanced Process Control.

OBJECTIVE: This research aims to elucidate critical impurities in process validation batches of tacrolimus injection formulations, focusing on identification and characterization of previously unreported impurity at RRT 0.42, identified as the tacrolimus alcohol adduct. The potential root causes for the formation of new impurity was determined using structured risk assessment by cause and effect fishbone diagram. The primary objective was to propose mitigation plan and demonstrate the control of impurities with 6 month accelerated stability results in development batches. METHODS: The investigation utilizes method validation and characterization studies to affirm the accuracy of quantifying the tacrolimus alcohol adduct. The research methodology employed different characterization techniques like rotational rheometer, ICP‒MS, MALDI-MS, 1H NMR, 13C NMR, and DEPT-135 NMR for structural elucidation. Additionally, the exact mass of the impurity is validated using electrospray ionization mass spectra. RESULTS: Results indicate successful identification and characterization of the tacrolimus alcohol adduct. The study further explores the transformation of Tacrolimus monohydrate under various conditions, unveiling the formation of Tacrolimus hydroxy acid and proposing the existence of a novel degradation product, the Tacrolimus alcohol adduct. Six-month data from development lots utilizing Manufacturing Process II demonstrate significantly lower levels of alcohol adducts. CONCLUSIONS: Manufacturing Process II, selectively locates Tacrolimus within the micellar core of HCO-60, this prevent direct contact of ethanol with Tacrolimus which minimizes impurity alcohol adduct formation. This research contributes to the understanding of tacrolimus formulations, offering ways to safeguard product integrity and stability during manufacturing and storage.

The Role of Self-Nanoemulsifying Drug Delivery Systems of CDODA-Me in Sensitizing Erlotinib-Resistant Non-Small Cell Lung Cancer.

We have investigated the effects of combination treatment involving ERL (erlotinib) with a glycyrrhetinic acid analog, CDODA-Me in overcoming ERL resistance, providing efforts to improve the oral bioavailability of this treatment using self-nanoemulsifying drug delivery systems (SNEDDS). A Qbd (quality-by-design) approach was used to prepare CDMS (CDODA-SNEDDS, 2 µΜ), which was characterized using surface response methodology to optimize drug content, particle size, and drug release. CDMS/ERL combinations showed synergism in wild-type and resistant H1975 and HCC827 cell lines with combination index values less than 1. Increased apoptosis, mitochondrial membrane potential depletion, and enhanced intracellular ROS levels were also observed in combination therapy. Western blot analysis showed that combination therapy inhibited phosphorylation of epidermal growth factor receptor (EGFR) (p < 0.01 in all cell lines) and Met receptor tyrosine kinase (MET) (p < 0.01 in all cell lines). In vivo, the relative bioavailability of CDMS increased significantly from 22.13 to 151.76 µg/mL compared to the dosing of oral suspension (dose equivalent). Our results demonstrate that combination therapy involving ERL and CDODA-Me overcomes resistance through dual inhibition of p-EGFR and p-MET leading to the induction of apoptosis, intracellular ROS accumulation, and decreased mitochondrial potential. Furthermore, CDMS improved the oral bioavailability of CDODA-Me.

Formulation characteristics and in vitro release testing of cyclosporine ophthalmic ointments.

The aim of the present study was to investigate the relationship between formulation/process variables versus the critical quality attributes (CQAs) of cyclosporine ophthalmic ointments and to explore the feasibility of using an in vitro approach to assess product sameness. A definitive screening design (DSD) was used to evaluate the impact of formulation and process variables. The formulation variables included drug percentage, percentage of corn oil and lanolin alcohol. The process variables studied were mixing temperature, mixing time and the method of mixing. The quality and performance attributes examined included drug assay, content uniformity, image analysis, rheology (storage modulus, shear viscosity) and in vitro drug release. Of the formulation variables evaluated, the percentage of the drug substance and the percentage of corn oil in the matrix were the most influential factors with respect to in vitro drug release. Conversely, the process parameters tested were observed to have minimal impact. An evaluation of the release mechanism of cyclosporine from the ointment revealed an interplay between formulation (e.g. physicochemical properties of the drug and ointment matrix type) and the release medium. These data provide a scientific basis to guide method development for in vitro drug release testing of ointment dosage forms. These results demonstrate that the in vitro methods used in this investigation were fit-for-purpose for detecting formulation and process changes and therefore amenable to assessment of product sameness.

Molecular and Functional Diagnostic Tools in Precision Oncology for Urological Malignancies.

Urological malignancies, represented mainly by prostate, bladder, and renal cancers, are some of the leading causes of cancer-related mortalities worldwide. Despite various efforts over decades to develop early detection tests and effective therapeutic paradigms, the response rate to the existing treatments remains low for both primary and late stage/recurrent phases of these cancers. The evolving landscape of molecular diagnostics, aiming to make the diagnosis and treatment more patient-driven, underpins precision oncology and particularly intends to rationally profile individual tumors and highlight the mechanistic insight and complexity of tumor microenvironment in order to develop biomarkers of toxicity risks and response prediction in a clinically oriented dynamical setting. The present review is an effort to capture some of the recent developments in the area of molecular diagnostics and functional testing platforms and their potential application in clinical decision making in the premises of precision oncology of urological malignancies.

CDKN2A-p53 mediated antitumor effect of Lupeol in head and neck cancer.

PURPOSE: The tumor suppressor protein p53 is known to control cell cycle arrest and apoptosis. Lupeol is a phytochemical that has been found to induce apoptosis in different cancer types through the extrinsic pathway. As yet, however, its role in the induction of cell cycle arrest and apoptosis through the intrinsic pathway in head and neck cancer has not been investigated. Here, we aimed at understanding the mechanism underlying the antitumor effect of Lupeol in head and neck cancer. METHODS: The antitumor effect of Lupeol on oral and laryngeal carcinomas was assessed using two in vitro 2D cell line models (HEp-2, UPCI:SCC-131) and, subsequently, an ex vivo 3D tumor explant culture platform that maintains key features of the native tumor microenvironment. The mechanism underlying Lupeol-mediated antitumor responses was delineated using MTT, colony formation, flow cytometry, immunofluorescence, Western blotting and immunohistochemistry assays. RESULTS: We found that Lupeol induced an enhanced expression of p53 in both cell line models tested and, subsequently, cell cycle arrest at the G1 phase. In addition we found that, following Lupeol treatment, p53 induced Bax expression and activated the intrinsic apoptotic pathway (as measured by Caspase-3 cleavage). Interestingly, Lupeol was also found to trigger G1 cell cycle arrest through up-regulation of the expression of CDKN2A, but not p21, resulting in inhibition of CyclinD1. In an ex vivo platform Lupeol was found to impart a potent antitumor response as defined by inhibition of Ki67 expression, decreased cell viability and concomitant activation (cleavage) of Caspase-3. Finally, we found that Lupeol can re-sensitize primary head and neck squamous cell carcinoma (HNSCC) tumor samples that had clinically progressed under a Cisplatin treatment regimen. CONCLUSION: Together, our data indicate that Lupeol may orchestrate a bifurcated regulation of neoplastic growth and apoptosis in head and neck cancers and may serve as a promising agent for the management of tumors that have progressed on a platinum-based treatment regimen.

Combination Approach of YSA Peptide Anchored Docetaxel Stealth Liposomes with Oral Antifibrotic Agent for the Treatment of Lung Cancer.

Therapeutic efficacy of nanocarriers can be amplified by active targeting and overcoming the extracellular matrix associated barriers of tumors. The aim of the present study was to investigate the effect of oral antifibrotic agent (telmisartan) on tumor uptake and anticancer efficacy of EphA2 receptor targeted liposomes. Docetaxel loaded PEGylated liposomes (DPL) functionalized with nickel chelated phospholipid were prepared using a modified hydration method. DPL were incubated with various concentrations of histidine tagged EphA2 receptor specific peptide (YSA) to optimize particle size, zeta potential, and percentage YSA binding. Cellular uptake studies using various endocytosis blockers revealed that a caveolae dependent pathway was the major route for internalization of YSA anchored liposomes of docetaxel (YDPL) in A549 lung cancer cell line. Hydrodynamic diameter and zeta potential of optimized YDPL were 157.3 ± 11.8 nm and -3.64 mV, respectively. Orthotopic lung tumor xenograft (A549) bearing athymic nude mice treated with oral telmisartan (5 mg/kg) for 2 days showed significantly (p < 0.05) higher uptake of YDPL in tumor tissues compared to healthy tissue. Average lung tumor weight of the YDPL + telmisartan treated group was 4.8- and 3.8-fold lower than that of the DPL and YDPL treated groups (p < 0.05). Substantially lower expression (p < 0.05) of EphA2 receptor protein, proliferating cell nuclear antigen (PCNA), MMP-9, and collagen 1A level with increased E-cadherin and TIMP-1 levels in immunohistochemistry and Western blot analysis of lung tumor samples of the combination group confirmed antifibrotic effect with enhanced anticancer activity. Active targeting and ECM remodeling synergistically contributed to anticancer efficacy of YDPL in orthotopic lung cancer.

Immediate release tablets of telmisartan using superdisintegrant-formulation, evaluation and stability studies.

Telmisartan (anti-hypertensive) is insoluble in water; hence the drug may be slowly or incompletely dissolved in the gastro intestinal tract. So the rate of dissolution and therefore its bioavailability is less (bioavailability 42%). In the present study an attempt has been made to prepare immediate release tablets of telmisartan by using Polyplasdone XL-10 (Crosspovidone) at intragranular, extragranular and partly intra and extragranular level of addition to increase the rate of drug release from dosage form to increase the dissolution rate and hence its bioavailability. The prepared granules and tablets were evaluated for their physiochemical properties and in-vitro dissolution study was conducted for the prepared tablets. It was concluded that the immediate release tablets with proper hardness, disintegration time and with increase rate of dissolution can be made using Polyplasdone XL-10. Formuation-10 (F10) was selected for stability study and the in-vitro dissolution study showed that was no difference in percent of drug released between initial and sixth month sample.