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As the SARS-CoV-2 virus continues to spread and mutate, it remains important to focus not only on preventing spread through vaccination but also on treating infection with direct-acting antivirals (DAA). The approval of Paxlovid, a SARS-CoV-2 main protease (Mpro) DAA, has been significant for treatment of patients. A limitation of this DAA, however, is that the antiviral component, nirmatrelvir, is rapidly metabolized and requires inclusion of a CYP450 3A4 metabolic inhibitor, ritonavir, to boost levels of the active drug. Serious drug-drug interactions can occur with Paxlovid for patients who are also taking other medications metabolized by CYP4503A4, particularly transplant or otherwise immunocompromised patients who are most at risk for SARS-CoV-2 infection and the development of severe symptoms. Developing an alternative antiviral with improved pharmacological properties is critical for treatment of these patients. By using a computational and structure-guided approach, we were able to optimize a 100 to 250 µM screening hit to a potent nanomolar inhibitor and lead compound, Mpro61. In this study, we further evaluate Mpro61 as a lead compound, starting with examination of its mode of binding to SARS-CoV-2 Mpro. In vitro pharmacological profiling established a lack of off-target effects, particularly CYP450 3A4 inhibition, as well as potential for synergy with the currently approved alternate antiviral, molnupiravir. Development and subsequent testing of a capsule formulation for oral dosing of Mpro61 in B6-K18-hACE2 mice demonstrated favorable pharmacological properties, efficacy, and synergy with molnupiravir, and complete recovery from subsequent challenge by SARS-CoV-2, establishing Mpro61 as a promising potential preclinical candidate. [ABSTRACT FROM AUTHOR]

A hallmark of effective cancer treatment is the prevention of tumor reoccurrence and metastasis to distal organs, which are responsible for most cancer deaths. However, primary tumor resection is expected to be curative as most solid tumors have been shown both experimentally and clinically to accelerate metastasis to distal organs including the lungs. In this study, we evaluated the efficacy of our engineered nasal nano-vaccine (CpG-NP-Tag) in reducing accelerated lung metastasis resulting from primary tumor resection. Cytosine–phosphate–guanine oligonucleotide [CpG ODN]-conjugated nanoparticle [NP] encapsulating tumor antigen [Tag] (CpG-NP-Tag) was manufactured and tested in vivo using a syngeneic mouse mammary tumor model following intranasal delivery. We found that our nasal nano-vaccine (CpG-NP-Tag), compared to control NPs administered after primary mammary tumor resection, significantly reduced lung metastasis in female BALB/c mice subjected to surgery (surgery mice). An evaluation of vaccine efficacy in both surgery and non-surgery mice revealed that primary tumor resection reduces CD11b+ monocyte-derived suppressor-like cell accumulation in the lungs, allowing increased infiltration of vaccine-elicited T cells (IFN-γ CD8+ T cells) in the lungs of surgery mice compared to non-surgery mice. These findings suggest that the combination of the target delivery of a nasal vaccine in conjunction with the standard surgery of primary tumors is a plausible adjunctive treatment against the establishment of lung metastasis. [ABSTRACT FROM AUTHOR]

Abstract Background Nanoparticle based delivery of anticancer drugs have been widely investigated. However, a very important process for Research & Development in any pharmaceutical industry is scaling nanoparticle formulation techniques so as to produce large batches for preclinical and clinical trials. This process is not only critical but also difficult as it involves various formulation parameters to be modulated all in the same process. Methods In our present study, we formulated curcumin loaded poly (lactic acid-co-glycolic acid) nanoparticles (PLGA-CURC). This improved the bioavailability of curcumin, a potent natural anticancer drug, making it suitable for cancer therapy. Post formulation, we optimized our process by Reponse Surface Methodology (RSM) using Central Composite Design (CCD) and scaled up the formulation process in four stages with final scale-up process yielding 5 g of curcumin loaded nanoparticles within the laboratory setup. The nanoparticles formed after scale-up process were characterized for particle size, drug loading and encapsulation efficiency, surface morphology, in vitro release kinetics and pharmacokinetics. Stability analysis and gamma sterilization were also carried out. Results Results revealed that that process scale-up is being mastered for elaboration to 5 g level. The mean nanoparticle size of the scaled up batch was found to be 158.5 ± 9.8 nm and the drug loading was determined to be 10.32 ± 1.4%. The in vitro release study illustrated a slow sustained release corresponding to 75% drug over a period of 10 days. The pharmacokinetic profile of PLGA-CURC in rats following i.v. administration showed two compartmental model with the area under the curve (AUC0-∞) being 6.139 mg/L h. Gamma sterilization showed no significant change in the particle size or drug loading of the nanoparticles. Stability analysis revealed long term physiochemical stability of the PLGA-CURC formulation. Conclusions A successful effort towards formulating, optimizing and scaling up PLGA-CURC by using Solid-Oil/Water emulsion technique was demonstrated. The process used CCD-RSM for optimization and further scaled up to produce 5 g of PLGA-CURC with almost similar physicochemical characteristics as that of the primary formulated batch.

The gastrointestinal disease cryptosporidiosis, caused by the genus Cryptosporidium , is a common cause of diarrheal diseases in children, particularly in developing countries and frequently fatal in immunocompromised individuals. Cryptosporidium hominis ( Ch )-specific bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) has been a molecular target for inhibitor design. ( Note that this bifunctional enzyme has also been referred to as TS-DHFR in previous literature since the functional biochemical reaction first involves the conversion of methylene tetrahydrofolate to dihydrofolate at the TS site .) While nanomolar inhibitors of Ch DHFR-TS have been identified at the biochemical level, effective delivery of these compounds to achieve anticryptosporidial activity in cell culture and in vivo models of parasite infection remains a major challenge in developing new therapies. Previous studies, using a nanotherapy approach, have shown a promising Ch DHFR-TS inhibitor, 906, that can successfully target Cryptosporidium parasites in cell culture with nanomolar anticryptosporidial activity. This formulation utilized poly(lactic- co -glycolic acid) (PLGA) nanoparticles (NPs) loaded with 906 (NP-906) and conjugated with a Cryptosporidium monoclonal antibody (MAb) on the nanoparticle surface to specifically target the glycoprotein GP25-200 in excysting oocysts. However, a limitation for in vivo use is antibody susceptibility to gastric acidity. To address this gap, a prodrug diethyl ester form of 906 (MAb-NP-Prodrug) was synthesized that allowed higher compound loading in the MAb-coated PLGA nanoparticles. An oral formulation was prepared by loading lyophilized MAb-NP-Prodrug into gelatin capsules with an enteric coating for gastric stability. Proof-of-concept studies with this oral formulation demonstrated antiparasitic activity in a chronic mouse model of Cryptosporidium infection. Efficacy was observed after a low daily dose of 2 × 8 mg kg -1 for 5 days, when examined 6 and 20 days postinfection, offering a new avenue of drug delivery to be further explored., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Aim: To generate and characterize a telomerase-immortalized human retinal microvascular endothelial cell (HREC) line. This cell line may be utilized as an in vitro model to study the molecular basis of several diseases of the human retina., Materials and Methods: Primary retinal neuronal cells were isolated and transfected with plasmid encoding full-length human telomerase reverse transcriptase (hTERT). Transfected cells were selected and characterized to determine telomerase activity, karyotype, proliferative capacity and functionality., Results: HREC-hTERT cells appear morphologically similar to primary endothelial cells and have an extended in vitro life-span. HREC-hTERT cells express the progenitor/stem cell marker nestin. They have active telomerase and a high proliferative capacity. These cells also maintain a diploid karyotype. The HREC-hTERT cells showed high colony-formation capacity and plating efficiency compared to the primary cells. These cells are capable of differentiation into neuronal and glial cell phenotypes and the differentiated cells express the astrocyte marker glial fibrillary acidic protein (GFAP) and the neuronal marker microtubule-associated protein-2 (MAP2), respectively., Conclusion: The in vitro life-span of human retinal neuronal endothelial cells can be extended by ectopic expression of hTERT without altering the genetic stability and functionality of these cells. These cells will be a valuable tool to further our understanding on the role of HRECs in the human blood-retinal-barrier and in angiogenesis and neovascularization.

Background: Liposome-based drug delivery has been successful in the past decade, with some formulations being Food and Drug Administration (FDA)-approved and others in clinical trials around the world. The major disadvantage associated with curcumin, a potent anticancer agent, is its poor aqueous solubility and hence low systemic bioavailability. However, curcumin can be encapsulated into liposomes to improve systemic bioavailability., Materials and Methods: We determined the antitumor effects of a liposomal curcumin formulation against human MiaPaCa pancreatic cancer cells both in vitro and in xenograft studies. Histological sections were isolated from murine xenografts and immunohistochemistry was performed., Results: The in vitro (IC50) liposomal curcumin proliferation-inhibiting concentration was 17.5 μM. In xenograft tumors in nude mice, liposomal curcumin at 20 mg/kg i.p. three-times a week for four weeks induced 42% suppression of tumor growth compared to untreated controls. A potent antiangiogenic effect characterized by a reduced number of blood vessels and reduced expression of vascular endothelial growth factor and annexin A2 proteins, as determined by immunohistochemistry was observed in treated tumors., Conclusion: These data clearly establish the efficacy of liposomal curcumin in reducing human pancreatic cancer growth in the examined model. The therapeutic curcumin-based effects, with no limiting side-effects, suggest that liposomal curcumin may be beneficial in patients with pancreatic cancer.

Background: The neuropathic side-effects of trauma, stroke or therapeutic radiation of the brain for life-threatening neoplastic diseases are the result of damage to normal tissues resulting in defects in cognition and memory. Based upon published preclinical data of curcumin activity application of parenteral curcumin formulations may prove to be to be promising chemotherapy for disorders following neuropathic insults. Studies in in vitro and animal models suggest curcumin may be an effective remediative agent for brain damage. The initial steps in curcumin development for clinical applications to neuropathic disorders are formulating it for intravenous administration, determining the formulated product passes the blood-brain barrier and reaches therapeutic amounts in damaged areas in the brain with tolerable safety. Following intravenous administration of liposomal curcumin, polymeric nanocurcumin and polylactic glycolic acid co-polymer (PLGA)-curcumin in rats, these formulations were observed to cross the blood-brain barrier using a sensitive HPLC assay. All three formulations localized in specific sites in the brain without observable adverse events. One hour following intravenous injection of 5 mg/kg nanocurcumin, or 20 mg/kg PLGA-curcumin, or liposomal curcumin, up to 0.5% of the injected material localized in the brain stem, the striatum, and the hippocampus with varied accumulation and clearance rates., Conclusion: These data indicate that curcumin does localize in putative damaged brain tissues and suggest therapeutic trials be explored with all three formulations in animal models with pre- and post traumatic states.