A Quality by Design Approach for Developing SNEDDS Loaded with Vemurafenib for Enhanced Oral Bioavailability.

Vemurafenib (VMF) is a practically insoluble (< 0.1 µg/mL) and least bioavailable (1%) drug. To enhance its oral bioavailability and solubility, we formulated a reliable self-nano emulsifying drug delivery system (SNEDDS). A Quality by Design (QbD) approach was used to optimize the ratio of Capryol 90, Tween 80, and Transcutol HP. VMF-loaded SNEDDS was characterized for its size, polydispersity index (PDI), zeta potential, drug content, and transmittance. The in vitro release profile of the drug loaded in SNEDDS was compared to the free drug in two media, pH 6.8 and 1.2, and the data obtained were analyzed with different mathematical models. A reverse-phase ultra-pressure liquid chromatography (UPLC) technique with high sensitivity and selectivity was developed and validated for the quantification of VMF in analytical and bioanalytical samples. Dissolution efficiency for SNEDDS was estimated using different models, which proved that the developed novel SNEDDS formulation had a better in vitro dissolution profile than the free drug. A 2.13-fold enhanced oral bioavailability of VMF-loaded SNEDDS compared to the free drug demonstrates the superiority of the developed formulation. This work thus presents an overview of VMF-loaded SNEDDS as a promising alternative to improve the oral bioavailability of the drug.

Preparation and Evaluation of Low-Dose Calcitriol Dry Powder Inhalation as Host-Directed Adjunct Therapy for Tuberculosis.

BACKGROUND: It is unclear whether Vitamin D is efficacious as a host-directed therapy (HDT) for patients of tuberculosis (TB). We investigated pulmonary delivery of the active metabolite of Vitamin D3, i.e., 1, 25-dihydroxy vitamin D3 (calcitriol) in a mouse model of infection with Mycobacterium tuberculosis (Mtb). METHODS: We optimized a spray drying process to prepare a dry powder inhalation (DPI) of calcitriol using a Quality by Design (QbD) approach. We then compared outcomes when Mtb-infected mice were treated with inhaled calcitriol at 5 ng/kg as a stand-alone intervention versus DPI as adjunct to standard oral anti-tuberculosis therapy (ATT). RESULTS: The DPI with or without concomitant ATT markedly improved the morphology of the lungs and mitigated histopathology in both the lungs and the spleens. The number of nodular lesions on the lung surface decreased from 43.7 ± 3.1 to 22.5 ± 3.9 with the DPI alone and to 9.8 ± 2.5 with DPI + ATT. However, no statistically significant induction of host antimicrobial peptide cathelicidin or reduction in bacterial burden was seen with the DPI alone. DPI + ATT did not significantly reduce the bacterial burden in the lungs compared to ATT alone. CONCLUSIONS: We concluded that HDT using the low dose calcitriol DPI contributed markedly to mitigation of pathology, but higher dose may be required to evoke significant induction of bactericidal host response and bactericidal activity in the lung.

Optimizing the fabrication of electrospun nanofibers of prochlorperazine for enhanced dissolution and permeation properties.

Despite being an approved antiemetic for more than five decades, the clinical usefulness of prochlorperazine is limited by its low solubility and inconsistent absorption in the gastrointestinal tract, which presents challenges for nanotherapeutic interventions. Here, we report the preparation of a highly soluble and permeable nanofiber formulation of prochlorperazine using the Quality-by-Design approach. The final nanofiber formulation with drug entrapment of 88.02 ± 1.14 % was obtained at 20.0 kV, with a flow rate of 0.5 ml/h and tip-to-collector distance of 19.9 cm. Physio-mechanical properties, such as thickness (0.42 ± 0.02 mm), pH resistance (7.04 ± 0.08), folding endurance (54 ± 5), and tensile strength (0.244 ± 0.02 N.mm-2), were appropriate for packaging and application to oromucosal surfaces. The content uniformity (93.48-106.63 %) and weight variation (<1.8 mg) of the optimal nanofiber formulation were within the permissible limits prescribed for orodispersible films. Microscopical investigations confirm a randomly deposited and dense network of woven nanofibers with an average diameter of 363 ± 5.66 nm. The drug particles were embedded homogeneously on the fiber in the nanoform (4.27 ± 1.34 nm). The spectral analysis using TEM-EDS shows diffraction peaks of sulfur and chlorine, the elemental constituents of prochlorperazine. The drug was amorphized in the nanofiber formulation, as led by the decline of the crystallinity index from 87.25 % to 7.93 % due to electrostatic destabilization and flash evaporation of the solvent. The enthalpy of fusion values of the drug in the nanofiber mat decreased significantly to 23.6 J/g compared to its pristine form, which exhibits a value of 260.7 J/g. The nanofibers were biocompatible with oral mucosal cells, and there were no signs of mucosal irritation compared to 1 % sodium lauryl sulfate. The fiber mats rapidly disintegrated within <1 s and released ≈91.49 ± 2.1 % of the drug within 2 min, almost 2-fold compared to the commercial Stemetil MD® tablets. Similarly, the cumulative amount of the drug permeated across the unit area of the oromucosal membrane was remarkably high (31.28 ± 1.30 µg) compared to 10.17 ± 1.11 µg and 13.10 ± 1.79 µg from the cast film and drug suspension. Our results revealed these nanofiber formulations have the potential to be fast-dissolving oromucosal delivery systems, which can result in enhanced bioavailability with an early onset of action due to rapid disintegration, dissolution, and permeation.

Transcriptional regulation of suppressors of cytokine signaling during infection with Mycobacterium tuberculosis in human THP-1-derived macrophages and in mice.

Mycobacterium tuberculosis (Mtb) infection leads to upregulation of Suppressors of Cytokine signaling (SOCS) expression in host macrophages (Mϕ). SOCS proteins inhibit cytokine signaling by negatively regulating JAK/STAT. We investigated this host-pathogen dialectic at the level of transcription. We used phorbol-differentiated THP-1 Mϕ infected with Mtb to investigate preferential upregulation of some SOCS isoforms that are known to inhibit signaling by IFN-γ, IL-12, and IL-6. We examined time kinetics of likely transcription factors and signaling molecules upstream of SOCS transcription, and survival of intracellular Mtb following SOCS upregulation. Our results suggest a plausible mechanism that involves PGE2 secretion during infection to induce the PKA/CREB axis, culminating in nuclear translocation of C/EBPß to induce expression of SOCS1. Mtb-infected Mϕ secreted IL-10, suggesting a mechanism of induction of STAT3, which may subsequently induce SOCS3. We provide evidence of temporal variation in SOCS isoform exspression and decay. Small-interfering RNA-mediated knockdown of SOCS1 and SOCS3 restored the pro-inflammatory milieu and reduced Mtb viability. In mice infected with Mtb, SOCS isoforms persisted across Days 28-85 post infection. Our results suggest that differential temporal regulation of SOCS isoforms by Mtb drives the host immune response towards a phenotype that facilitates the pathogen's survival.

Transient, inhaled gene therapy with gamma interferon mitigates pathology induced by host response in a mouse model of tuberculosis.

Transient transfection of the respiratory mucosa of mice infected with Mycobacterium tuberculosis (Mtb) with gamma interferon (IFN-γ) promises benefits in disease therapy. We investigated preclinical efficacy of a dry powder inhalation (DPI) as a stand-alone versus adjunct to oral anti-tuberculosis (TB) chemotherapy in mice. We observed that this host-directed therapy mitigates the gross organ pathology and histopathology of lung and spleen tissue of infected mice receiving the DPI, either alone or as adjunct therapy. However, no statistically significant reduction in Mtb colony forming units (CFU) occurred if mice were given only DPI; but not drugs. We compared one and three doses a week of the DPI over four weeks; with or without concomitant oral drugs. There was no significant difference in lung CFU after four or 12 doses of the DPI alone, but, surprisingly, four doses were qualitatively better than 12 doses in mitigating lung pathology. Nodular lesions on the lung surface and the area occupied by these was significantly reduced after four doses of the DPI, even without oral drugs. Transient transfection with IFN-γ did not induce pathological inflammation of the lungs and airways. We conclude that IFN-γ, as expected of host-directed therapy, 'heals the host; ' but does not 'kill the bug.'

Transient Transfection of the Respiratory Epithelium with Gamma Interferon for Host-Directed Therapy in Pulmonary Tuberculosis.

Nebulized gamma interferon (IFN-γ) protein has been studied for clinical safety and efficacy against pulmonary tuberculosis (TB). The protein is expensive, requires a cold chain, and is difficult to deploy in limited-resource, high-incidence settings. We generated a preclinical proof of concept (PoC) for a dry powder inhalation (DPI) containing DNA constructs to transiently transfect the lung and airway epithelium of mice with murine IFN-γ. Bacterial colony-forming units (CFU) in the lungs of mice infected with Mycobacterium tuberculosis (Mtb) reduced from about 106/g of tissue to ~104 after four doses given once a week. Nodular inflammatory lesions in the lungs reduced significantly in number. Immunohistochemistry of infected lung sections for LC3-1 and LAMP-1 indicated autophagy induction between 18 and 48 h after inhalation. ELISA on bronchoalveolar lavage (BAL) fluid showed differences in kinetics of IFN-γ concentrations in the epithelial lining fluid of healthy versus infected mice. Uninfected mice receiving DNA constructs expressing a fluorescent protein were live-imaged. The fluorescence signals from the intracellular protein peaked at about 36 h after inhalation and declined by 48 h. These results establish preclinical PoC of the efficacy of a DPI and dosing regimen as a host-directed and transient gene therapy of experimental pulmonary TB in mice, justifying preclinical development.

Dehydroascorbate-derivatized chitosan particles for targeting antimalarial agents to infected erythrocytes.

The mammalian glucose transporter GLUT-1 and Plasmodium falciparum hexose transporter PfHT1 are overexpressed on human RBC infected with the parasite (iRBC), presumably for enhanced glucose uptake. Dehydroascorbic acid (DHA) competes out glucose in GLUT-1 binding. We prepared particles containing chloroquine phosphate using novel derivatives of chitosan (CSN). CSN was either pre-derivatized with DHA (PRE) or particles made of CSN were derivatized by surface-grafting DHA (POST). The optimized formulations were analyzed for size (170-200nm) drug content (about 40%) entrapment efficiency (50-57%), in vitro drug release (80% in 72h, Higuchi's model), hemolysis on exposure to whole blood or RBC at 5% hematocrit, cytotoxicity towards cultured HEK 293T (kidney) and HepG2 (hepatic) cells, targeting iRBC and in vitro efficacy against P. falciparum. PRE particles were superior to POST CSN particles in terms of uptake and extent of preferential targeting to iRBCs than RBCs. Unlike starch particles reported earlier, dextrose did not competitively inhibit uptake of DHA-derivatized CSN particles. Both formulations significantly induced parasite inhibition at 1nM while free drug showed comparable activity at 100nM. Both PRE and POST particles were superior to free drug in efficacy. Targeting with high efficiency promises dose reduction and possibility of overcoming efflux-based drug resistance.