Preparation and evaluation of sublingual film of ketorolac tromethamine.

OBJECTIVE: This research aimed to formulate fast-dissolving sublingual films of Ketorolac tromethamine to improve therapeutic efficacy, patient compliance and overcome the drug's gastrointestinal side effects by avoiding direct contact with the gastric mucosa. METHODS: This research produced Ketorolac tromethamine sublingual film by solvent casting method using a variable ratio of polymer and plasticizer but a fixed quantity of other excipients and solvent ratio to evaluate the effect of these components on the overall formulation. Total 9 (F1 to F9) formulations were prepared where the ratio of Kollicoat®IR as polymer and Polyethylene glycol 400 as plasticizer were 2.0:1, 3.0:1, 4.0:1, 4.0:1, 4.8:1, 5.6:1, 5.33:1, 6.0:1, 6.66:1 respectively. The prepared films were evaluated through morphological and organoleptic properties, weight uniformity, folding endurance, surface pH, thickness, percentage of moisture loss, dispersion, dissolution, and drug content uniformity. Also, API-excipients compatibility was evaluated by FTIR spectroscopy. RESULTS: Formulation-2 (F2) demonstrated better film with optimum folding endurance where the ratio of Kollicoat®IR and Polyethylene glycol 400 was 3.0:1. The film's surface and distribution of polymers and drugs were examined by trinocular microscopic imaging where drug molecule showed uniform distribution which was supported by the assay (100.1%) and content uniformity (100.1 ± 1.97%). Performed dissolution studies showed 99.3% of drug dissolution occurred in just 3 min at pH 6.8. CONCLUSION: Prepared films were found to have thin, fast dispersion and dissolution properties. Therefore, the patients can use the sublingual film to get rapid relief of pain with minimal side effects in the gastrointestinal tract.

Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation.

We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus during ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas.

Mutation Spectrum in TPO Gene of Bangladeshi Patients with Thyroid Dyshormonogenesis and Analysis of the Effects of Different Mutations on the Structural Features and Functions of TPO Protein through In Silico Approach.

Although thyroid dyshormonogenesis (TDH) accounts for 10-20% of congenital hypothyroidism (CH), the molecular etiology of TDH is unknown in Bangladesh. Thyroid peroxidase (TPO) is most frequently associated with TDH and the present study investigated the spectrum of TPO mutations in Bangladeshi patients and analyzed the effects of mutations on TPO protein structure through in silico approach. Sequencing-based analysis of TPO gene revealed four mutations in 36 diagnosed patients with TDH including three nonsynonymous mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, and one synonymous mutation p.Pro715Pro. Homology modelling-based analysis of predicted structures of MPO-like domain (TPO142-738) and the full-length TPO protein (TPO1-933) revealed differences between mutant and wild type structures. Molecular docking studies were performed between predicted structures and heme. TPO1-933 predicted structure showed more reliable results in terms of interactions with the heme prosthetic group as the binding energies were -11.5 kcal/mol, -3.2 kcal/mol, -11.5 kcal/mol, and -7.9 kcal/mol for WT, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, respectively, implying that p.Ala373Ser and p.Thr725Pro mutations were more damaging than p.Ser398Thr. However, for the TPO142-738 predicted structures, the binding energies were -11.9 kcal/mol, -10.8 kcal/mol, -2.5 kcal/mol, and -5.3 kcal/mol for the wild type protein, mutant proteins with p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro substitutions, respectively. However, when the interactions between the crucial residues including residues His239, Arg396, Glu399, and His494 of TPO protein and heme were taken into consideration using both TPO1-933 and TPO142-738 predicted structures, it appeared that p.Ala373Ser and p.Thr725Pro could affect the interactions more severely than the p.Ser398Thr. Validation of the molecular docking results was performed by computer simulation in terms of quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulation. In conclusion, the substitutions mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, had been involved in Bangladeshi patients with TDH and molecular docking-based study revealed that these mutations had damaging effect on the TPO protein activity.