Intracellular delivery of reactive oxygen species generating polypeptide-drug conjugates for cancer therapy

Auto-oxidation of D-penicillamine (D-pen), FDA registered for the treatment of Wilson's disease and rheumatoid arthritis, generates reactive oxygen species (ROS), a reaction catalyzed by transition metal ions (TMIs). D-pen has anti-proliferative and anti-angiogenic effects and is known to modulate several signaling pathways. However, an exact mechanism of action is not known. A reactive thiol group, strong plasma protein binding, rapid clearance, higher effective concentrations and cell impermeability challenge the development of D-pen as an anticancer agent. This dissertation work investigates poly([alpha])-L-glutamic acid (PGA) conjugates of D-pen for enhanced delivery to cancer cells and overcome the challenges mentioned above. Complete biodegradability, ability to carry large payloads of D-pen, reversible conjugation, biocompatibility, longer circulation and passive tumor accumulation make PGA an ideal drug carrier. Evidence is presented that PGA-D-pen conjugates enhance the intracellular uptake of D-pen. Upon release from the conjugate, D-pen causes significant elevation in ROS levels leading to apoptotic cell death in murine and human leukemia, and breast cancer cells. Treatment with PGA-D-pen improves the survival of CD2F1 mice bearing intra-peritoneal (i.p.) leukemia with no apparent adverse events. Idarubicin (Ida), an anthracycline chemotherapeutic, has shown efficacy as first-line treatment in acute leukemia and other cancers. ROS elevation that plays a major role in mediating its cytotoxicity is dependent on and augmented in the presence of low molecular weight thiols (LMWTs) like D-pen. We hypothesized that a combination of Ida and D-pen formulated as dual drug conjugates (DDCs) will provide co-delivery leading to enhanced anticancer effects while increasing the therapeutic index of Ida. Targeting to sigma-1 receptors, known to be over-expressed in many different cancers, to further enhance the efficacy and specificity was also examined. It was shown that stable DDCs could be synthesized with programmed drug release properties. The conjugates were successfully targeted in-vitro to sigma-1 receptor over-expressing non-small cell lung cancer (NSCLC) cells with a novel benzamide derivative, trivalent anisamide, as the ligand. DDCs showed prolonged circulation, enhanced tumor accumulation, reduced cardiac exposure of Ida, and improved tumor efficacy and survival in athymic nu/nu mice bearing NSCLC tumor xenografts

Plutajuće mikrosfere cimetidina: Priprava, karakterizacija i in vitro evaluacija

The present study involves preparation and evaluation of floating microspheres with cimetidine as model drug for prolongation of gastric residence time. The microspheres were prepared by the solvent evaporation method using polymers hydroxypropylmethyl cellulose and ethyl cellulose. The shape and surface morphology of prepared microspheres were characterized by optical and scanning electron microscopy, respectively. In vitro drug release studies were performed and drug release kinetics was evaluated using the linear regression method. Effects of stirring rate during preparation, polymer concentration, solvent composition and dissolution medium on the size of microspheres and drug release were also observed. The prepared microspheres exhibited prolonged drug release (~ 8 h) and remained buoyant for > 10 h. The mean particle size increased and drug release rate increased at higher polymer concentration. In vitro studies demonstrated diffusion-controlled drug release from the microspheres. No significant effect of the stirring rate during preparation on drug release was observed.U radu je opisana priprava i evaluacija plutajućih mikrosfera za produljeno zadržavanje u želucu. Cimetidin je izabran kao model lijeka. Mikrosfere su pripravljene metodom uparavanja otapala koristeći hidroksipropilmetilcelulozu i etilcelulozu. Optičkom, odnosno pretražnom elektronskom mikroskopijom karakterizirani su oblik i morfologija površine mikrosfera. Kinetika oslobađanja ljekovite tvari in vitro evaluirana je pomoću metode linearne regresije. Proučavan je utjecaj brzine miješanja tijekom priprave, koncentracije polimera, vrste otapala i medija za oslobađanje na veličinu mikrosfera, odnosno oslobađanje lijeka. Iz pripravljenih mikrosfera lijek se produljeno oslobađa (~ 8 h), a mikrosfere ostaju plutati više od 10 h. Veće mikrosfere dobivene su upotrebom veće koncentracije polimera. Pokusi in vitro ukazuju da je oslobađanje ljekovite tvari studies kontrolirano difuzijom. Brzina miješanja tijekom priprave nema utjecaja na oslobađanje ljekovite tvari

Hybrid deep learning model for sarcasm detection in Indian indigenous language using word-emoji embeddings

Automated sarcasm detection is deemed as a complex natural language processing task and extending it to a morphologically-rich and free-order dominant indigenous Indian language Hindi is another challenge in itself. The scarcity of resources and tools such as annotated corpora, lexicons, dependency parser, Part-of-Speech tagger and benchmark datasets engorge the linguistic challenges of sarcasm detection in low-resource languages like Hindi. Furthermore, as context incongruity is imperative to detect sarcasm, various linguistic, aural and visual cues can be used to predict target utterance as sarcastic. While pre-trained word embeddings capture the meanings, semantic relationships and different types of contexts in the form of word representations, emojis can also render useful contextual information, analogous to human facial expressions, for gauging sarcasm. Thus, the goal of this research is to demonstrate the use of a hybrid deep learning model trained using two embeddings, namely word and emoji embeddings to detect sarcasm. The model is validated on a Hindi tweets dataset, Sarc-H, manually annotated with sarcastic and non-sarcastic labels. The preliminary results clearly depict the importance of using emojis for sarcasm detection, with our model attaining an accuracy of 97.35% with an F-score of 0.9708. The research validates that automated feature engineering facilitates efficient and repeatable predictive model for detecting sarcasm in indigenous, low-resource languages

Lipid nanocapsule as vaccine carriers for his-tagged proteins: Evaluation of antigen-specific immune responses to HIV I His-Gag p41 and systemic inflammatory responses

The purpose of this study was to design novel nanocapsules (NCs) with surface-chelated nickel (Ni-NCs) as a vaccine delivery system for histidine (His)-tagged protein antigens. Ni-NCs were characterized for binding His-tagged model proteins through high affinity non-covalent interactions. The mean diameter and zeta potential of the optimized Ni-NCs was 214.9 nm and - 14.8 mV, respectively. The optimal binding ratio of His-tagged Green Fluorescent Protein (His-GFP) and His-tagged HIV-1 Gag p41 (His-Gag p41) to the Ni-NCs was 1:221 and 1:480 w/w, respectively. Treatment of DC2.4 cells with Ni-NCs did not result in significant loss in the cell viability up to 24 h

A meta-analysis on efficacy and safety: single-balloon vs. double-balloon enteroscopy

Background and aim: Double-balloon enteroscopy (DBE) and single-balloon enteroscopy (SBE) are new techniques capable of providing deep enteroscopy. Results of individual studies comparing these techniques have not been able to identify a superior strategy. Our aim was to systematically pool all available studies to compare the efficacy and safety of DBE with SBE for evaluation of the small bowel. Methods: Databases were searched, including PubMed, Embase, and the Cochrane Central Register of Controlled Trials. The main outcome measures were complete small-bowel visualization, diagnostic yield, therapeutic yield, and complication rate. Statistical analysis was performed using Review Manager (RevMan version 5.2). Meta-analysis was performed using fixed-effect or random-effect methods, depending on the absence or presence of significant heterogeneity. We used the χ2 and I2 test to assess heterogeneity between trials. Results were expressed as risk ratios (RR) or mean differences with 95% confidence intervals (CI). Results: Four prospective, randomized, controlled trials with a total of 375 patients were identified. DBE was superior to SBE for visualization of the entire small bowel [pooled RR = 0.37 (95% CI: 0.19–0.73; P = 0.004)]. DBE and SBE were similar in ability to provide diagnosis [pooled RR = 0.95 (95% CI: 0.77–1.17; P = 0.62)]. There was no significant difference between DBE and SBE in therapeutic yield [pooled RR = 0.78 (95% CI: 0.59–1.04; P = 0.09)] and complication rate [pooled RR = 1.08 (95% CI: 0.28–4.22); P = 0.91]. Conclusions: DBE was superior to SBE with regard to complete small bowel visualization. DBE was similar to SBE with regard to diagnostic yield, ability to provide treatment and complication rate, but these results should be interpreted with caution as they is based on very few studies and the overall quality of the evidence was rated as low to moderate, due to the small sample size

In vitro and in vivo assessment of targeting lipid-based nanoparticles to the epidermal growth factor-receptor (EGFR) using a novel Heptameric ZEGFR domain

Lipid-based oil-filled nanoparticles (NPs) with a high concentration of surface-chelated nickel (Ni-NPs) were successfully prepared using a Brij78-NTA-Ni conjugate synthesized with Brij 78 (Polyoxyethylene (20) stearyl ether) and nitrilotriacetic acid (NTA). The facile incorporation of the Brij 78-NTA-Ni conjugate into the NP formulation allowed up to 90% Ni incorporation, which was a significant improvement over the previously used standard agent DOGS-NTA-Ni which led to ~6% Ni incorporation. The Ni-NPs were targeted to the highly epidermal growth factor receptor (EGFR)-overexpressing epidermoid carcinoma cells A431. This was accomplished using a novel high affinity histidine×6-tagged EGFR-binding Z domain (heptameric ZEGFR domain). In vitro cell uptake studies showed enhanced internalization (up to 90%) of the targeted Ni-NPs in A431 cells with only ≤10% internalization of the of untargeted Ni-NPs. ICP-MS analysis used to quantify the amount of Ni in the cells were in close agreement with flow cytometry studies, which showed a dose dependent increase in the amount of Ni with the targeted Ni-NPs. Cell uptake competition studies showed that internalization of the targeted Ni-NPs within the cells was competed off with free heptameric ZEGFR domain at concentrations of 8.75 ng/mL or higher. In vivo studies were carried out in nude mice bearing A431 tumors to determine the biodistribution and intracellular delivery. Near Infrared (NIR) optical imaging studies using Alexa750-labeled heptameric ZEGFR domain showed localization of 19% of the total detected fluorescence intensity in the tumor tissue, 28% in the liver and 42% in the kidneys 16 h post i.v. injection. ICP-MS analysis showed almost a two-fold increase in the amount of intracellular Ni with the targeted Ni-NPs. These new Ni-NPs could be a very useful tool for targeting and drug delivery to a wide range of EGFR positive cancers

DESIGN AND PERFORMANCE VERIFICATION OF NEWLY DEVELOPED DISPOSABLE STATIC DIFFUSION CELL FOR DRUG DIFFUSION/PERMEABILITY STUDIES

Objectives: The present study describes a disposable static diffusion cell for in vitro diffusion studies to achieve better results as compared to well existing Franz diffusion cell (FDC) in terms of the absence of bubbles, variable receptor compartment, ease of handling, and faster results.Materials and Methods: The cell consists of a cup-shaped donor compartment made of semi permeable that could be either cellophane membrane or, animal skin fitted to a rigid frame, which is supported on a plastic plate that contains a hole for the sample withdrawal. The receptor compartment is a separate unit, and it could be any container up to 500ml volume capacity. The most preferred receptor compartment is glass beaker. In the present study, goatskin was used as semi-permeable membrane and verification of its performance was carried out through diffusion studies using gel formulations of one each of the four-selected biopharmaceutical classification system (BCS) class drugs. Metronidazole, diclofenac sodium, fluconazole, and sulfadiazine were used as model drugs for BCS Class I, II, III, and IV, respectively.Results: The newly developed diffusion cell (NDDC) was found to provide faster and more reproducible results as compared to FDC. At the time interval of 24 h, the cell was found to exhibit a higher diffusion of metronidazole, diclofenac sodium, fluconazole, and sulfadiazine by 0.65, 0.65, 0.32, and 0.81 folds, respectively. The faster release obtained with NDDC was attributed to a larger surface area of skin as compared to that in FDC.Conclusion: It was concluded that better reproducibility of results could be achieved with NDDC

Cardiac arrhythmia in Wilson’s disease: An oversighted and overlooked entity!

Wilson’s disease is a multisystem disorder which manifests with hepatic, neurological, musculoskeletal, hematological, renal, and cardiac symptoms. The hepatic and neurological manifestations often overshadow the other system involvement including cardiac symptoms and signs, which may prove fatal. We report a case of a young female who presented with progressive parkinsonian features and dystonia for around 4 months followed 2 months later by the complaint of episodes of light-headedness. She was diagnosed to have Wilson’s disease based on the presence of Kayser–Fleischer ring and laboratory parameters of copper metabolism. Electrocardiography of the patient incidentally revealed 2nd degree Mobitz type-1 atrioventricular block explaining her episodes of light-headedness. She was started on penicillamine and trihexyphenidyl. The heart block improved spontaneously. Cardiac autonomic function tests including blood pressure response to standing and heart rate response to standing were observed to be normal. We review the literature on cardiac manifestations of Wilson’s disease and emphasize that patients with Wilson’s disease should be assessed for cardiac arrhythmia and cardiac dysfunction as these may have therapeutic and prognostic implications