Clozapine-laden carbon dots delivered to the brain via an intranasal pathway: Synthesis, characterization, ex vivo, and in vivo studies.

Clozapine, which is widely used to treat schizophrenia, shows low bioavailability due to poor solubility and high first-pass metabolism. The study aimed to design clozapine-loaded carbon dots (CDs) to enhance availability of the clozapine to the brain via intranasal pathway. The CDs were synthesized by pyrolysis of citric acid and urea at 200 °C by hydrothermal technique and characterized by photoluminescence, transmission electron microscopy (TEM), X-ray Photoelectron Spectrometer (XPS), and Fourier transform infrared spectrum (FTIR). The optimized clozapine-loaded CDs (CLZ-CDs-1:3-200) showed a quasi-spherical shape (9-12 nm) with stable blue fluorescence. The CDs showed high drug solubilization capacity (1.5 mg drug in 1 mg/ml CDs) with strong electrostatic interaction with clozapine (drug loading efficiency = 94.74%). The ex vivo release study performed using nasal goat mucosa showed sustained release of clozapine (43.89%) from CLZ-CDs-1:3-200 for 30 h. The ciliotoxicity study (histopathology) confirmed no toxicity to the nasal mucosal tissues using CDs. In the rat model (in vivo pharmacokinetic study), when CDs were administrated by the intranasal route, a significantly higher concentration of clozapine in the brain tissue (Cmax = 58.07 ± 5.36 µg/g and AUCt (µg/h*g) = 105.76 ± 12.31) was noted within a short time (tmax = 1 h) compared to clozapine suspension administered by intravenous route (Cmax = 20.99 ± 3.91 µg/g, AUC t (µg/h*g) = 56.89 ± 12.31, and tmax = 4 h). The high value of drug targeting efficiency (DTE, 486%) index and direct transport percentage (DTP, 58%) indicates the direct entry of clozapine-CDs in the brain via the olfactory route. In conclusion, designed CDs demonstrated a promising dosage form for targeted nose-to-brain delivery of clozapine for the effective treatment of schizophrenia.

In vitro and in vivo evaluation of cyclosporine-graphene oxide laden hydrogel contact lenses.

Drug-eluting contact lens can substitute the multiple eye drop therapy. However, loading hydrophobic drug like cyclosporine in the contact lens is very challenging, due to low drug uptake (via soaking method); and alteration in the swelling and optical properties which restricts its clinical application. To address the above issues, graphene oxide (GO, large surface area with oxygen containing functional groups) was incorporated in the contact lenses during fabrication. These GO-laden contact lenses (SM-GO-Cys) as well as blank contact lenses (SM-Cys) were soaked in the solution of cyclosporine. Alternatively, cyclosporine-laden contact lenses (DL-Cys-20) and cyclosporine-GO-laden contact lenses (DL-Cys-20-GO) were fabricated by adding drug and drug-GO (at various level of GO) during fabrication, respectively. Contact angle and swelling data showed increase in water holding capacity of GO laden contact lenses. Optical property was significantly improved due to molecular dispersion of drug on the surface of GO sheets. The drug uptake and in vitro release profile was improved with GO-laden contact lenses by soaking method (SM-GO-Cys-400n) due to hydrophobic interactions between GO and drug. Adding cyclosporine-GO (DL-Cys-20-GO-800n) during fabrication significantly improved drug release kinetics with higher drug leaching (during extraction and sterilization) due to increased swelling, improved dissolution and molecular dispersion of drug on GO sheets. Ocular irritation and histopathological studies demonstrated the safety of GO-contact lens. The in vivo drug release studies in the rabbit eye showed significant improvement in mean residence time (MRT) and area under the curve (AUC) using DL-Cys-20-GO-800n contact lens compared to eye drop solution with reduction in protein adherence value. The study demonstrated that the incorporation of GO into the contact lens can control the release of cyclosporine as well as improved the lens swelling and transmittance properties.

Proton pump inhibitors and osteoporosis risk: exploring the role of TRPM7 channel.

BACKGROUND: Long-term use of proton pump inhibitors (PPIs) has been linked to an increased risk of osteoporosis, with various indirect mechanisms so far identified. Although no direct underlying mechanism for effect on bone cells have been investigated with the use of PPIs. Melastatin-like transient receptor potential 7 (TRPM7)channel has been engaged in the proliferation of bone cells. TRPM7 channel is regulated by extracellular Mg2+ and Ca2+ level, that further encourages to analyse if any imbalance with pantoprazole usage could alter bone remodelling process mediated by TRPM7. OBJECTIVES: The present study was conducted to investigate the effect of pantoprazole on the calcium and magnesium level, the cations involved in the bone remodelling process, as well as role of the TRPM7 channel in the proliferation of bone cells. METHODS: A cytotoxicity study was carried out to study the effect of pantoprazole on the bone cell using MC3T3-E1 cell line, together with the expression of TRPM7 was determined post-pantoprazole treatment. An in vivo study in rats was carried out for estimation of Ca2+, Mg2+ and Ca2+/Mg2+ ratio as well as bone strength was measured over a duration of 4 weeks and 8 weeks with the treatment of pantoprazole. A pilot-scale clinical study was carried out in patients with a fracture to support the evidence of preliminary findings from in-vitro and in vivo studies. RESULTS: MC3T3-E1 cell line treated with pantoprazole showed decreased cell viability in a dose-dependent manner and reduced expression of TRPM7 channel, evidencing interaction of TRPM7 and pantoprazole in the bone remodelling process. A pilot study conducted on 12 patients having major fractures showed changes in serum Mg2+ and Ca2+ levels over a period of 1 month as well as the animal study also showed ionic imbalance over 8-week treatment with pantoprazole. Bone density measured for the patient at the end of the 1-month treatment was found to be in the osteopenic category, together with the animal study which showed a decrease in femur bone strength for the animal treated with pantoprazole over a period of 8 weeks. CONCLUSION: The study findings proved a negative impact of pantoprazole use on Ca2+ and Mg2+ levels, which can impact TRPM7-mediated bone remodelling which serves to be a possible mechanism for osteoporosis upon pantoprazole use.

Nose to brain delivery of tailored clozapine nanosuspension stabilized using (+)-alpha-tocopherol polyethylene glycol 1000 succinate: Optimization and in vivo pharmacokinetic studies.

Clozapine is widely used to treat schizophrenia as an atypical antipsychotic. Low solubility, poor dissolution rate, degradation in the gastrointestinal tract, high hepatic first-pass metabolism, and eventually less drug transfer in the brain are all issues with oral clozapine administration. On account of this poor pharmacokinetic parameters, the authors aimed to develop clozapine nanosuspension using (+)-alpha-tocopherol polyethylene glycol 1000 succinate (TPGS) and polyvinylpyrrolidone K-30 (PVP K-30) and deliver it through the intranasal route. The nanosuspension was prepared by the high-speed homogenization method with 32 full factorial design for optimization of the product. Quality Target Product Profile (QTPP) was enlisted before the product development. The amount of TPGS and speed of homogenizer were selected as independent variables whereas, particle size and drug permeation profile after 24 h (Y2, %) were selected as dependent variables. As per the results of optimization, amount of TPGS and speed of homogenizer were chosen as 0.1% and 7000 rpm, respectively. The particle size of the optimized nanosuspension of clozapine was found to be 281 nm. The conversion of clozapine crystals to an amorphous form was verified by characterization studies (XRD and DSC). The drug permeability study showed 96.15% and 41.12% clozapine release after 24 h from nanosuspension and conventional suspension, respectively. The study of nasal cilio-toxicity (histopathological studies) demonstrated the appropriateness of nanosuspension for intranasal purposes. The single-dose in vivo pharmacokinetic analysis in the rat model showed a substantial increase in the therapeutic concentration of clozapine in the brain tissue in the case of intranasal nanosuspension (dose = 0.05 mg drug/0.1 mL, Cmax = 8.62 ± 0.45 µg/g, tmax = 1 h) compared to conventional oral clozapine suspension (dose = 26.43 mg drug/0.158 mL, Cmax = 1.14 ± 0.12 µg/g, tmax = 1 h).Ultimately, in the case of an intranasal route, a 3.56-fold increase in brain drug concentration was observed with a 528-fold lower drug dose compared with oral administration. The results suggest that clozapine nanosuspension may be used for successful nose-to-brain delivery.

Identification, isolation and characterization of impurities of clindamycin palmitate hydrochloride.

Clindamycin palmitate hydrochloride is a water soluble hydrochloride salt of the ester of clindamycin and palmitic acid. It is inactive in vitro, rapid in vivo hydrolysis converts this compound to the antibacterially active clindamycin. Total 12 impurities at levels ranging from 0.05% to 0.5% were detected by isocratic reverse-phase high performance liquid chromatography (HPLC) using RI detector. The molecular weights of impurities were determined by LC-MS analysis. Two impurities were starting materials and the remaining impurities were isolated from crude samples/enriched mother liquors using reverse-phase preparative HPLC. Based on the spectral data the structures of these impurities were characterized as, clindamycin palmitate sulphoxides alpha-/beta-isomers (impurity I); clindamycin laurate (impurity II); lincomycin palmitate (impurity III); clindamycin myristate (impurity IV); epiclindamycin palmitate (impurity V); clindamycin palmitate 3-isomer (impurity VI); clindamycin pentadecanoate (impurity VII); clindamycin B-palmitate (impurity VIII); clindamycin heptadecanoate (impurity IX) and clindamycin stearate (impurity X). Structural elucidation of all impurities by spectral data ((1)H NMR, (13)C NMR, MS and IR) and formation of these impurities have been discussed in detail.

Mineral content of water and food in fluorotic villages and prevalence of dental fluorosis.

The fluoride (F), calcium (Ca), magnesium (Mg), zinc (Zn), copper (Cu), and phosphorus (P) content in potable water and food samples from endemic and nonendemic villages for fluorosis were analyzed. It was found that the F content in water was significantly higher (p<0.01) in endemic villages (4.20+/-1.6 ppm) than control villages (0.63+/-0.15 ppm), whereas the Ca, Cu, and Mg contents were found to be significantly lower (p<0.05) in endemic villages compared to control villages. However, there was no significant difference in Zn and P contents between the villages. Foods grown in endemic villages contained significantly higher (p<0.01) fluoride content as compared to control villages. There was no significant difference in Ca, Mg, P, and Zn contents in food grown in endemic and control villages. Copper content in cereals (p<0.05), pulses (p<0.01), and vegetables (p<0.01) in endemic villages was found significantly higher as compared to control villages. The overall prevalence of dental fluorosis in six endemic villages was 97.4% in boys and 96% in girls, which was significantly higher (p<0.01) than that of control villages, where it was 10.5% in boys and 8.3% in girls. The prevalence of dental fluorosis was positively correlated (r=0.125, p<0.01) to fluoride and negatively correlated to Ca and Cu content in drinking water in endemic villages.

Phosphorylation of profilin regulates its interaction with actin and poly (L-proline).

Activation of bovine platelets with thrombin and phorbol 12,13-dibutyrate (PDBu) resulted in phosphorylation of profilin on serine. The phosphorylation was inhibited when platelets were pretreated with the PI 3-kinase inhibitor, LY294002, indicating that profilin phosphorylation is a downstream event with respect to PI 3-kinase activation. Phosphorylation of profilin resulted in significant decrease in actin polymerization (16.5%), indicating an increased affinity of phosphoprofilin towards actin. The critical actin monomer concentration (Cc) increased to 260 nM in the presence of phosphoprofilin in comparison with 200 nM in the presence of profilin. The interaction of phosphoprofilin with phosphatidylinositol 4,5-bisphosphate [PI (4,5)-P2] and poly (L-proline) (PLP) was examined by monitoring the quenching of tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no difference in PI (4,5)-P2 binding between profilin and phosphoprofilin (Kd=20.4 microM), while poly (L-proline)-binding studies indicated a sixfold decrease (27.34 microM for profilin and 4.73 microM for phosphoprofilin) in Kd with phosphoprofilin. In vivo studies with platelets indicated an increased association of p85alpha, the regulatory subunit of PI 3-kinase with phosphoprofilin over profilin. Overall, the data presented conclude that profilin phosphorylated under in vivo conditions and phosphorylation depends upon activation of PI 3-kinase. Phosphoprofilin exhibited increased affinity to poly (L-proline) sequences both in vitro and in vivo.

Phosphatidylinositol 3-kinase binds to profilin through the p85 alpha subunit and regulates cytoskeletal assembly.

Phosphatidylinositol 3-kinase (PI3-K), endowed with catalytic (110kDa) and regulatory (85kDa) subunits co-precipitates with anti-tyrosine antibodies in mitogen-activated cells. Association of PI3-K with cytoskeleton activates its catalytic activity through undeciphered mechanisms. Recently Singh et al., (Biochemistry, 35, 16544-16549, 1996) have shown that profilin activates PI3-K activity in a concentration-dependent manner. Consequently, we investigated the interaction between the PI3-K and profilin employing the GSTp85 alpha fusion protein and the results indicate a specific interaction between profilin and p85 alpha. The effect of p85 alpha/profilin complex on polymerization of actin monomers was monitored fluorimetrically employing pyrene-labelled actin monomers. It was noted that p85 alpha/profilin complex inhibits actin polymerization suggesting that profilin can simultaneously bind to actin as well as to p85 alpha. The affinity of p85 alpha/profilin complex to actin increases in the presence of p85 alpha subunit of PI3-K as compared to profilin itself.