Aswathi R. Hegde

Influence of peptide dendrimers and sonophoresis on the transdermal delivery of ketoprofen

The aim of this study was to determine the individual and combined effects of peptide dendrimers and low frequency ultrasound on the transdermal permeation of ketoprofen. Arginine terminated peptide dendrimers of varying charges (4+, 8+ and 16+, named as A4. A8 and A16 respectively) were synthesized and characterized. Ketoprofen was subjected to passive, peptide dendrimer-assisted and sonophoretic permeation studies (with and without dendrimer application) across Swiss albino mouse skin, both in vitro and in vivo. The studies revealed that the synthesized peptide dendrimers considerably increased the transdermal permeation of ketoprofen and displayed enhancement ratios of up to 3.25 (with A16 dendrimer), compared to passive diffusion of drug alone in vitro. Moreover, the combination of peptide dendrimer treatment and ultrasound application worked in synergy and gave enhancement ratios of up to 1369.15 (with ketoprofen-A16 dendrimer complex). In vivo studies demonstrated that dendrimer and ultrasound-assisted permeation of drug achieved much higher plasma concentration of drug, compared to passive diffusion. Comparison of transdermal and oral absorption studies revealed that transdermal administration of ketoprofen with A8 dendrimer showed comparable absorption and plasma drug levels with oral route. The excised mouse skin after in vivo permeation study with dendrimers and ultrasound did not show major toxic reactions. This study demonstrates that arginine terminated peptide dendrimers combined with sonophoresis can effectively improve the transdermal permeation of ketoprofen.

Nano-transfersomal formulations for transdermal delivery of asenapine maleate: in vitro and in vivo performance evaluations

Abstract Context: Asenapine maleate (ASPM) is an antipsychotic drug for the treatment of schizophrenia and bipolar disorder. Extensive metabolism makes the oral route inconvenient for ASPM. Objective: The objective of this study is to increase ASPM bioavailability via transdermal route by improving the skin permeation using combined strategy of chemical and nano-carrier (transfersomal) based approaches. Materials and methods: Transfersomes were prepared by the thin film hydration method using soy-phosphatidylcholine (SPC) and sodium deoxycholate (SDC). Transfersomes were characterized for particle size, polydispersity index (PDI), zeta potential (ZP), entrapment efficiency, surface morphology, and in vitro skin permeation studies. Various chemical enhancers were screened for skin permeation enhancement of ASPM. Optimized transfersomes were incorporated into a gel base containing suitable chemical enhancer for efficient transdermal delivery. In vivo pharmacokinetic study was performed in rats to assess bioavailability by transdermal route against oral administration. Results and discussion: Optimized transfersomes with drug:SPC:SDC weight ratio of 5:75:10 were spherical with an average size of 126.0 nm, PDI of 0.232, ZP of −43.7 mV, and entrapment efficiency of 54.96%. Ethanol (20% v/v) showed greater skin permeation enhancement. The cumulative amount of ASPM permeated after 24 h (Q24) by individual effect of ethanol and transfersome, and in combination was found to be 160.0, 132.9, and 309.3 μg, respectively, indicating beneficial synergistic effect of combined approach. In vivo pharmacokinetic study revealed significant (p < 0.05) increase in bioavailability upon transdermal application compared with oral route. Conclusion: Dual strategy of permeation enhancement was successful in increasing the transdermal permeation and bioavailability of ASPM.

Peptide dendrimer-conjugates of ketoprofen: Synthesis and ex vivo and in vivo evaluations of passive diffusion, sonophoresis and iontophoresis for skin delivery

&NA; The aim of this study was to evaluate skin delivery of ketoprofen when covalently tethered to mildly cationic (2+ or 4+) peptide dendrimers prepared wholly by solid phase peptide synthesis. The amino acids glycine, arginine and lysine formed the dendrimer with ketoprofen tethered either to the lysine side‐arm (N&egr;) or periphery of dendrimeric branches. Passive diffusion, sonophoresis‐ and iontophoresis‐assisted permeation of each peptide dendrimer‐drug conjugate (D1–D4) was studied across mouse skin, both in vitro and in vivo. In addition, skin toxicity of dendrimeric conjugates when trialed with iontophoresis or sonophoresis was also evaluated. All dendrimeric conjugates improved aqueous solubility at least 5‐fold, compared to ketoprofen alone, while also exhibiting appreciable lipophilicity. In vitro passive diffusion studies revealed that ketoprofen in its native form was delivered to a greater extent, compared with a dendrimer‐conjugated form at the end of 24 h (Q24 h (&mgr;g/cm2): ketoprofen (68.06 ± 3.62) > D2 (49.62 ± 2.92) > D4 (19.20 ± 0.89) > D1 (6.45 ± 0.40) > D3 (2.21 ± 0.19). However, sonophoresis substantially increased the skin permeation of ketoprofen‐dendrimer conjugates in 30 min (Q30 min (&mgr;g/cm2): D4 (122.19 ± 7.14) > D2 (66.74 ± 3.86) > D1 (52.10 ± 3.22) > D3 (41.66 ± 3.22)) although ketoprofen alone again proved superior (Q30 min: 167.99 ± 9.11 &mgr;g/cm2). Next, application of iontophoresis was trialed and shown to considerably increase permeation of dendrimeric ketoprofen in 6 h (Q6 h (&mgr;g/cm2): D2 (711.49 ± 39.14) > D4 (341.23 ± 16.43) > D3 (89.50 ± 4.99) > D1 (50.91 ± 2.98), with a Q6 h value of 96.60 ± 5.12 &mgr;g/cm2 for ketoprofen alone). In vivo studies indicated that therapeutically relevant concentrations of ketoprofen could be delivered transdermally when iontophoresis was paired with D2 (985.49 ± 43.25 ng/mL). Further, histopathological analysis showed that the dendrimeric approach was a safe mode as ketoprofen alone. The present study successfully demonstrates that peptide dendrimer conjugates of ketoprofen, when combined with non‐invasive modalities, such as iontophoresis can enhance skin permeation with clinically relevant concentrations achieved transdermally. Graphical abstract Figure. No caption available.

Liposome encapsulated soy lecithin and cholesterol can efficiently replace chicken egg yolk in human semen cryopreservation medium

Abstract Cryopreservation of spermatozoa plays a significant role in reproductive medicine and fertility preservation. Chicken egg yolk is used as an extender in cryopreservation of human spermatozoa using glycerol egg yolk citrate (GEYC) buffered medium. Even though 50% survival of spermatozoa is generally achieved with this method, the risk of high levels of endotoxins and transmission pathogens from chicken egg yolk is a matter of concern. In the present study we attempted to establish a chemically defined cryopreservation medium which can replace the chicken egg yolk without affecting sperm survival. Ejaculates from 28 men were cryopreserved with GEYC based freezing medium or liposome encapsulated soy lecithin-cholesterol based freezing medium (LFM). The semen samples were subjected to rapid thawing after 14 days of storage in liquid nitrogen. Post-thaw analysis indicated significantly higher post-thaw motility and sperm survival in spermatozoa cryopreserved with LFM compared to conventional GEYC freezing medium. The soy lecithin and cholesterol at the ratio of 80:20 with sucrose showed the highest percentage of post-thaw motility and survival compared to the other compositions. In conclusion, chemically defined cryopreservation medium with liposome encapsulated soy lecithin and cholesterol can effectively replace the chicken egg yolk from human semen cryopreservation medium without compromising post-thaw outcome.

Indian propolis ameliorates the mitomycin C-induced testicular toxicity by reducing DNA damage and elevating the antioxidant activity

Development of excellent curative therapy for most of the malignancies has resulted in a growing population of cancer survivors who are at increased risk for a variety of health problems including infertility. Therefore, fertility preservation has become an important issue during cancer treatment in recent years. Combination therapy with natural agents such as vitamins, antioxidants, dietary supplements, and plant products are considered as an attractive option to mitigate normal tissue toxicity imparted by chemotherapy. The aim of the present study was to explore the beneficial effect of hydroethanolic extract of Indian propolis (HEIP) on mitigating mitomycin C (MMC)-induced testicular damage and its mechanism of action. Healthy adult male mice were injected intraperitoneally with saline, MMC, HEIP and HEIP followed by MMC after 1h. The animals were dissected at 35days after various treatments to analyze testicular function. MMC administration resulted in significant reduction in testicular function in a dose-dependent manner at 35days after treatment which significantly improved by HEIP pre-treatment. At 24h after treatment, MMC induced significant increase in oxidative stress, γ-H2AX foci and expression of RAD51 and KU80 in testicular cells. Prior treatment with HEIP decreased the oxidative stress, reduced DNA damage and restored the testicular testosterone and inhibin B level. In conclusion, co-administration of Indian propolis extract may play a promising beneficial role in fertility preservation of males undergoing chemotherapy.

Peptide Dendrimers in Delivery of Bioactive Molecules to Skin

The development of dendrimers as potential drug carriers or scaffolds is currently one of the most active areas of pharmaceutical and biomedical sciences. Peptide dendrimers, an alternative class of dendrimers, are wedge-like molecules of high molecular weight, composed of basic amino acids associated through amide and peptide bonds; these bonds are present both inside the branching core and on their external surface. Delivery of drugs through skin has many advantages compared to the traditional routes (ie, reduced adverse effects, noninvasiveness and resulting patient compliance, avoidance of first-pass metabolism, and allowing for sustained drug delivery). Unlike acrylate dendrimers, peptide dendrimers are broken down to simple and harmless amino acids, possess low toxicity, and are cost-effective to prepare and purify in bulk when synthesized using solid-phase peptide synthesis. Hence, amino acid–based peptide dendrimers are being regarded as highly favorable scaffolds for the efficient and safe delivery of drugs or genes. In this regard, their future in the transdermal delivery of therapeutics is certainly necessary.

Sunscreen creams containing naringenin nanoparticles: Formulation development and in vitro and in vivo evaluations

The aim of this study was to develop sunscreen creams containing polymeric nanoparticles (NPs) of naringenin for photoprotective and antioxidant effects.

Liposome-encapsulated diacyl glycerol and inositol triphosphate-induced delayed oocyte activation and poor development of parthenotes

Objective: To explore the ability of diacyl glycerol (DAG) and inositol triphosphate (IP3), two major secondary messengers in the calcium signaling pathway, in activating oocytes. Material and Methods: Oocyte cumulus complex obtained from superovulated Swiss albino mice were incubated in M16 medium with liposome-encapsulated 1,2-Dipalmitoyl-sn-glycerol (LEDAG) and/or IP3 for 3 h. Strontium chloride was used as positive control. The activation potential, ploidy status, and blastocyst rate was calculated. Results: Both DAG and IP3, individually, induced activation in ~98% of oocytes, which was significantly higher (p<0.01) than activation induced by strontium chloride (60%). Delayed pronucleus formation and a higher percentage of diploid parthenotes was observed in oocytes activated with LEDAG and/or IP3. However, these embryos failed to progress beyond the 6-8–cell stage. Only when the medium was supplemented with LEDAG (5 μg/mL) and IP3 (10 μg/mL) could activated oocytes progress till the blastocyst stage (5.26%), which was lower than the blastocyst rate in the positive controls (13.91%). Conclusion: The results of the present study indicate that DAG and IP3 can induce delayed oocyte activation and poor development of parthenotes in vitro.

Full Factorial Experimental Design for Development and Validation of a RP-HPLC Method for Estimation of Letrozole in Nanoformulations

Background: Letrozole (LTZ) is a potent aromatase inhibitor which blocks estrogen synthesis in post-menopausal women. Suitable analytical methods are required to quantify letrozole in bulk and its nanoformulations. Hence a sensitive and robust reverse-phase high performance liquid chromatographic method (RP-HPLC) was developed for the estimation of LTZ.Methods: The method was developed using Kinetex C18 column (250 mm�4.6 mm; 5�) and mobile phase comprising acetonitrile (ACN): acetate buffer (pH 4.5) mixture (50:50% v/v) was used to affect the chromatographic separation at 0.8 mL/min flow rate. The responses were measured at 240 nm.Force degradation studies were done by exposing LTZ to acid- and alkali-induced hydrolysis, oxidation (H2O2), thermal and photolysis. Two-level factorial design was used to validate the method as per ICH Q2 (R1) using Design-Expert® software. The effects of independent variables on flow rate, pH, acetonitrile content and column temperature were recorded as responses.Results: Method was linear over the concentration range of 5-2500 ng/mL with a correlation coefficient (R2) of 1.000. Force degradation studies revealed that LTZ was stable to all stress agents except the basic conditions. The inter-day precision results were reproducible with relative standard deviation (% RSD) of 0.078. The peak area RSD and mean recovery of LTZ was found to be <1.0% and 99 102% in drug solution and <1.0% and 98-106% in nanoformulations respectively. Deliberate changes in independent chromatographic parameters analyzed using analysis of variance (ANOVA) indicated that the model was significant (p<0.0001).Conclusion: The developed analytical method was successfully utilized to quantify LTZ in bulk and its nanoformulations.