Nanotechnology and pharmaceutical inhalation aerosols

Pharmaceutical inhalation aerosols have been playing a crucial role in the health and well being of millions of people throughout the world for many years. The technology's continual advancement, the ease of use and the more desirable pulmonary-rather-than-needle delivery for systemic drugs has increased the attraction for the pharmaceutical aerosol in recent years. But administration of drugs by the pulmonary route is technically challenging because oral deposition can be high, and variations in inhalation technique can affect the quantity of drug delivered to the lungs. Recent advances in nanotechnology, particularly drug delivery field have encouraged formulation scientists to expand their reach in solving tricky problems related to drug delivery. Moreover, application of nanotechnology to aerosol science has opened up a new category of pharmaceutical aerosols (collectively known as nanoenabled-aerosols) with added advantages and effectiveness. In this review, some of the latest approaches of nano-enabled aerosol drug delivery system (including nano-suspension, trojan particles, bioadhesive nanoparticles and smart particle aerosols) that can be employed successfully to overcome problems of conventional aerosol systems have been introduced.     

Detection of two loci involved in (1-->3)-beta-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene

Genes essential for the production of a linear, bacterial (1-->3)-beta- glucan, curdlan, have been cloned for the first time from Agrobacterium sp. ATCC31749. The genes occurred in two, nonoverlapping, genomic fragments that complemented different sets of curdlan( crd )-deficient transposon-insertion mutations. These were detected as colonies that failed to stain with aniline blue, a (1-->3)-beta-glucan specific dye. One fragment carried a biosynthetic gene cluster (locus I) containing the putative curdlan synthase gene, crdS, and at least two other crd genes. The second fragment may contain only a single crd gene (locus II). Determination of the DNA sequence adjacent to several locus I mutations revealed homology to known sequences only in the cases of crdS mutations. Complete sequencing of the 1623 bp crdS gene revealed highest similarities between the predicted CrdS protein (540 amino acids) and glycosyl transferases with repetitive action patterns. These include bacterial cellulose synthases (and their homologs), which form (1-->4)-beta-glucans. No similarity was detected with putative (1-->3)- beta-glucan synthases from yeasts and filamentous fungi. Whatever the determinants of the linkage specificity of these beta-glucan synthases might be, these results raise the possibility that (1-->3)-beta-glucans and (1-->4)-beta-glucans are formed by related catalytic polypeptides.      

Changes in thylakoid membrane thickness associated with the reorganization of photosystem II light harvesting complexes during photoprotective energy dissipation

Using freeze-fracture electron microscopy we have recently shown that non-photochemical quenching (NPQ), a mechanism of photoprotective energy dissipation in higher plant chloroplasts, involves a reorganization of the pigment-protein complexes within the stacked grana thylakoids.¹ Photosystem II light harvesting complexes (LHCII) are reorganized in response to the amplitude of the light driven transmembrane proton gradient (ΔpH) leading to their dissociation from photosystem II reaction centers and their aggregation within the membrane.¹ This reorganization of the PSII-LHCII macrostructure was found to be enhanced by the formation of zeaxanthin and was associated with changes in the mobility of the pigment-protein complexes therein.¹ We suspected that the structural changes we observed were linked to the ΔpH-induced changes in thylakoid membrane thickness that were first observed by Murikami and Packer.^2,3 Here using thin-section electron microscopy we show that the changes in thylakoid membrane thickness do not correlate with ΔpH per se but rather the amplitude of NPQ and is thus affected by the de-epoxidation of the LHCII bound xanthophyll violaxanthin to zeaxanthin. We thus suggest that the change in thylakoid membrane thickness occurring during NPQ reflects the conformational change within LHCII proteins brought about by their protonation and aggregation within the membrane.Key words: nonphotochemical quenching, photoprotection, LHCII, photosystem II, thylakoid membrane     

Biochemical differentiation in bile duct cestodes and their marsupial hosts

Isozyme electrophoresis was used to assess possible cospeciation of parasites (cestodes of the Progamotaenia festiva complex) and their hosts (Australian diprotodont marsupials) and to compare the extent of interspecific genetic diversity of the parasites and their hosts. On the basis of morphology, there are three species in the complex, although electrophoresis revealed 14 distinct genetic types, most of which were host specific, although there were three cases of apparent host switching. The evolutionary relationships among the parasites were only partially concordant with those among the hosts. Moreover, the extent of electrophoretic diversity among the parasites was much higher than that among hosts.      

Danazol-beta-cyclodextrin binary system: a potential application in emergency contraception by the oral route

This study explored the potential of beta-cyclodextrin to improve the aqueous solubility and dissolution of danazol, investigated a simple and less expensive method for preparation of a danazol-beta-cyclodextrin binary system, and explored the potential application of a danazol-beta-cyclodextrin binary system as a single-dose emergency contraceptive. Phase solubility analysis indicated formation of a first-order soluble complex with stability constant 972.03 M(-1), while Job's plot affirmed 1:1 stoichiometry. The hyperchromic shift in the UV-Vis spectrum of danazol in the presence of beta-cyclodextrin indicated solubilization capability of beta-cyclodextrin for danazol. The extrinsic Cotton effect with a negative peak at 280.7 nm confirmed the inclusion of danazol in the asymmetric locus of beta-cyclodextrin. (1)H-nuclear magnetic resonance analysis suggested that the protons of the steroidal skeleton of danazol display favorable interactions with the beta-cyclodextrin cavity. The danazol-beta-cyclodextrin binary system was prepared by kneading, solution, freeze-drying, and milling methods. The extent of the enhancement of dissolution rate was found to be dependent on the preparation method. Dissolution studies showed a similar relative dissolution rate (2.85) of the danazol-beta-cyclodextrin binary system prepared by the freeze-drying and milling (in the presence of 13% moisture) methods. In a mouse model, the danazol-beta-cyclodextrin binary system at 51.2 mg/kg (equivalent to a 400-mg human dose) showed 100% inhibition of implantation when given postcoitally. Moreover, the danazol-beta-cyclodextrin binary system is safe up to 2000 mg/kg in the mouse (15.52 g/70 kg human) as a single oral dose. Thus, the danazol-beta-cyclodextrin binary system could serve as a new therapeutic application: an oral emergency contraceptive at a physiologically acceptable single dose.     

<Emphasis Type="Italic">In Vivo</Emphasis> Anticancer Efficacy and Toxicity Studies of a Novel Polymer Conjugate <Emphasis Type="Italic">N</Emphasis>-Acetyl Glucosamine (NAG)–PEG–Doxorubicin for Targeted Cancer Therapy

A novel polymer–drug conjugate, polyethylene glycol–N-(acetyl)-glucosamine–doxorubicin (PEG-NAG-DOX) was evaluated in this study for its in vivo potential for treatment of tumours demonstrating improved efficacy and reduced toxicity. The proposed polymer–drug conjugate comprised of polyethylene glycol–maleimide (mPEG-MAL, 30000 Da) as a carrier, doxorubicin (DOX) as an anticancer drug and N-acetyl glucosamine (NAG) as a targeting moiety as well as penetration enhancer. Doxorubicin has a potent and promising anticancer activity; however, severe cardiotoxicity limits its application in cancer treatment. By modifying DOX in PEG-NAG-DOX prodrug conjugate, we aimed to eliminate this limitation. In vivo anticancer efficacy of the conjugate was evaluated using BDF mice-induced skin melanoma model by i.v. administration of DOX conjugates. Anticancer efficacy studies were done by comparing tumour volume, body weight, organ index and percent survival rate of the animals. Tumour suppression achieved by PEG-NAG-DOX at the cumulative dose of 7.5 mg/kg was two-fold better than that achieved by DOX solution. Also, the survival rate for PEG-NAG-DOX conjugate was >70% as compared to <50% survival rate for DOX solution. In addition, toxicity studies and histopathological studies revealed that while maintaining its cytotoxicity towards tumour cells, PEG-NAG-DOX conjugate showed no toxicities to major organs. Therefore, PEG-NAG-DOX conjugate can be suggested as a desirable candidate for targeted cancer therapy.     

Soluble Itraconazole in Tablet Form Using Disordered Drug Delivery Approach: Critical Scale-up Considerations and Bio-equivalence Studies

The present research work explores formulation design, critical scale-up considerations and bio-equivalence studies of soluble itraconazole (ITZ) in a tablet form using disordered drug delivery approach. Disordered system of ITZ with a lower viscosity grade of hydroxypropyl methyl cellulose (Pharmacoat 603) was developed for the first time and extensively characterised at three different stages, namely development of glass system, pellet coating and tablet compression using advanced analytical techniques. Complete molecular embedment of ITZ resulting in amorphisation was observed and found to be sustained until end of the real-time and accelerated stability studies. Developed formulation exhibited comparative in vitro dissolution profile (similarity factor >70) with reference product (Sporanox, Janssen Pharmaceutica) in simulated gastric fluid without enzymes. Formulation was scaled up in three batches (50,000 tablets/batch) with detailed validation of critical process parameters using process capability index method. Critical scale-up considerations like control of residual solvent content, effect of pellet size on dissolution, process variables in pellet coating, compressibility of coated pellets and cushioning effect required for desired compressibility were thoroughly discussed. Bioequivalence study of single dose of test and reference product in seven healthy human volunteers under fed condition exhibited significant bioequivalence with results (AUC_last and AUC_∞) lying between 90% confidence interval. With increase in number of subjects to 24, a significant effect on pharmacokinetic parameters of both reference as well as developed ITZ tablets was observed.     

Nanolipidgel for Enhanced Skin Deposition and Improved Antifungal Activity

The purpose of the research was to prepare and evaluate a topical nanolipidgel (NLH) of terbinafine hydrochloride (TRB), an antimycotic agent, for enhanced skin deposition and improved antifungal activity. Topical solid lipid nanoparticles (SLN) based nanolipidgel was formulated and evaluated. TRB-loaded SLNs were formulated by high-pressure homogenization technique. The stable TRB SLN dispersion was incorporated into a gel using 1% Carbopol 980 NF. Rheological evaluation and texture analysis of the TRB NLH was carried out. Skin permeation, skin deposition, antifungal activity, and occlusivity studies of the nanolipidgel formulation were carried out. The safety of the TRB NLH gel was evaluated using acute skin irritation test on New Zealand White rabbits. The SLN dispersion containing 10% of glyceryl monostearate, 3% of Tween 80, and 1% Plurol Oleique was the most stable. The optimized TRB SLN had a particle size and zeta potential value of 148.6 ± 0.305 nm and −20.4 ± 1.2 mV, respectively. TRB NLH had excellent rheological and texture properties to facilitate its topical application. TRB NLH showed increased skin deposition of the drug over plain (3-fold) and marketed TRB formulation (2-fold). TRB NLH had significantly enhanced antifungal activity against Candida albicans. TRB NLH showed efficient occlusivity and was non-irritant to the rabbit skin with no signs of erythema or edema. Solid lipid nanoparticles-based topical nanolipidgel of terbinafine can be an efficient, industrially scalable, and cost-effective alternative to the existing conventional formulations.KEY WORDS: in vitro antifungal activity, rheological analysis of gel, solid lipid nanoparticles, terbinafine, texture analysis of gel     

RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis

Background

To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH).

Methods

Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test.

Results

We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase.

Conclusion

Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively.