Quality by design (QbD) approaches in current pharmaceutical set-up.

Introduction: Quality by design (QbD) encourages the pharmaceutical industry to use risk management and science-based manufacturing principles to gain process and product understanding and thus assures quality of the product. With the objective to curb the rising costs for development and regulatory barriers to innovation and creativity, QbD is being widely promoted by Food and Drug Administration (FDA) and International Conference on Harmonization (ICH).

Areas Covered: This review describes the elements, different design and tools of QbD as well as multidimensional applications of QbD ranging from dosage form and method development to meeting latest regulatory requirements.

Bilosomes in the context of oral immunization: development, challenges and opportunities.

Oral administration remains the most favored means of drug administration as far as patient compliance is concerned. For the majority of vaccines and biologics, the oral route is not a good choice because of diminished uptake of the administered dose. Vesicular carriers like liposomes and niosomes are among the potential candidates for vaccine delivery by the oral route but their instability in the gastrointestinal environment is a problem.

Alginate coated chitosan microparticles mediated oral delivery of diphtheria toxoid. Part A. Systematic optimization, development and characterization.

The current study was embarked upon to develop "optimized" alginate coated chitosan microparticles (ACMs) loaded with Diphtheria toxoid (DTx) employing formulation by design approach. The developed system was characterized for particle size, zeta potential, surface morphology, acidic degradation protection studies, in process stability studies, storage stability studies and in-vivo uptake studies. Microparticles with minimum of average size of 5 μm (PDI, 0.

The unusual mycobacterial chaperonins: evidence for in vivo oligomerization and specialization of function.

The pathogen Mycobacterium tuberculosis expresses two chaperonins, one (Cpn60.1) dispensable and one (Cpn60.2) essential.

Tamoxifen-loaded novel liposomal formulations: evaluation of anticancer activity on DMBA-TPA induced mouse skin carcinogenesis.

Purpose: Tamoxifen (TAM) is a non-steroidal estrogen receptor modulator known for its anticancer activity. Apart from marked breast cancer activity, this drug has also shown potential in treating other types of cancers including skin cancers. TAM is reported to be associated with serious side effects primarily due to its systemic distribution.

The proline rich homeodomain protein PRH/Hhex forms stable oligomers that are highly resistant to denaturation.

Background: Many transcription factors control gene expression by binding to specific DNA sequences at or near the genes that they regulate. However, some transcription factors play more global roles in the control of gene expression by altering the architecture of sections of chromatin or even the whole genome. The ability to form oligomeric protein assemblies allows many of these proteins to manipulate extensive segments of DNA or chromatin via the formation of structures such as DNA loops or protein-DNA fibres.

Novel stain-free lecithinized coal tar formulation for psoriasis.

Background: Coal tar has been a very popular traditional treatment for various types of psoriasis for over a century. It is the first-line treatment for scalp, hand, and foot psoriasis. However, the application of coal tar on hair invariably causes staining, which results in a high degree of patient non-compliance, especially in patients with non-black hair.

Significant systemic and mucosal immune response induced on oral delivery of diphtheria toxoid using nano-bilosomes.

Background And Purpose: Over the last decade apprehension has been growing over the effectiveness of existing parenteral vaccines for diphtheria and has created an interest in the development of a mucosally active vaccine. Oral immunization appears to be an effective alternative, but is not without the inherent disadvantages of antigen destruction and tolerance. Therefore, our objective was to investigate the incorporation of diphtheria toxoid (DTx) into bilosomes, which could provide protection as well as aid transmucosal uptake and subsequent immunization.

Novel dithranol phospholipid microemulsion for topical application: development, characterization and percutaneous absorption studies.

The objective of this study was to develop and characterize a novel dithranol-containing phospholipid microemulsion systems for enhanced skin permeation and retention. Based on the solubility of dithranol, the selected oils were isopropyl myristate (IPM) and tocopherol acetate (TA), and the surfactants were Tween 80 (T80) and Tween 20 (T20). The ratios of cosurfactants comprising of phospholipids and ethanol (1 : 10) and surfactant to co-surfactant (1 : 1 and 2.

The homodimeric GBS1074 from Streptococcus agalactiae.

ESAT-6 is a well characterized secreted protein from Mycobacterium tuberculosis and represents the archetype of the WXG100 family of proteins. Genes encoding ESAT-6 homologues have been identified in the genome of the human pathogen Streptococcus agalactiae; one of these genes, esxA, has been cloned and the recombinant protein has been crystallized. In contrast to M.

DNA compaction by the higher-order assembly of PRH/Hex homeodomain protein oligomers.

Protein self-organization is essential for the establishment and maintenance of nuclear architecture and for the regulation of gene expression. We have shown previously that the Proline-Rich Homeodomain protein (PRH/Hex) self-assembles to form oligomeric complexes that bind to arrays of PRH binding sites with high affinity and specificity. We have also shown that many PRH target genes contain suitably spaced arrays of PRH sites that allow this protein to bind and regulate transcription.

M-cell targeted delivery of recombinant hepatitis B surface antigen using cholera toxin B subunit conjugated bilosomes.

The present study aims to improve upon our earlier findings with bilosomes as potential delivery vehicle through oral route for recombinant hepatitis B surface antigen (HBsAg). The work entails the conjugation of bilosomal system with cholera toxin B subunit (CTB) to increase transmucosal uptake via M-cell specific delivery approach. The study encompasses the development and characterization of HBsAg-loaded CTB-conjugated system for percent antigen entrapment, size, shape, and stability in SGF (USP, pH 1.

Oral immunization against hepatitis B using bile salt stabilized vesicles (bilosomes).

Purpose: Most of the presently available vaccines including hepatitis B vaccines administered through parenteral route, fail to induce a mucosal antibody response. Therefore, oral immunization appears to be an effective and attractive alternative to parenteral immunization. However, the problem of degradation of antigen in the harsh and hostile environment of the gastrointestinal tract consequently requires larger doses and more frequent dosing of antigen.

Folding behavior of a backbone-reversed protein: reversible polyproline type II to beta-sheet thermal transitions in retro-GroES multimers with GroES-like features.

The structural consequences of the reversal of polypeptide backbone direction (retro modification) remain insufficiently explored. Here, we describe the behavior of an engineered, backbone-reversed form of the 97 residues-long GroES co-chaperonin of Escherichia coli. FTIR and far-UV CD spectroscopy suggest that retro-GroES adopts a mixed polyproline type II (PPII)-beta-strand structure with a beta(II) type CD spectrum similar to that of GroES.

Confocal spectrofluorimetric evidence for the hetero-aggregation of sequence-scrambled forms of two model all-beta sheet proteins.

Can two unrelated proteins with deliberately compromised folding abilities, marked propensities to aggregate upon increase of protein concentration, and proclivities towards beta sheet formation, be caused to hetero-aggregate? We address this question here using the 'designer' backbone-reversed forms of two model all-beta sheet proteins, E. coli CspA and C. elegans HSP12.

Putative structure and characteristics of a red water-soluble pigment secreted by Penicillium marneffei.

The dimorphic fungus, Penicillium marneffei, produces and secretes a brick red pigment, during growth at temperatures below 30 degrees C. It generally diffuses into commonly used media like Sabouraud dextrose agar and malt extract agar. The pigment was purified by reverse-phase liquid chromatography and subjected to structural determination by elemental and spectral analysis using atomic absorption (AAS), ultra violet and visible (UV-VIS), fluorescence, infra red (IR), nuclear magnetic resonance (NMR) and tandem mass spectrometry (MS-MS).

Identification of a 4-mer peptide inhibitor that effectively blocks the polymerization of pathogenic Z alpha1-antitrypsin.

alpha(1)-Antitrypsin (AT) is a major proteinase inhibitor within the lung. The Z variant of AT (E342K) polymerizes within the liver and lung, resulting in hepatic aggregation of AT and tissue deficiency, predisposing to early onset of cirrhosis and emphysema, respectively. Polymerization of the aberrant protein can be prevented in vitro by specific peptides such as FLEAIG.

The excised heat-shock domain of alphaB crystallin is a folded, proteolytically susceptible trimer with significant surface hydrophobicity and a tendency to self-aggregate upon heating.

The lens protein, alpha-crystallin, is a molecular chaperone that prevents the thermal aggregation of other proteins. The C-terminal domain of this protein (homologous to domains present in small heat-shock proteins) is implicated in chaperone function, although the domain itself has been reported to show no chaperone activity. Here, we show that the domain can be excised out of the intact alphaB polypeptide and recovered directly in pure form through the transfer of CNBr digests of whole lens homogenates into urea-containing buffer, followed by dialysis-based refolding of digests under acidic conditions and a single gel-filtration purification step.

A novel UV laser-induced visible blue radiation from protein crystals and aggregates: scattering artifacts or fluorescence transitions of peptide electrons delocalized through hydrogen bonding?

Proteins lacking prosthetic groups and/or cofactors are known to undergo electronic excitation transitions only upon exposure to UV-C (< 280 nm) and UV-B (280-320 nm), but not UV-A (320-400 nm) photons. Here, we report the discovery of a novel excitation that peaks at approximately 340 nm and yields visible violet-blue radiation with apparent band maxima at approximately 425, 445, 470, and 500 nm. All proteins and large polypeptides examined in solid form, and in solutions, display this quenchable and photobleachable radiation which can be established not owing to aromatic sidechains.

Folding of beta/alpha-unit scrambled forms of S. cerevisiae triosephosphate isomerase: Evidence for autonomy of substructure formation and plasticity of hydrophobic and hydrogen bonding interactions in core of (beta/alpha)8-barrel.

The (beta/alpha)(8)-barrel domain consists of eight topologically equivalent supersecondary structural motifs known as beta/alpha-units. Each unit consists of a single beta-strand, an alpha-helix, and two loops. Evidence collected in recent years indicates that the (beta/alpha)(8)-barrel motif may not be a single, autonomously-folding domain, as was previously assumed.

A backbone-reversed all-beta polypeptide (retro-CspA) folds and assembles into amyloid nanofibres.

The backbone-reversed or 'retro', form of a model all-beta-sheet protein, Escherichia coli CspA, was produced from a synthetic gene in E.coli in fusion with an N-terminal affinity tag. Following purification under denaturing conditions and dialysis-based removal of urea, the protein was found to fold into a soluble, poorly structured multimer.

A backbone-reversed form of an all-beta alpha-crystallin domain from a small heat-shock protein (retro-HSP12.6) folds and assembles into structured multimers.

The structural consequences of polypeptide backbone reversal ("retro" modification) remain largely unexplored, in particular, for the retro forms of globular all-beta-sheet proteins. To examine whether the backbone-reversed form of a model all-beta-sheet protein can fold and adopt secondary and tertiary structure, we created and examined the recombinant retro form of a 110-residue-long polypeptide, an alpha-crystallin-like small heat-shock protein, HSP12.6, from C.

Single-step purification of a protein-folding catalyst, the SlyD peptidyl prolyl isomerase (PPI), from cytoplasmic extracts of Escherichia coli.

The protein-folding catalyst SlyD, a peptidyl prolyl cis-trans isomerase regulated by metal binding, was initially discovered as a major contaminant of non-denaturing immobilized metal-affinity chromatography (IMAC)-based procedures for the purification of heterologously expressed 6xHis-tagged proteins from Escherichia coli. Given its ability to bind weakly to nickel-nitrilotriacetic acid (Ni(2+)-NTA), protocols for the purification of SlyD currently use an initial non-denaturing IMAC step, followed by an ion-exchange chromatographic step and occasionally also other chromatographic steps. Here we demonstrate that using denaturing conditions instead of non-denaturing conditions, and by processing of large quantities of culture through small volumes of Ni(2+)-NTA resin to increase competition for binding, single-step purification of SlyD to homogeneity can be achieved directly from E.

Peptide scanning-based identification of regions of gamma-II crystallin involved in thermal aggregation: evidence of the involvement of structurally analogous, helix-containing loops from the two double Greek key domains of the molecule.

Gamma crystallin is one of three structural proteins present in great abundance in the fiber cells of the vertebrate eye lens. The protein displays a tendency to aggregate readily in the course of heating, cooling, being exposed to ultraviolet radiation, or rapid refolding. To investigate the molecular mechanisms underlying such aggregation, we have employed a peptide-scanning approach aimed at identifying regions of bovine gamma-II crystallin that may be involved in intermolecular interactions leading to aggregation, using assays that measure the competitive inhibition of such aggregation by reagents drawn from a group of contiguous (overlapping) peptides derived from the sequence of the protein itself.