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. EXPERT OPINION: The understanding of a process is facilitated by proper identification of sources of variation, management of variability by process design, and prediction of product quality attributes using design space. The pharmaceutical industry is rapidly adopting the QbD principles for fabrication of safe, effective and quality products; however, we are still on a journey and the process of gathering all experience and metrics required for connecting and demonstrating QbD benefits to all stakeholders is still in progress. Understanding the formulation and process parameters with the philosophy of QbD will be useful for the optimization of complex drug delivery systems in the near future.

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. Bilosomes represent a key advance in oral vaccine delivery because they are more resistant to disruption by gastric acid as well as enzymes. Here, we focus on different aspects of bilosomes including composition, developmental techniques, stability, transitional modifications and scale-up - emphasizing their biomedical potential in oral immunization against various diseases.

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, 0.184) were chosen after optimizing the composition and process conditions. The optimized chitosan microparticles were subjected to alginate coating for better protection of loaded antigen till it reached to uptake site i.e. M cells in the Peyer's patches (PPs) and transport of higher amount antigen to the PPs. The zeta-potential values for uncoated chitosan microparticles and ACMs were found to be +29 ± 3.3 mV and -32.6 ± 4.2 mV, respectively. This change of zeta potential, for uncoated to coated, can be explained by the fact that the coating of alginate on chitosan microparticles led to negative side of the zeta potential by virtue of its predominance on the surface. The developed ACMs were able to transport the antigen effectively to the M cell as revealed by confocal laser scanning microscopy. Further, DTx-loaded ACMs demonstrated significant immune responses at serum IgG as well as mucosal sIgA level.

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. These proteins have been reported not to form oligomers despite the fact that oligomerization of chaperonins is regarded as essential for their function. We show here that the Cpn60.2 homologue from Mycobacterium smegmatis also fails to oligomerize under standard conditions. However, we also show that the Cpn60.2 proteins from both organisms can replace the essential groEL gene of Escherichia coli, and that they can function with E. coli GroES cochaperonin, as well as with their cognate cochaperonin proteins, strongly implying that they form oligomers in vivo. We show that the Cpn60.1 proteins, but not the Cpn60.2 proteins, can complement for loss of the M. smegmatis cpn60.1 gene. We investigated the oligomerization of the Cpn60.2 proteins using analytical ultracentrifugation and mass spectroscopy. Both form monomers under standard conditions, but they form higher order oligomers in the presence of kosmotropes and ADP or ATP. Under these conditions, their ATPase activity is significantly enhanced. We conclude that the essential mycobacterial chaperonins, while unstable compared to many other bacterial chaperonins, do act as oligomers in vivo, and that there has been specialization of function of the mycobacterial chaperonins following gene duplication.

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 localized delivery of this drug in this regard would be highly beneficial with respect to safety as well as efficacy. METHODS: In the current studies, an endeavor has been made to investigate the efficacy of topically applied liposome-encapsulated TAM on skin cancer model. The drug was encapsulated in phospholipid-based vesicular systems viz. conventional liposomes and elastic liposomes. Incidence of papillomas and histopathological examination were employed to determine the efficacy of the tested formulations. RESULTS: The results demonstrated carrier-dependent strong inhibition of skin carcinogenesis with encapsulated drug vis-à-vis drug in the solution form. The encouraging findings from the current work construe immense potential of the TAM-loaded liposomal systems in the management of skin cancer.

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. PRINCIPAL FINDINGS: Here we show that the proline rich homeodomain protein PRH/Hhex forms predominantly octameric and/or hexadecameric species in solution as well as larger assemblies. We show that these assemblies are highly stable resisting denaturation by temperature and chemical denaturants. CONCLUSION: These data indicate that PRH is functionally and structurally related to the Lrp/AsnC family of proteins, a group of proteins that are known to act globally to control gene expression in bacteria and archaea.

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. Thus, the treatment of scalp psoriasis with a topical coal tar formulation requires that special concern be paid to product esthetics. OBJECTIVE: This study aimed to evaluate the hair-staining characteristics of a novel lecithinized coal tar (LCT) formulation on different types of mammalian hair. METHODS: Samples of hair from different mammals, including human, sheep, rabbit, and goat, were repeatedly exposed to the LCT formulation over 14 days. The color of hair samples treated with LCT was compared with that of untreated control hair samples. RESULTS: The study revealed the distinct non-staining potential of the LCT formulation. CONCLUSIONS: This LCT formulation lacks the propensity to stain hair and thus has excellent potential to be exploited in the treatment of scalp psoriasis.

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. Another objective was to determine the oral dose that will produce serum antibody titres comparable with those produced by i.m. doses of DTx. EXPERIMENTAL APPROACH: Bilosomes containing DTx were prepared by thin film hydration and characterized in vitro for their shape, size, percent antigen entrapment and stability. In the in vivo study the anti-DTx IgG and anti-DTx sIgA response was estimated using elisa, in serum and in various body secretions, respectively, following oral immunization with different doses of DTx entrapped in nano-bilosomes. KEY RESULTS: High dose loaded nano-bilosomes (DTxNB3, 2Lf) produced comparable anti-DTx IgG levels in serum to those induced by i.m. alum-adsorbed DTx (0.5Lf). In addition, all the nano-bilosomal preparations elicited a measurable anti-DTx sIgA response in mucosal secretion, whereas i.m. alum-adsorbed DTx (0.5Lf) was unable to elicit this response. CONCLUSIONS AND IMPLICATIONS: The orally administered nano-bilosomal DTx formulation produced comparable serum antibody titres to i.m.alum-adsorbed DTx, at a fourfold higher dose and without the induction of tolerance. This approach will provide an effective and comprehensive immune protection against diphtheria with better patient compliance.

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 2.75 : 1) were fixed for the phase diagram construction. Selected microemulsions were evaluated for globule size, zeta potential, viscosity, refractive index, per cent transmittance, stability (freeze thaw and centrifugation), ex vivo skin permeation and retention. The microemulsion systems composed of IPM and T80 with mean particle diameter of 72.8 nm showed maximum skin permeation (82.23%), skin permeation flux (0.281 mg/cm²/h) along with skin retention (8.31%) vis-à-vis systems containing TA and T20. The results suggest that the developed novel lecithinized microemulsion systems have a promising potential for the improved topical delivery of dithranol.

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. tuberculosis ESAT-6, the crystal structure of GBS1074 reveals a homodimeric structure similar to homologous structures from Staphylococcus aureus and Helicobacter pylori. Intriguingly, GBS1074 forms elongated fibre-like assemblies in the crystal structure.

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. Here, we use analytical ultracentrifugation and electron microscopy to further characterize PRH oligomers. We use the same techniques to show that PRH oligomers bound to long DNA fragments self-associate to form highly ordered assemblies. Electron microscopy and linear dichroism reveal that PRH oligomers can form protein-DNA fibres and that PRH is able to compact DNA in the absence of other proteins. Finally, we show that DNA compaction is not sufficient for the repression of PRH target genes in cells. We conclude that DNA compaction is a consequence of the binding of large PRH oligomers to arrays of binding sites and that PRH is functionally and structurally related to the Lrp/AsnC family of proteins from bacteria and archaea, a group of proteins formerly thought to be without eukaryotic equivalents.

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 1.2), SIF (USP, pH 7.5) and in bile salt solutions. Biological activity of CTB, subsequent to conjugation, was verified by hemagglutination test. Anti-HBsAg IgG response in serum and anti-HBsAg sIgA in various body secretions were estimated using ELISA, following oral immunization with 10 microg dose-loaded CTB-conjugated bilosomes (CTB2) and 20 microg dose-loaded CTB-conjugated bilosomes (CTB1) in BALB/c mice. The results showed that CTB1 produced anti-HBsAg IgG antibody titre response comparable to that of the intramuscular (i.m.) injection of 10 microg of alum-adsorbed HBsAg. Moreover, all the bilosomal preparations elicited measurable sIgA vis-à-vis negligible response with i.m. administered HBsAg. Thus, HBsAg-loaded CTB-conjugated bilosomes provide a promising potential for targeted oral immunization against hepatitis B.

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. Furthermore, much larger doses can induce antigen tolerance. Therefore the purpose of the present study was firstly to overcome these problems by the use of bile salt stabilized vesicles (bilosomes) and HBsAg as the model antigen,which could provide both protection to the antigen as well as enable transmucosal uptake and subsequent immunization. Another purpose of this study was to determine the dose that could produce serum antibody titres against hepatitis B via the oral route compared to those following intramuscular immunization. METHODS: In the present study bilosomes containing recombinant hepatitis B surface antigen were prepared by a lipid cast film method. HBsAg loaded bilosomeswere characterized in vitro for their shape, size, percent antigen entrapment and stability. Fluorescence microscopy was carried out to confirm the uptake of bilosomes by gut associated lymphoid tissues (GALT). The in vivo part of the study comprised estimation of anti-HBsAg IgG response in serum and anti-HBsAg sIgA in various body secretions using specific ELISA techniques following oral immunization with low dose loaded bilosomes (B1, 10 microg), intermediate dose loaded bilosomes (B2, 20 microg) and high dose loaded bilosomes (B3, 50 microg) in BALB/c mice. RESULTS: Fluorescence microscopy suggested that there was an increase in fluorescence intensity following the uptake of bilosomes entrapped FITC-BSA in gut associated lymphoid tissues. The high dose HBsAg bilosomes (B3, 50 microg) produced comparable anti-HBsAg IgG levels in serum to those observed in the case of intramuscular administration of alum adsorbed HBsAg (10 microg). In addition, the bilosomal preparations elicited measurable sIgA in mucosal secretions, where the highest responses were observed with high dose HBsAg bilosomes (B3,50 microg) and as expected, intramuscular administered alum adsorbed HBsAg (10 microg) failed to elicit such responses. CONCLUSIONS: HBsAg loaded bilosomes produced both systemic as well as mucosal antibody responses upon oral administration. Furthermore, bilosomes with a five times higher dose upon oral administration produced comparable serum antibody titres to those obtained after intramuscular immunization without the induction of systemic tolerance.

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. Gel-filtration chromatography reveals that the protein adopts trimeric and/or pentameric quaternary structures, with solubility retained up to concentrations of 5.0-5.5 mg/ml in aqueous solutions. Mutations inserting a single tryptophan residue as a spectroscopic probe at three different sites cause no perturbation in the protein's CD spectral characteristics, or in its quaternary structural status. The protein is cooperatively dissociated, and non-cooperatively unfolded, by both guanidine hydrochloride and urea. Intriguingly, unlike with GroES, retro-GroES is not unfolded by heat. Instead, there is a reversible structural transition involving conversion of PPII structure to beta sheet structure, upon heating, with no attendant aggregation even at 90 degrees C. Retro-GroES does not bind GroEL. In summary, some structure-forming characteristics of GroES appear to be conserved through the backbone reversal process, although the differential conformational behavior upon heating also indicates differences.

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 HSP12.6, both earlier created and characterized by our group. These were covalently labeled with fluorescent dyes of well-resolved spectral characteristics [retro-CspA with FITC, and retro-HSP12.6 with TRITC] and then allowed to aggregate within the same reaction vessel. The resultant aggregates are shown by spectrofluorimetry-coupled confocal laser scanning microscopy to constitute uniform mixtures of both proteins, existing within every cylindrical volume element of approximately 200nm diameter, and comparable height, in all sections of the co-aggregated material suggesting that the two proteins do not selectively associate with copies of themselves during aggregation. Thus, it would appear that aggregation can occur without reference to protein molecular identity.

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). The pigment showed a buffering ability in aqueous solutions, maintaining an alkaline pH of 8.0. It behaved as a colorimetric pH indicator over a wide acidic and alkaline pH range, with discoloration occurring ostensibly through hydrolysis of key chemical groups at extremely acidic pH ( approximately 2.0). The pigment was found to have some structural resemblance with the copper-colored pigment (herquinone) produced by Penicillium herquei as both pigments contain the phenalene carbon framework. The notable differences between herquinone and the pigment produced by P. marneffei are (i) the latter's apparent dimerization through a sulphur-sulphur (disulfide) bond and (ii) the presence of 1,1,3,3-tetramethyl-2,3-dihydropyrrole moiety in the latter instead of 2,3,3-trimethyl-2,3-dihydrofuran moiety found in the former. The delineation of the structure of the pigment produced by Penicillium marneffei may help in understanding certain aspects of the biology of this pathogenic fungus.

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. This peptide serves as a lead molecule to design a shorter peptide that may be effective as a therapeutic agent. In this study we employed a systematic chemical approach using alanine scanning of Ac-FLEAIG-OH and subsequent peptide shortening to study the binding of shorter peptides to Z-AT. While two additional 6-mer peptides Ac-FLAAIG-OH and Ac-FLEAAG-OH were found to bind to Z-AT, their daughter peptides Ac-FLEAA-NH(2) and Ac-FLAA-NH(2) also bound avidly to Z-AT and prevented polymerization of the protein. Further comparative studies revealed that the binding of Ac-FLAA-NH(2) was more specific for Z-AT. The peptide-AT complex formation was enhanced by the presence of C-terminal amide group on the peptide, and circular dichroism analysis demonstrated that a random coil rather than a beta-helical conformation favored binding of the peptide to AT. In summary, this study has identified novel small peptides that inhibit Z-AT polymerization, and are a significant advance towards the treatment of Z-AT-related cirrhosis and emphysema.

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. The folded (beta sheet) domain thus obtained is found to be (a) predominantly trimeric, and to display (b) significant surface hydrophobicity, (c) a marked tendency to undergo degradation, and (d) a tendency to aggregate upon heating, and on exposure to UV light. Thus, the twin 'chaperone' features of multimericity and surface hydrophobicity are clearly seen to be insufficient for this domain to function as a chaperone. Since alpha-crystallin interacts with its substrates through hydrophobic interactions, the hydrophobicity of the excised domain indicates that separation of domains may regulate function; at the same time, the fact is also highlighted that surface hydrophobicity is a liability in a chaperone since heating strengthens hydrophobic interactions and can potentially promote self-aggregation. Thus, it would appear that the role of the N-terminal domain in alpha-crystallin is to facilitate the creation of a porous, hollow structural framework of >/=24 subunits in which solubility is effected through increase in the ratio of exposed surface area to buried volume. Trimers of interacting C-terminal domains anchored to this superstructure, and positioned within its interior, might allow hydrophobic surfaces to remain accessible to substrates without compromising solubility.

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. As a note of caution, we wish to state that we have not been able to completely eliminate the possibility that the radiation may be an artifact owing to second order effects such as, e.g., Raman scattering of Raman-scattered photons; however, we assert that all our experiments indicate that the radiation actually owes to some form of fluorescence. We propose that peptide electrons that have been delocalized through intramolecular or intermolecular hydrogen bond formation display these long-wavelength electronic transitions. If confirmed by future studies, this preliminary discovery may turn out to have important implications for biomolecular spectroscopy, protein crystallography, and materials science.