A randomized, active-controlled, multi-centric, phase-II clinical study to assess safety and immunogenicity of a fully liquid DTwP-HepB-IPV-Hib hexavalent vaccine (HEXASIIL®) in Indian toddlers.

BACKGROUND: Combination vaccines are effective in simplifying complex vaccination schedules involving multiple vaccines. A fully liquid hexavalent diphtheria (D)-tetanus (T)-whole-cell pertussis (wP)- hepatitis B (HepB)-inactivated poliovirus (IPV)-Haemophilus influenzae b (Hib) vaccine (HEXASIIL®), manufactured by Serum Institute of India Pvt. Ltd. was tested for safety and immunogenicity following booster vaccination. METHODS: This was a phase-II/III, open label, multicentric, controlled trial in toddlers (phase II) and infants (phase III) in India. This manuscript presents results of phase II. Healthy toddlers aged 12-24 months were randomized (1:1) to receive a 0.5 ml booster dose of HEXASIIL® or comparator Pentavac SD + Poliovac, intramuscularly and followed for 28 days for safety assessment. Blood samples were collected pre-vaccination and 28 days post-vaccination to assess immunogenicity. Descriptive summary statistics were provided for safety and immunogenecity analyses. RESULTS: A total of 223 subjects were randomized. One subject droped out prior to dosing, due to consent withdrawal. Thus, 222 subjects received study vaccine (110 HEXASIIL® and 112 comparator). Frequency of solicited adverse events was comparable between HEXASIIL® and comparator (85.5 % vs 90.2 %). Most local and systemic solicited AEs were mild to moderate in severity. All events resolved completely without any sequelae and none led to subject discontinuation. No vaccine related serious AE was reported. Post vaccination, seroprotection rates against tetanus, Hib and polio type 1 and 3 were 100 % in both the groups. Seroprotection rates for diphtheria (99.1 % vs 100 %) and polio type 2 (98.2 % vs 100 %) were observed in HEXASIIL® and comparator group, respectively. For Hepatitis B, seroprotection was >99 % in both groups. Seroconversion observed for Bordetella Pertussis (94.5 % vs 95.4 %) and Pertussis Toxin (77.1 % vs 87.2 %) in HEXASIIL® and comparator group, respectively. CONCLUSION: HEXASIIL® vaccine was found to be safe and immunogenic in toddlers and supported its further clinical development in infants. Clinical Trial Registration - CTRI/2019/11/022052.

A phase III randomized-controlled study of safety and immunogenicity of DTwP-HepB-IPV-Hib vaccine (HEXASIIL®) in infants.

A fully liquid hexavalent containing Diphtheria (D), Tetanus (T) toxoids, whole cell Pertussis (wP), Hepatitis B (Hep B), type 1, 2, 3 of inactivated poliovirus (IPV) and Haemophilus influenzae type b (Hib) conjugate vaccine (DTwP-HepB-IPV-Hib vaccine, HEXASIIL®) was tested for lot-to-lot consistency and non-inferiority against licensed DTwP-HepB-Hib + IPV in an open label, randomized Phase II/III study. In Phase III part, healthy infants received DTwP-HepB-IPV-Hib or DTwP-HepB-Hib + IPV vaccines at 6, 10 and 14 weeks of age. Blood samples were collected prior to the first dose and 28 days, post dose 3. Non inferiority versus DTwP-HepB-Hib + IPV was demonstrated with 95% CIs for the treatment difference for seroprotection/seroconversion rates. For DTwP-HepB-IPV-Hib lots, limits of 95% CI for post-vaccination geometric mean concentration ratios were within equivalence limits (0.5 and 2). Vaccine was well-tolerated and no safety concerns observed.Clinical Trial Registration - CTRI/2019/11/022052.

Method development and validation of HPLC tandem/mass spectrometry for quantification of perindopril arginine and amlodipine besylate combination in bulk and pharmaceutical formulations.

A well-characterized and fully validated ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometric (UHPLC-ESI-MS/MS) method was developed to reliably analyze combination of perindopril arginine and amlodipine besylate in bulk and tablet formulations. The chromatographic separation was achieved on a Waters ACQUITY UPLC® BEH C18 column with 1.7 µm particle packing which enabled the higher peak capacity, greater resolution, increased sensitivity, and higher speed of analysis using a volatile mobile phase ideally being at least 2 pH units below and above the perindopril arginine and amlodipine besylate pKa, respectively. Mass spectrometric detection was performed using electrospray ion source in positive ion polarity to profile the abundances of perindopril arginine and amlodipine besylate, using the transitions m/z 369 → m/z 172, and m/z 409 → m/z 238 for perindopril arginine and amlodipine besylate, respectively. Calibration curve was constructed over the range 0.25 - 500 ng/mL and 1.0 - 100 ng/mL for perindopril arginine and amlodipine besylate, respectively. The method was precise and accurate, and provided recovery rates > 80% for both compounds. Furthermore, the intra- and inter-assay precision in terms of % RSD was in between 0.1 - 3.7 for both perindopril arginine and amlodipine besylate. A specific, accurate, and precise UHPLC-MS/MS method for the determination of perindopril arginine and amlodipine besylate in bulk and tablet formulation.

Mucoadhesive glycol chitosan nanoparticles for intranasal delivery of hepatitis B vaccine: enhancement of mucosal and systemic immune response.

In this study, for the first time, glycol chitosan (GC) nanoparticles (NPs) were prepared and evaluated to obtain systemic and mucosal immune responses against nasally administered hepatitis B surface antigen (HBsAg). Size, zeta potential and morphology of the NPs were investigated as a function of preparation method. NPs with high loading efficacy ( > 95%) and positively charged surface were obtained with an average particle size of approximately 200 nm. The structural integrity of HBsAg in NPs was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and further confirmed by measuring the in vitro antigenicity using an enzyme immunoassay. During in vivo studies, GC NPs showed the lowest nasal clearance rate and better mucosal uptake when compared with chitosan (CS) NPs. The immunogenicity of NPs-based delivery system(s) was assessed by measuring anti-HBsAg antibody titer in mice serum and secretions after intranasal administration. The alum-based HBsAg vaccine injected subcutaneously was used as positive control. Results indicated that alum-based HBsAg induced strong humoral but negligible mucosal immunity. However, GC NPs induced stronger immune response at both of the fronts as compared to generated by CS NPs. This study demonstrates that this newly developed system has potential for mucosal administration of vaccines.

Aerosolized liposome-based delivery of amphotericin B to alveolar macrophages.

The present study was aimed at preparation, characterization, and performance evaluation of amphotericin B (Amp B)-loaded aerosolized liposomes for their selective presentation to lungs (alveolar macrophages), that being the densest site of Aspergillosis infection. Egg phosphatidylcholine (PC)- and cholesterol (Chol)-based liposomes were modified by coating them with alveolar macrophage-specific ligands (O-palmitoyl mannan, OPM, and O-polmitoyl pullulan, OPP). The prepared formulations were characterized in vitro for vesicle morphology, mean vesicle size, vesicle size distribution and percent drug entrapment. Pressurized packed systems based on preformed liposomal formulations in chlorofluorocarbon aerosol propellants were prepared. In vitro airways penetration efficiency of the liposomal aerosols was determined by percent dose reaching the peripheral airways, it was recorded 1.4-1.6 times lower as compared to plain drug solution-based aerosol. In vivo tissue distribution studies on albino rats suggested the preferential accumulation of OPM- and OPP-coated formulations in the lung macrophages. Higher lung drug concentration was recorded in case of ligand-anchored liposomal aerosols as compared to plain drug solution and plain liposome-based aerosols. The drug was estimated in the lung in high concentration even after 24 h. The drug-localization index calculated after 6 h was nearly 1.42-, 4.47-, and 4.16-fold, respectively, for plain, OPM-, and OPP-coated liposomal aerosols as compared to plain drug solution-based aerosols. These results suggest that the ligand anchored liposomal aerosols are not only effective in rapid attainment of high-drug concentration in lungs with high population of alveolar macrophages but also maintain the same over prolonged period of time. The significance of targeting potential of the developed systems was established.

Development of a single dose tetanus toxoid formulation based on polymeric microspheres: a comparative study of poly(D,L-lactic-co-glycolic acid) versus chitosan microspheres.

Stable polymeric microspheres capable of controlled release of tetanus toxoid (TT) for periods ranging from days to over months were developed. TT was stabilized, encapsulated in microspheres prepared from poly(D,L)-lactide-co-glycolide (PLGA) and chitosan by using protein stabilizer (trehalose) and its immune response was compared. The influence of co-encapsulated protein stabilizer on tetanus toxoid's stability and release from the microspheres was studied. The protein stabilizer (trehalose) prevented structural losses and aggregation of microencapsulated TT. To neutralize the acids liberated by the biodegradable lactic/glycolic acid-based polymer, we also co-incorporated into the polymer an antacid, (Mg(OH)2), which neutralized the acidity during degradation of the polymer and also prevented TT structural losses and aggregation. The in vitro release experiments with PLGA and chitosan microspheres were performed and the release of TT was increased up to 80-90%. The antigen integrity was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by coomassie brilliant blue staining. The SDS-PAGE analysis confirmed that antigen integrity was not affected by the encapsulation procedure. In addition, the immunogenicity of PLGA and chitosan microspheres based single dose vaccine was evaluated in guinea pigs and compared with multiple doses of alum adsorbed TT. Results indicated that a single injection of PLGA and chitosan microspheres containing TT could maintain the antibody response at a level comparable to the booster injections of conventional alum adsorbed vaccines. The both PLGA and chitosan based stable vaccine formulations produced an equal immune response. Hence chitosan can be used to replace the expensive polymer PLGA. This approach should have potential application in the field of vaccine delivery.

Osmotically regulated asymmetric capsular systems for simultaneous sustained delivery of anti-tubercular drugs.

Sustained release asymmetric membrane capsular systems were developed for simultaneous oral delivery of rifampicin and isoniazid sodium in order to reduce the problems associated with the multi drug therapy of tuberculosis. Dense semipermeable membrane coating capsules were also prepared for the delivery of these drugs by adopting two different filling approaches. In vitro release studies were carried out for both types of systems and the results were compared. Asymmetric membrane capsules provided sustained release of rifampicin associated with initial burst release, where isoniazid release rates were comparatively high due to higher aqueous solubility. Dense semipermeable membrane systems provided controlled release of both drugs but were devoid of initial burst release of isoniazid. To overcome these drawbacks, a modified asymmetric system was developed by adding appropriate amount of hydrophilic polymer mixture with isoniazid. The system provided satisfactory sustained release of rifampicin and isoniazid with initial burst release may be sufficient to achieve minimum effective concentration in blood. In vitro dissolution kinetics of the systems followed first order kinetics and statistical analysis of release rate data proved that modified asymmetric system was better amongst the developed systems.

Modified push-pull osmotic system for simultaneous delivery of theophylline and salbutamol: development and in vitro characterization.

An oral osmotic system which can deliver theophylline and salbutamol sulphate simultaneously for extended period of time was developed and characterized in a view to reduce the problems associated with the multidrug therapy of asthma. Simple controlled porosity osmotic pump contained both drugs (in freely soluble form) did not provide satisfactory extended release of theophylline. A modified two-layered, push-pull osmotic system was developed by using the basic designs of various oral osmotic pumps, such as controlled porosity osmotic pump (CPOP), elementary osmotic pump (EOP) and push-pull osmotic pump (PPOP). Scanning electron microscopy of cellulose acetate coating membrane after dissolution revealed that 25% (w/w) of sorbitol can be used as an optimized concentration of pore forming agent with 25% (w/w) of plasticizer, which was kept constant. Formulations were initially developed for theophylline and the release was optimized by using two different soluble forms of theophylline with varying amount of hydrophilic polymer mixture in upper layer and polyethylene oxide (expandable hydrogel) in lower layer. Further, the release of salbutamol sulphate was optimized by keeping the drug in upper or lower layer or both layers. In vitro release studies showed satisfactory controlled release profiles of both drugs. The release profiles of both drug statistically compared with respective marketed controlled release formulations. An optimized system was selected to study the effect of concentration of pore forming agent and orifice diameter on the release of both drugs.

Cholera toxin B subunit conjugated bile salt stabilized vesicles (bilosomes) for oral immunization.

Bile salt stabilized vesicles, bilosomes appear to be a promising and potential carrier system for oral delivery of peptides and proteins. Bilosomes containing bovine serum albumin (BSA), a model antigen, were prepared and conjugated with cholera toxin B subunit (CTB) in order to enhance their affinity towards M cells of Peyer's patches. Stability studies were undertaken to ascertain the effect of simulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH 7.5) and different concentrations of bile salts. Intactness and biological activity of CTB were checked by hemagglutination test. A single oral dose of CTB-conjugated bilosomes produced almost equivalent response compared to parenteral administration of antigen with Freund's complete adjuvant (FCA). However, in contrast to FCA, oral administration of bilosomes is convenient and devoid of any adverse effects that are observed with parenteral administration of FCA. Serum IgG titers after single administration were significantly better (P < 0.05) than oral administration of antigen with other systems for 3 consecutive days, suggesting an effective stimulation of systemic immune response. Mucosal IgA titers obtained advocated a possible application of CTB-conjugated bilosomes as oral vaccine delivery system.

Development of a single-dose stabilized poly(D,L-lactic-co-glycolic acid) microspheres-based vaccine against hepatitis B.

The purpose of this study was to develop a stable single-dose vaccine based on recombinant hepatitis B surface antigen (HBsAg) in poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, in which HBsAg was stabilized by a protein stabilizer (trehalose) and an antacid (Mg(OH)2). The microspheres were prepared by the double emulsion method and characterized by scanning electron microscopy. To neutralize the acids liberated by the biodegradable lactic/glycolic acid based polymer, we coincorporated into the polymer an antacid, Mg(OH)2, which neutralized the acidity during degradation of the polymer and also prevented HBsAg structural losses and aggregation. The antigen integrity after encapsulation was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by silver staining, isoelectric focusing and Western blotting techniques, which confirmed that antigen remained intact after encapsulation. In-vitro release experiments were performed in phosphate-buffered saline (pH 7.4) and the release of antigen was found to be improved by the protein stabilizer (trehalose). In stability studies, performed at 37 degrees C, the microspheres were found to be stable for 16 days. The immunogenicity of stable microsphere formulations bearing HBsAg was compared with the conventional alum-absorbed HBsAg vaccine in a guinea-pig model. The antibody titre indicated that a single injection of stabilized HBsAg-PLGA microspheres produced a better immune response than two injections of alum-formulated HBsAg vaccine. The findings suggest that recombinant HBsAg can be stabilized by use of a protein stabilizer and antacid during entrapment, and this stabilized preparation can be useful for antigen delivery.

Development and characterization of surface modified PLGA nanoparticles for nasal vaccine delivery: effect of mucoadhesive coating on antigen uptake and immune adjuvant activity.

In this study, the efficacy of mucoadhesive polymers, i.e., chitosan and glycol chitosan as a mucoadhesive coating material in nasal vaccine delivery was investigated. The Hepatitis B surface Antigen (HBsAg) encapsulated PLGA, chitosan coated PLGA (C-PLGA), and Glycol chitosan coated PLGA (GC-PLGA) nanoparticles (NPs) were prepared. The formulations were characterized for particle size, shape, surface charge, and entrapment efficiency. The mucoadhesive ability of coated and non-coated NPs was determined using in vitro mucoadhesion and nasal clearance test. In addition, the systemic uptake and bio-distribution were also evaluated to understand the fate of NPs following nasal delivery. The immuno-adjuvant ability of various formulations was compared by measuring specific antibody titer in serum and secretory. The results indicated that PLGA NPs exhibit negative surface charge, whereas C-PLGA and GC-PLGA NPs exhibited positive surface charge. The GC-PLGA NPs demonstrated lower clearance and better local and systemic uptake compared to chitosan coated and uncoated PLGA NPs. In vivo immunogenicity studies indicated that GC-PLGA NPs could induce significantly higher systemic and mucosal immune response compared to PLGA and C-PLGA NPs. In conclusion, GC-PLGA NPs could be a promising carrier adjuvant for the nasal vaccine delivery for inducing a potent immune response at mucosal surface(s) and systemic circulation.

Pharmaceutical and immunological evaluation of mucoadhesive nanoparticles based delivery system(s) administered intranasally.

Tri-methyl chitosan synthesis accompanies polymer chain scission, which may affect the carrier and adjuvant properties of the polymer. The main objective of this study was to synthesize the tri-methylated chitosan using mild (TMC-M) and conventional (TMC) method and compare their efficacy as nasal vaccine delivery vehicle. During in vitro studies TMC-M nanoparticles showed the lowest nasal clearance rate when compared with chitosan (CS) and TMC nanoparticles. The immunogenicity of nanoparticles based delivery system(s) was assessed by measuring anti-HBsAg antibody titer in mice serum and secretions after intranasal administration. The alum based HBsAg vaccine injected subcutaneously was used as positive control. Results indicated that alum based HBsAg induced strong humoral but negligible mucosal immunity. However, TMC-M nanoparticles induced stronger immune response at both of the fronts as compared to generated by CS or TMC nanoparticles. Present study demonstrates that TMC-M can be a better carrier adjuvant for nasal subunit vaccines.

Comparison of humoral and cell-mediated immune responses to cationic PLGA microspheres containing recombinant hepatitis B antigen.

Presently available marketed alum adsorbed hepatitis B vaccine used for prophylactic immunization, can effectively elicit humoral immunity but is poor inducer of cell-mediated immunity (CMI). Besides, conventional alum-adjuvant vaccines require multiple injections to achieve long-lasting protective immune responses. Therefore, as a result of insufficient immunization, infections are still the leading killer among diseases. The present investigation was therefore, aimed at developing "single-shot" HBsAg adsorbed microspheres of poly (DL)-lactide-co-glycolide (PLGA) (L/G 50:50 and 75:25) and their capability to stimulate the cell mediated immune response against hepatitis B surface antigen. These microspheres were characterized in vitro for their size, shape polydispersity index, percentage HBsAg adsorption efficiency and in vitro release profile. The immune-stimulating activities were also studied following subcutaneous injection of HBsAg adsorbed PLGA microspheres (single-dose on day 0) and compared with alum adsorbed vaccines (two-doses on 0 and 28 days) in Balb/c mice. Specific cell-mediated immune responses such as lymphocyte transformation assay (stimulation-index) including release of interferon-gamma (IFN-γ), interleukin-2 (IL-2) and nitric-oxide were determined. Cellular responses in case of alum adsorb HBsAg vaccine was very low. These studies demonstrate the potential of cationic polymeric microspheres based vaccine in stimulating cell mediated immune response along with humoral response against hepatitis B.

Humoral and cell-mediated immune-responses after administration of a single-shot recombinant hepatitis B surface antigen vaccine formulated with cationic poly(l-lactide) microspheres.

The present investigations were aimed to compare the humoral and cell-mediated immune responses between recombinant hepatitis B surface antigens (HBsAg) adsorbed L-PLA microspheres (Ms) vaccine (single-shot) and marketed alum-HBsAg vaccine (two-doses). The blank cationic (cetyltrimethyammoniumbromide) microspheres were prepared by the double emulsion (w/o/w) solvent evaporation technique. The HBsAg was adsorbed onto the surface of blank cationic microspheres. These microspheres were characterized in vitro for their size, shape, adsorption-efficiency, in-process stability, and HBsAg release studies. Specific humoral immune responses (IgM and IgG) and cell-mediated immune responses (cellular-proliferation) assay including release of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and nitric oxide (NO) from host's cells stimulated with HBsAg or lipopolysaccharide (LPS)/ concanavalin A (con A) in-vitro were determined. Based on these findings, it was concluded that the single injection (using subcutaneous-route) of the polymeric microspheres produced better immune response (both humoral and cell-mediated) than two injections of a conventional alum-HBsAg vaccine. These data demonstrate high potential of polymeric microspheres for their use as a carrier adjuvant for hepatitis B vaccine.

Strong systemic and mucosal immune responses to surface-modified PLGA microspheres containing recombinant hepatitis B antigen administered intranasally.

Surface-modified DL-lactide/glycolide copolymer (PLGA) microspheres with chitosan (CS) were developed for nasal immunization using recombinant Hepatitis B (HBsAg) surface protein for the induction of humoral, cellular and mucosal immunity. Modified PLGA microspheres were characterized in vitro for their size, shape, entrapment efficiency and zeta potential. The nasal clearance rate was evaluated by gamma scintigraphy in rabbits. The antigen integrity, in vitro release and its stability at 37 degrees C were also evaluated. The designed cationic microspheres possessed 27.2 mV zeta potential and an average size less than 10 microm with antigen loading efficiency of 80+/-5%. However, zeta potential of unmodified PLGA microspheres was measured to be negative (-8.7 mV). The modified PLGA microspheres showed the lowest nasal clearance rate when compared with unmodified PLGA microspheres and lactose powder. The antigen integrity was retained intact in encapsulated form as well as on release. The immune-stimulating activity was studied by measuring anti-HBsAg titre, secretory IgA level in serum, vaginal, nasal and salivary secretions (mucosal secretions) and cytokine level (interleukin-2 (IL-2) and interferon-gamma (IFN-gamma)) in spleen homogenates following nasal administration of modified PLGA microspheres in Balb/c mice and compared with alum-HBsAg vaccine injected subcutaneously. The serum anti-HBsAg titre obtained after nasal administration of modified PLGA microspheres was comparable with titre recorded after alum-HBsAg was administered subcutaneously. Moreover, alum-HBsAg vaccine did not elicit sIgA in mucosal secretions as it was induced and measured in the case of nasal administration of modified PLGA microspheres. Similarly, there was no cellular response (cytokine level) in case of alum-HBsAg vaccine. Modified PLGA microspheres (cationic microspheres) thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. These data demonstrate high potential of modified PLGA microspheres for their use as a carrier adjuvant for nasal subunit vaccines.

Controlled porosity osmotic pumps of highly aqueous soluble drug containing hydrophilic polymers as release retardants.

Controlled porosity osmotic pumps (CPOPs) are devoid of delivery orifice to release core contents and essentially possess pore-forming agent(s) in coating composition. When the pump comes in contact with aqueous media, pore-forming agent(s) generate pores through which core contents are delivered. Diltiazem hydrochloride (DLTZ) is a freely water-soluble drug and the release rates of DLTZ are higher from oral osmotic pumps including CPOPs, in which the drug release is controlled by concentration of pore-forming agents. The effect of appropriate concentration of hydroxypropyl methyl cellulose and sodium carboxy methyl cellulose mixture on the release of DLTZ from CPOPs was studied. In vitro drug release profiles were compared with that of different marketed controlled release formulations and statistically analysed to examine the suitability of CPOP for twice or once daily administration. Dissolution models were applied to drug release data in order to establish the mechanism of drug release and kinetics. Drug release from the CPOPs was effectively modified with the concentration of pore-forming agent in membrane and concentration of hydrophilic polymers in the core. CPOPs showed minimum 65% of consistent DLTZ release at 16 h. Statistical analysis confirmed that with an increase in the amount of hydrophilic polymers release rate decreased. Drug release from the systems follows Hixson-Crowell cube root model and mechanism of release follow non-Fickian diffusion.