Clinically proven specification setting for a meningococcal serogroup a conjugate vaccine.

GSK is currently working to improve the commercial presentation of the licensed quadrivalent conjugate vaccine (Menveo) for use against meningococcal serogroup A, C, W, Y (MenACWY) infections. Menveo consists of a primary, lyophilized vial, containing the serogroup A antigen that is reconstituted with the content of a second, liquid, vial that contains the serogroup C, W, Y antigens, to give the final liquid MenACWY product. Since the MenA structure is prone to hydrolytic degradation in liquid formulations, we used mathematical models to rationally design a clinical Phase 2 development plan and provide end of shelf-life (EoSL) and release specification setting for the MenACWY liquid product. By using development and clinical stability data, statistical models were built and used to predict both the MenA free saccharide (FS) and O-Acetyl (OAc) content during long-term storage conditions at 5 °C and stressed (accelerated) stability studies at 15 °C, 22.5 °C, 25 °C, 37 °C and 50 °C. This approach allowed us to define an aging plan for the clinical material to reach at least the required levels of MenA FS and OAc levels at product EoSL. The clinical material was then exposed to a temperature of 22.5 ± 2.5 °C for 59 days to generate FS OAc content of about 35% and 40%, respectively, which was then delivered to the patients in the clinical trial. To the best of our knowledge, this work represents the first example in the field of vaccine research where statistical models have been used to rationally design tailored lots, with the goal of setting EoSL and release specification limits based on data collected on artificially aged clinical material, in which the FS and OAc levels tested were intended to support a product shelf-life of at least 24 months.

Flow cytometry: an alternative method for direct quantification of antigens adsorbed to aluminum hydroxide adjuvant.

Flow cytometry (FC) has been widely used in biological research; however, its use for vaccine characterization has been very limited. Here we describe the development of an FC method for the direct quantification of two Neisseria meningitidis vaccine antigens, in mono- and multivalent formulations, while still adsorbed on aluminum hydroxide (AH) suspension. The antibody-based method is specific and sensitive. Because FC allows microscopic particle examination, the entire aluminum suspension carrying adsorbed antigen(s) can be analyzed directly. In addition to determining antigen concentration and identity, the assay is able to determine the distribution of the antigens on AH. High correlation coefficients (r(2)) were routinely achieved for a broad range of antigen doses from 0 to 150 µg/dose. Traditional assays for quantitative and qualitative antigen characterization on AH particles involve either complete aluminum dissolution or antigen desorption from the adjuvant. Because our direct method uses the whole AH suspension, the cumbersome steps used by traditional methods are not required. Those steps are often inefficient in desorbing the antigens and in some cases can lead to protein denaturation. We believe that this novel FC-based assay could circumvent some of the complex and tedious antigen-adjuvant desorption methods.

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclo- propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 2.

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.

DPP-IV-resistant, long-acting oxyntomodulin derivatives.

Obesity is one of the major risk factors for type 2 diabetes, and the development of agents, that can simultaneously achieve glucose control and weight loss, is being actively pursued. Therapies based on peptide mimetics of the gut hormone glucagon-like peptide 1 (GLP-1) are rapidly gaining favor, due to their ability to increase insulin secretion in a strictly glucose-dependent manner, with little or no risk of hypoglycemia, and to their additional benefit of causing a modest, but durable weight loss. Oxyntomodulin (OXM), a 37-amino acid peptide hormone of the glucagon (GCG) family with dual agonistic activity on both the GLP-1 (GLP1R) and the GCG (GCGR) receptors, has been shown to reduce food intake and body weight in humans, with a lower incidence of treatment-associated nausea than GLP-1 mimetics. As for other peptide hormones, its clinical application is limited by the short circulatory half-life, a major component of which is cleavage by the enzyme dipeptidyl peptidase IV (DPP-IV). SAR studies on OXM, described herein, led to the identification of molecules resistant to DPP-IV degradation, with increased potency as compared to the natural hormone. Analogs derivatized with a cholesterol moiety display increased duration of action in vivo. Moreover, we identified a single substitution which can change the OXM pharmacological profile from a dual GLP1R/GCGR agonist to a selective GLP1R agonist. The latter finding enabled studies, described in detail in a separate study (Pocai A, Carrington PE, Adams JR, Wright M, Eiermann G, Zhu L, Du X, Petrov A, Lassman ME, Jiang G, Liu F, Miller C, Tota LM, Zhou G, Zhang X, Sountis MM, Santoprete A, Capitò E, Chicchi GG, Thornberry N, Bianchi E, Pessi A, Marsh DJ, SinhaRoy R. Glucagon-like peptide 1/glucagon receptor dual agonism reverses obesity in mice. Diabetes 2009; 58: 2258-2266), which highlight the potential of GLP1R/GCGR dual agonists as a potentially superior class of therapeutics over the pure GLP1R agonists currently in clinical use.

Determination of carbohydrate-binding preferences of human galectins with carbohydrate microarrays.

Galectins are a class of carbohydrate-binding proteins named for their galactose-binding preference and are involved in a host of processes ranging from homeostasis of organisms to immune responses. As a first step towards correlating the carbohydrate-binding preferences of the different galectins with their biological functions, we determined carbohydrate recognition fine-specificities of galectins with the aid of carbohydrate microarrays. A focused set of oligosaccharides considered relevant to galectins was prepared by chemical synthesis. Structure-activity relationships for galectin-sugar interactions were determined, and these helped in the establishment of redundant and specific galectin actions by comparison of binding preferences. Distinct glycosylations on the basic lactosyl motifs proved to be key to galectin binding regulation--and therefore galectin action--as either high-affinity ligands are produced or binding is blocked. High-affinity ligands such as the blood group antigens that presumably mediate particular functions were identified.

Surface characterization of carbohydrate microarrays.

Carbohydrate microarrays are essential tools to determine the biological function of glycans. Here, we analyze a glycan array by time-of-flight secondary ion mass spectrometry (ToF-SIMS) to gain a better understanding of the physicochemical properties of the individual spots and to improve carbohydrate microarray quality. The carbohydrate microarray is prepared by piezo printing of thiol-terminated sugars onto a maleimide functionalized glass slide. The hyperspectral ToF-SIMS imaging data are analyzed by multivariate curve resolution (MCR) to discern secondary ions from regions of the array containing saccharide, linker, salts from the printing buffer, and the background linker chemistry. Analysis of secondary ions from the linker common to all of the sugar molecules employed reveals a relatively uniform distribution of the sugars within the spots formed from solutions with saccharide concentration of 0.4 mM and less, whereas a doughnut shape is often formed at higher-concentration solutions. A detailed analysis of individual spots reveals that in the larger spots the phosphate buffered saline (PBS) salts are heterogeneously distributed, apparently resulting in saccharide concentrated at the rim of the spots. A model of spot formation from the evaporating sessile drop is proposed to explain these observations. Saccharide spot diameters increase with saccharide concentration due to a reduction in surface tension of the saccharide solution compared to PBS. The multivariate analytical partial least squares (PLS) technique identifies ions from the sugars that in the complex ToF-SIMS spectra correlate with the binding of galectin proteins.

ApoA-I mimetic peptides promote pre-ß HDL formation in vivo causing remodeling of HDL and triglyceride accumulation at higher dose.

Reverse cholesterol transport promoted by HDL-apoA-I is an important mechanism of protection against atherosclerosis. We have previously identified apoA-I mimetic peptides by synthesizing analogs of the 22 amino acid apoA-I consensus sequence (apoA-I(cons)) containing non-natural aliphatic amino acids. Here we examined the effect of different aliphatic non-natural amino acids on the structure-activity relationship (SAR) of apoA-I mimetic peptides. These novel apoA-I mimetics, with long hydrocarbon chain (C(5-8)) amino acids incorporated in the amphipathic α helix of the apoA-I(cons), have the following properties: (i) they stimulate in vitro cholesterol efflux from macrophages via ABCA1; (ii) they associate with HDL and cause formation of pre-ß HDL particles when incubated with human and mouse plasma; (iii) they associate with HDL and induce pre-ß HDL formation in vivo, with a corresponding increase in ABCA1-dependent cholesterol efflux capacity ex vivo; (iv) at high dose they associate with VLDL and induce hypertriglyceridemia in mice. These results suggest our peptide design confers activities that are potentially anti-atherogenic. However a dosing regimen which maximizes their therapeutic properties while minimizing adverse effects needs to be established.

Natural Sphingomonas glycolipids vary greatly in their ability to activate natural killer T cells.

Mouse natural killer T (NKT) cells expressing an invariant T cell antigen receptor (TCR) recognize glycosphingolipids (GSLs) from Sphingomonas bacteria. The synthetic antigens previously tested, however, were designed to closely resemble the potent synthetic agonist alpha-galactosyl ceramide (alphaGalCer), which contains a monosaccharide and a C18:0 sphingosine lipid. Some Sphingomonas bacteria, however, also have oligosaccharide-containing GSLs, and they normally synthesize several GSLs with different sphingosine chains including one with a cyclopropyl ring-containing C21:0 (C21cycl) sphingosine. Here we studied the stimulation of NKT cells with synthetic GSL antigens containing natural tetrasaccharide sugars, or the C21cycl sphingosine. Our results indicate that there is a great degree of variability in the antigenic potency of different natural Sphingomonas glycolipids, with the C21cycl sphingosine having intermediate potency and the oligosaccharide-containing antigens exhibiting limited or no stimulatory capacity.

The Preparation and Physicochemical Characterization of Aluminum Hydroxide/TLR7a, a Novel Vaccine Adjuvant Comprising a Small Molecule Adsorbed to Aluminum Hydroxide.

Adjuvants are necessary to enable vaccine development against a significant number of challenging pathogens for which effective vaccines are not available. We engineered a novel small-molecule immune potentiator, a benzonaphthyridine agonist targeting toll-like receptor 7 (TLR7), as a vaccine adjuvant. TLR7 agonist (TLR7a) was engineered to be adsorbed onto aluminum hydroxide (AlOH), and the resulting AlOH/TLR7a was evaluated as a vaccine adjuvant. AlOH/TLR7a exploits the flexibility of AlOH formulations, has an application in many vaccine candidates, and induced good efficacy and safety profiles against all tested antigens (bacterial- and viral-derived protein antigens, toxoids, glycoconjugates, and so forth) in many animal models, including nonhuman primates. In this article, we describe the outcome of the physicochemical characterization of AlOH/TLR7a. Reverse-phase ultra performance liquid chromatography, confocal microscopy, flow cytometry, zeta potential, and phosphophilicity assays were used as tools to demonstrate the association of TLR7a to AlOH and to characterize this novel formulation. Raman spectroscopy, nuclear magnetic resonance, and mass spectroscopy were also used to investigate the interaction between TLR7a and AlOH (data not shown). This pivotal work paved the way for AlOH/TLR7a to progress into the clinic for evaluation as an adjuvant platform for vaccines against challenging preventable diseases.

The preparation and characterization of PLG nanoparticles with an entrapped synthetic TLR7 agonist and their preclinical evaluation as adjuvant for an adsorbed DTaP vaccine.

The design of safe and potent adjuvants able to enhance and modulate antigen-specific immunity is of great interest for vaccine research and development. In the present study, negatively charged poly(lactide-co-glycolide) (PLG) nanoparticles have been combined with a synthetic immunepotentiator molecule targeting the Toll-like receptor 7. The selection of appropriate preparation and freeze-drying conditions resulted in a PLG-based adjuvant with well-defined and stable physico-chemical properties. The adjuvanticity of such nanosystem has later been evaluated in the mouse model with a diphtheria-tetanus-pertussis (DTaP) vaccine, on the basis of the current need to improve the efficacy of acellular pertussis (aP) vaccines. DTaP antigens were adsorbed onto PLG nanoparticles surface, allowing the co-delivery of TLR7a and multiple antigens through a single formulation. The entrapment of TLR7a into PLG nanoparticles resulted in enhanced IgG and IgG2a antibody titers. Notably, the immune potentiator effect of TLR7a was less evident when it was used in not-entrapped form, indicating that co-localization of TLR7a and antigens is required to adequately stimulate immune responses. In conclusion, the rational selection of adjuvants and formulation here described resulted as a highly valuable approach to potentiate and better tailor DTaP vaccine immunogenicity.

Novel adjuvant Alum-TLR7 significantly potentiates immune response to glycoconjugate vaccines.

Although glycoconjugate vaccines are generally very efficacious, there is still a need to improve their efficacy, especially in eliciting a strong primary antibody response. We have recently described a new type of vaccine adjuvant based on a TLR7 agonist adsorbed to alum (Alum-TLR7), which is highly efficacious at enhancing immunogenicity of protein based vaccines. Since no adjuvant has been shown to potentiate the immune response to glycoconjugate vaccines in humans, we investigated if Alum-TLR7 is able to improve immunogenicity of this class of vaccines. We found that in a mouse model Alum-TLR7 greatly improved potency of a CRM197-MenC vaccine increasing anti-MenC antibody titers and serum bactericidal activity (SBA) against MenC compared to alum adjuvanted vaccine, especially with a low dose of antigen and already after a single immunization. Alum-TLR7 also drives antibody response towards Th1 isotypes. This adjuvant was also able to increase immunogenicity of all polysaccharides of a multicomponent glycoconjugate vaccine CRM197-MenACWY. Furthermore, we found that Alum-TLR7 increases anti-polysaccharide immune response even in the presence of a prior immune response against the carrier protein. Finally, we demonstrate that Alum-TLR7 adjuvant effect requires a functional TLR7. Taken together, our data support the use of Alum-TLR7 as adjuvant for glycoconjugate vaccines.

Rational design of small molecules as vaccine adjuvants.

Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants.

Aluminum adjuvant dose guidelines in vaccine formulation for preclinical evaluations.

Aluminum (Al) salt-based adjuvants are present in a large variety of licensed vaccines and their use is widely considered for formulations in clinical trials. Although the regulatory agencies have clearly stated the acceptable levels of Al salts in vaccines for human use, there are no general indications for preclinical research. This brief commentary reviews the current status of Al concentrations in licensed vaccines, the related potential toxicity in preclinical species, and proposes a general guideline for selection of suitable Al salt levels in preclinical models, focusing on the formulation development for recombinant protein antigens. A table with conversion factors is included in order to provide a tool for calculation of doses with different Al salts.

Effect of tryptophan oligopeptides on the size distribution of POPC liposomes: a dynamic light scattering and turbidimetric study.

A chemical regulation of POPC liposome size distribution was investigated, based on the affinity of indole-containing compounds for phosphocholine membranes. In particular, tryptophan oligopeptides have shown interesting properties of size regulation, both when liposomes were formed in their presence and when the peptides were added to a preformed liposome suspension. Combining dynamic light scattering (DLS) and turbidimetric data, it was possible to show how such peptides had an influence on the size distribution of spontaneously formed liposomes prepared by the thin film hydration, reverse-phase evaporation and ethanol (or methanol) injection methods. In the presence of Trp-Trp or Trp-Trp-Trp, a disappearance of large vesicle aggregates was observed, as suggested also by light microscopy analysis. On the contrary, no effect was detected using extruded vesicles. Turbidimetric titration allowed the determination of the relative efficacy of the size regulators, Trp-Trp-Trp being about 20 times more powerful than the dimer, while the monomer had no effect. In addition, other indole-containing compounds and the antimicrobial peptide indolicidin were tested, showing similar behaviours. Discussing the results according to the current knowledge about the preference of Trp residues for interfacial regions in lecithin bilayers, this study confirms the relevant role of tryptophan in the biomembrane binding properties of many peptides and introduces a new behavior in the field of liposomes-peptides interactions.

Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection method.

Small-sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug-containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) then on the novel L-carnitine derivative PUCE (palmitoyl-(R)-carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of "multiple injection method" was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan-containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI-H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no-liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water-insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.