Safety and efficacy of an oral insulin (Capsulin) in patients with early-stage type 2 diabetes: A dose-ranging phase 2b study.

AIM: To compare the pharmacodynamic properties of different doses of regular human insulin administered in capsule form twice daily in a randomised twelve-week open-label study. METHODS: A total of 100 individuals (48 males, 52 females) with type 2 diabetes on metformin completed the study according to the protocol. The mean (SD) age was 48.5 (6.7) years, body mass index 25.7 (2.8) kg/m2 and HbA1c 8.10% (0.65%). Subjects randomized upon admission were assigned to one of three groups receiving formulated regular insulin at dose levels of 75 iu BD, 150 iu insulin BD, or 300 iu BD, all in enteric-coated capsules. The primary and secondary endpoints were change from baseline in HbA1c and fasting plasma glucose (FPG), respectively. RESULTS: The study met its primary clinical endpoint of a decrease in HbA1c of 0.5% or higher (least square mean decrease 0.52%; P = .004, median decrease 0.6%) in the dose group receiving 150 iu BD. In a subset of this population, with starting HbA1c values of 9% to 9.5%, an average decrease of 1.575% was observed. In the total population, least square mean decreases in HbA1c for the 75 and 300 iu BD groups were -0.11% and -0.42%, respectively. Mean change in FPG in the 150 iu BD dose group was -18.8 mg/dl (P = .017) and -14.8 and -2.7 mg/dl for the 75 and 300 iu BD groups, respectively. A decrease of 20% for triglycerides (-40 mg/dl) was observed in the 150 iu BD dose group. No significant increases in body weight were observed, and significant decreases in systolic blood pressure were seen in all groups. No serious treatment-related adverse events were recorded, and no incidence of hypoglycaemia was reported throughout the entire 12-week study period. CONCLUSIONS: Capsulin oral insulin administered twice per day at a dose of 150 iu per capsule is safe, with no confirmed treatment-linked hypoglycaemic events, and results in significant decreases from baseline in HbA1c, FPG and triglycerides.

A Global Review on Short Peptides: Frontiers and Perspectives.

Peptides are fragments of proteins that carry out biological functions. They act as signaling entities via all domains of life and interfere with protein-protein interactions, which are indispensable in bio-processes. Short peptides include fundamental molecular information for a prelude to the symphony of life. They have aroused considerable interest due to their unique features and great promise in innovative bio-therapies. This work focusing on the current state-of-the-art short peptide-based therapeutical developments is the first global review written by researchers from all continents, as a celebration of 100 years of peptide therapeutics since the commencement of insulin therapy in the 1920s. Peptide "drugs" initially played only the role of hormone analogs to balance disorders. Nowadays, they achieve numerous biomedical tasks, can cross membranes, or reach intracellular targets. The role of peptides in bio-processes can hardly be mimicked by other chemical substances. The article is divided into independent sections, which are related to either the progress in short peptide-based theranostics or the problems posing challenge to bio-medicine. In particular, the SWOT analysis of short peptides, their relevance in therapies of diverse diseases, improvements in (bio)synthesis platforms, advanced nano-supramolecular technologies, aptamers, altered peptide ligands and in silico methodologies to overcome peptide limitations, modern smart bio-functional materials, vaccines, and drug/gene-targeted delivery systems are discussed.

Binding Interactions of Peptide Aptamers.

Peptide aptamers are short amino acid chains that are capable of binding specifically to ligands in the same way as their much larger counterparts, antibodies. Ligands of therapeutic interest that can be targeted are other peptide chains or loops located on the surface of protein receptors (e.g., GCPR), which take part in cell-to-cell communications either directly or via the intermediary of hormones or signalling molecules. To confer on aptamers the same sort of conformational rigidity that characterises an antibody binding site, aptamers are often constructed in the form of cyclic peptides, on the assumption that this will encourage stronger binding interactions than would occur if the aptamers were simply linear chains. However, no formal studies have been conducted to confirm the hypothesis that linear peptides will engage in stronger binding interactions with cyclic peptides than with other linear peptides. In this study, the interaction of a model cyclic decamer with a series of linear peptide constructs was compared with that of a linear peptide with the same sequence, showing that the cyclic configuration does confer benefits by increasing the strength of binding.

Efficacy of Bioactive Cyclic Peptides in Rheumatoid Arthritis: Translation from In Vitro to In Vivo Models.

Using a novel drug discovery technology reported in previous issues of this journal cyclic peptides have been created which are able to down-regulate secretion of inflammatory cytokines, in vitro, by stimulated cells of the macrophage cell line J774. The cytokines in question, TNF-alpha and IL-6, are strongly implicated in etiology of diseases such as rheumatoid arthritis. Studies are reported here using the CAIA animal model for rheumatoid arthritis, which show that the peptides identified are indeed able to impact on inflammation of joints, induced in vivo. The results suggest that these peptides are effective at a dose which could be viable in man, and at which no adverse side effects are evident in the short term.

Design and optimisation of bioactive cyclic peptides: generation of a down-regulator of TNF secretion.

Although strong binding interactions between protein receptor and ligand do not require the participation of a large number of amino acids in either site, short peptide chains are generally poor at recreating the types of protein-protein interactions which take place during cell recognition and signalling process, probably because their flexible backbones prevent the side chains from forming sufficiently rigid and stable epitopes, which can take part in binding with the desired strength and specificity. In a recently-reported study, it was shown that a proto-epitope containing F, R and S amino acids has the ability to down-regulate TNF secretion by macrophages. This paper extends these findings, putting those amino acids into a short cyclic peptide scaffold, and determining the optimal configuration required to overcome the problems of conformational instability, and give rise to molecules which have potential as therapeutic agents in human disease, such as rheumatoid arthritis.

Use of mixed micelles for presentation of building blocks in a new combinatorial discovery methodology: proof-of-concept studies.

We describe a new method of combinatorial screening in which building blocks, instead of being linked together chemically, are placed on the surface of nanoparticles. Two- or three-dimensional structures form on the surface of these particles through the close approach of different building blocks, with sufficient flexibility to be able to adapt and interact with putative binding sites in biological systems. The particles assemble without the need for formation of chemical bonds, so libraries comprised of many structures can be prepared rapidly, with large quantities of material available for testing. Screening methods can include solid and solution-phase binding assays, or tissue culture models, for example looking for structures which can change the behaviour of cells in a disease-modifying manner.

Vaxcine(TM): an oil-based adjuvant for influenza vaccines.

Vaccination is the method of choice for the prevention of influenza infection. However, the quantity of the antigen available, especially in the case of pandemics, often fails to meet the global demand. However, improved adjuvants can overcome this problem. Preliminary results obtained in this study revealed that one year after a single subcutaneous immunisation with influenza A H3N2 virus in an oil-based carrier, Vaxcine(TM), outbreed mice produced a high immunoglobulin G response that lasted for up to one year and exhibited less variation in titre compared with the response of the control group treated with alum. The haemagglutination-inhibition titres induced by Vaxcine(TM) were also higher than those generated by alum. These data indicate that Vaxcine(TM) is a good adjuvant candidate for seasonal influenza vaccines.

The First Insight Into the Supramolecular System of D,L-α-Difluoromethylornithine: A New Antiviral Perspective.

Targeting the polyamine biosynthetic pathway by inhibiting ornithine decarboxylase (ODC) is a powerful approach in the fight against diverse viruses, including SARS-CoV-2. Difluoromethylornithine (DFMO, eflornithine) is the best-known inhibitor of ODC and a broad-spectrum, unique therapeutical agent. Nevertheless, its pharmacokinetic profile is not perfect, especially when large doses are required in antiviral treatment. This article presents a holistic study focusing on the molecular and supramolecular structure of DFMO and the design of its analogues toward the development of safer and more effective formulations. In this context, we provide the first deep insight into the supramolecular system of DFMO supplemented by a comprehensive, qualitative and quantitative survey of non-covalent interactions via Hirshfeld surface, molecular electrostatic potential, enrichment ratio and energy frameworks analysis visualizing 3-D topology of interactions in order to understand the differences in the cooperativity of interactions involved in the formation of either basic or large synthons (Long-range Synthon Aufbau Modules, LSAM) at the subsequent levels of well-organized supramolecular self-assembly, in comparison with the ornithine structure. In the light of the drug discovery, supramolecular studies of amino acids, essential constituents of proteins, are of prime importance. In brief, the same amino-carboxy synthons are observed in the bio-system containing DFMO. DFT calculations revealed that the biological environment changes the molecular structure of DFMO only slightly. The ADMET profile of structural modifications of DFMO and optimization of its analogue as a new promising drug via molecular docking are discussed in detail.

Oral Delivery of Biologics via the Intestine.

Biologics are currently one of the most promising avenues for therapeutic interventions in conditions such as metabolic disease, ageing and inflammatory disorders, and for chronic ailments, oral delivery of such medicines has for years been recognised as an important goal. Despite decades of intensive research, oral delivery of biologics is only just starting to prove feasible. There has been much talk about the barriers to uptake of biologics, and indeed, one function of the intestine is to prevent, in one way or another, passage of unwanted materials across the gut, and yet, grams of biological agents both large and small pass across the intestinal cell wall every day. This review first describes the functioning of the gut under normal circumstances, then identifies the principle biological mechanisms which have been harnessed successfully, to date, to achieve oral uptake, outlining the pros and cons of each approach. Examples with different biologics are given, and information on result of the latest clinical trials is provided, where available.

Inhibition of HIV Virus by Neutralizing Vhh Attached to Dual Functional Liposomes Encapsulating Dapivirine.

Although highly active antiretroviral therapy (HAART) has greatly improved the life expectancy of HIV/AIDS patients, the treatment is not curative. It is a global challenge which fosters an urgent need to develop an effective drug or neutralizing antibody delivery approach for the prevention and treatment of this disease. Due to the low density of envelope spikes with restricted mobility present on the surface of HIV virus, which limit the antibody potency and allow virus mutation and escape from the immune system, it is important for a neutralizing antibody to form bivalent or multivalent bonds with the virus. Liposome constructs could fulfil this need due to the flexible mobility of the membrane with its attached antibodies and the capacity for drug encapsulation. In this study, we evaluated the neutralization activity of a range of liposome formulations in different sizes coated with anti-gp120 llama antibody fragments (Vhhs) conjugated via either non-covalent metal chelation or a covalent linkage. The non-covalent construct demonstrated identical binding affinity to HIV-1 envelope glycoprotein gp120 and neutralizing ability for HIV virus as free Vhh. Although covalently linked Vhh showed significant binding affinity to gp120, it unexpectedly had a lower neutralization potency. This may be due to the comparability in size of the viral and liposome particles restricting the number which can be bound to the liposome surface so involving only a fraction of the antibodies, whereas non-covalently attached antibodies dissociate from the surface after acting with gp120 and free the remainder to bind further viruses. Covalently conjugated Vhh might also trigger the cellular uptake of a liposome-virion complex. To explore the possible ability of the antibody-coated liposomes to have a further function, we encapsulated the hydrophobic antiviral drug dapivirine into both of the non-covalently and covalently conjugated liposome formulations, both of which revealed high efficacy in reducing viral replication in vitro. Thus, dual function liposomes may lead to a novel strategy for the prophylaxis of HIV/AIDS by combining the neutralizing activity of Vhh with antiviral effects of high drug concentrations.

A universal polysaccharide conjugated vaccine against O111 E. coli.

E. coli O111 strains are responsible for outbreaks of blood diarrhea and hemolytic uremic syndrome throughout the world. Because of their phenotypic variability, the development of a vaccine against these strains which targets an antigen that is common to all of them is quite a challenge. Previous results have indicated, however, that O111 LPS is such a candidate, but its toxicity makes LPS forbidden for human use. To overcome this problem, O111 polysaccharides were conjugated either to cytochrome C or to EtxB (a recombinant B subunit of LT) as carrier proteins. The O111-cytochrome C conjugate was incorporated in silica SBA-15 nanoparticles and administered subcutaneously in rabbits, while the O111-EtxB conjugate was incorporated in Vaxcine(TM), an oil-based delivery system, and administered orally in mice. The results showed that one year post-vaccination, the conjugate incorporated in silica SBA-15 generated antibodies in rabbits able to inhibit the adhesion of all categories of O111 E. coli to epithelial cells. Importantly, mice immunized orally with the O111-EtxB conjugate in Vaxcine(TM) generated systemic and mucosal humoral responses against all categories of O111 E. coli as well as antibodies able to inhibit the toxic effect of LT in vitro. In summary, the results obtained by using 2 different approaches indicate that a vaccine that targets the O111 antigen has the potential to prevent diarrhea induced by O111 E. coli strains regardless their mechanism of virulence. They also suggest that a conjugated vaccine that uses EtxB as a carrier protein has potential to combat diarrhea induced by ETEC.

Synthesis of glycolipopeptidic building blocks for carbohydrate receptor discovery.

A class of glycolipopeptides for use as building blocks for a new type of dynamic combinatorial library is reported. The members of the library consist of a variable carbohydrate moiety, coded amino acids, and lipoamino acids in order to convert them into amphiphiles. Glycolipopeptidic amphiphiles interact through non-covalent bonding when mixed together in aqueous phase and form micelles in dynamic close-packed fluid mosaic arrays. The head groups of amphiphiles are exposed on the micelle surface, providing entities which could be screened in biological assays to find the most potent combination of building blocks in order to identify new bioactive carbohydrate ligands.

Reverse micelle-encapsulated recombinant baculovirus as an oral vaccine against H5N1 infection in mice.

Induction of mucosal immunity through oral immunization is an effective way to control influenza infection. In this study, baculovirus displaying influenza hemagglutinin was encapsulated within a reverse micelle structure of phosphatidylcholine and delivered into the gastrointestinal tract of mice to study its efficacy as an oral vaccine against cross-clade H5N1 infection. Mice vaccinated with encapsulated baculovirus displaying HA (En-BacHA) showed significantly enhanced HA specific serum IgG and mucosal IgA antibodies, and higher hemagglutination inhibition (HI) titers, when compared to its non-encapsulated form (BacHA). Estimation of serum neutralizing antibodies also indicated that En-BacHA formulation was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, clade 4.0 and clade 8.0). Further, mice vaccinated with En-BacHA alone were able to confer 100% protection against 5MLD50 of HPAI heterologous H5N1 strain (clade 1). Inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant in the vaccine formulation did not show any significant effect in both systemic and mucosal immune responses. Oral delivery of encapsulated recombinant H5 HA expressed on baculovirus surface is an effective way to prime the immune system against H5N1 infection in mice and will have no biosafety concerns associated with their production or administration.

Lipopolysaccharide as an antigen target for the formulation of a universal vaccine against Escherichia coli O111 strains.

A promising approach to developing a vaccine against O111 strains of diarrheagenic Escherichia coli that exhibit different mechanisms of virulence is to target either the core or the polysaccharide chain (O antigen) of their lipopolysaccharide (LPS). However, due to structural variations found in both these LPS components, to use them as antigen targets for vaccination, it is necessary to formulate a vaccine able to induce a humoral immune response that can recognize all different variants found in E. coli O111 strains. In this study, it was demonstrated that, despite differences in composition of oligosaccharide repeat units between O111ab and O111ac LPS subtypes, antibodies against one O111 subtype can recognize and inhibit the adhesion to human epithelial cells of all categories of O111 E. coli (enteropathogenic E. coli [EPEC], enterohemorrhagic E. coli [EHEC], and enteroaggregative E. coli [EAEC]) strains regardless of the nature of their flagellar antigens, mechanisms of virulence, or O111 polysaccharide subtypes. These antibodies were also able to increase the clearance of different strains of O111 E. coli by macrophages. PCR analyses of the pathways involved in O111 LPS core biosynthesis showed that all EAEC strains have core type R2, whereas typical EPEC and EHEC have core type R3. In contrast, atypical EPEC strains have core types R2 and R3. In summary, the results presented herein indicate that the O111 polysaccharide and LPS core types R2 and R3 are antigen targets for panspecific immunotherapy against all categories of O111 E. coli.

VaxcineTM: an oil-based adjuvant for influenza vaccines

Vaccination is the method of choice for the prevention of influenza infection. However, the quantity of the antigen available, especially in the case of pandemics, often fails to meet the global demand. However, improved adjuvants can overcome this problem. Preliminary results obtained in this study revealed that one year after a single subcutaneous immunisation with influenza A H3N2 virus in an oil-based carrier, VaxcineTM, outbreed mice produced a high immunoglobulin G response that lasted for up to one year and exhibited less variation in titre compared with the response of the control group treated with alum. The haemagglutination-inhibition titres induced by VaxcineTM were also higher than those generated by alum. These data indicate that VaxcineTM is a good adjuvant candidate for seasonal influenza vaccines.

Lipopolysaccharide as an Antigen Target for the Formulation of a Universal Vaccine against Escherichia coli O111 Strains

A promising approach to developing a vaccine against O111 strains of diarrheagenic Escherichia coli thatexhibit different mechanisms of virulence is to target either the core or the polysaccharide chain (O antigen)of their lipopolysaccharide (LPS). However, due to structural variations found in both these LPS components,to use them as antigen targets for vaccination, it is necessary to formulate a vaccine able to induce a humoralimmune response that can recognize all different variants found in E. coli O111 strains. In this study, it was demonstrated that, despite differences in composition of oligosaccharide repeat units between O111ab and O111ac LPS subtypes, antibodies against one O111 subtype can recognize and inhibit the adhesion to human epithelial cells of all categories of O111 E. coli(enteropathogenic E. coli [EPEC], enterohemorrhagic E. coli [EHEC], and enteroaggregative E. coli [EAEC]) strains regardless of the nature of their flagellar antigens, mechanisms of virulence, or O111 polysaccharide subtypes. These antibodies were also able to increase the clearance of different strains of O111 E. coli by macrophages. PCR analyses of the pathways involved in O111 LPS core biosynthesis showed that all EAEC strains have core type R2, whereas typical EPEC and EHEC havecore type R3. In contrast, atypical EPEC strains have core types R2 and R3. In summary, the results presentedherein indicate that the O111 polysaccharide and LPS core types R2 and R3 are antigen targets for panspecific immunotherapy against all categories of O111 E. coli.