A systematic and mechanistic evaluation of aspartic acid as filler for directly compressed tablets containing trimethoprim and trimethoprim aspartate

The generally accepted paradigm of 'inert' and 'mono functional' excipient in dosage form has been recently challenged with the development of individual excipients capable of exhibiting multiple functions (e.g. binder-disintegrants, surfactant which affect P-gp function). The proposed study has been designed within the realm of multifunctionality and is the first and novel investigation towards evaluation of aspartic acid as a filler and disintegration enhancing agent for the delivery of biopharmaceutical class IV model drug trimethoprim. The study investigated powder characteristics using angle of repose, laser diffractometry and scanning electron microscopy (SEM). The prepared tablets were characterised using Heckel analysis, disintegration time and tensile strength measurements. Although Heckel analysis revealed that both TMP and TMP aspartate salt have high elasticity, the salt form produced a stronger compact which was attributed to the formation of agglomerates. Aspartic acid was found to have high plasticity, but its incorporation into the formulations was found to have a negative impact on the compaction properties of TMP and its salt. Surface morphology investigations showed that mechanical interlocking plays a vital role in binding TMP crystals together during compaction, while the small particle size of TMP aspartate agglomerates was found to have significant impact on the tensile strength of the tablets. The study concluded that aspartic acid can be employed as filler and disintegrant and that compactability within tablets was independent of the surface charge of the excipients.

Dipeptidyl peptidase IV (DPP IV) and related molecules in type 2 diabetes

Dipeptidyl peptidase IV (DPP IV) is a widely distributed physiological enzyme that can be found solubilized in blood, or membrane-anchored in tissues. DPP IV and related dipeptidase enzymes cleave a wide range of physiological peptides and have been associated with several disease processes including Crohn's disease, chronic liver disease, osteoporosis, multiple sclerosis, eating disorders, rheumatoid arthritis, cancer, and of direct relevance to this review, type 2 diabetes. Here, we place particular emphasis on two peptide substrates of DPP IV with insulin-releasing and antidiabetic actions namely, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). The rationale for inhibiting DPP IV activity in type 2 diabetes is that it decreases peptide cleavage and thereby enhances endogenous incretin hormone activity. A multitude of novel DPP IV inhibitor compounds have now been developed and tested. Here we examine the information available on DPP IV and related enzymes, review recent preclinical and clinical data for DPP IV inhibitors, and assess their clinical significance.

Effect of cancer cachexia on the activity of tripeptidyl-peptidase II in skeletal muscle

The ubiquitin-proteasome proteolytic pathway plays a major role in degradation of myofibrillar proteins in skeletal muscle during cancer cachexia. The end-product of this pathway is oligopeptides and these are degraded by the extralysomal peptidase tripeptidyl-peptidase II (TPPII) together with various aminopeptidases to form tripeptides and amino acids. To investigate if a relationship exists between the activity of the proteasome and TPPII, functional activities have been measured in gastrocnemius muscle of mice bearing the MAC16 tumour, and with varying extents of weight loss. TPPII activity was quantitated using the specific substrate Ala-Ala-Phe-7-amido-4-methylcoumarin, while proteasome activity was determined as the 'chymotrypsin-like' enzyme activity. Both proteasome proteolytic activity and TPPII activity increased in parallel with increasing weight loss, reaching a maximum at 16% weight loss, after which there was a progressive decrease in activity for both proteases with increasing weight loss. In murine myotubes, proteolysis-inducing factor, which is a sulphated glycoprotein produced by cachexia-inducing tumours, induced an increase in activity of both proteasome and TPPII, with an identical dose-response curve, and both activities were inhibited by eicosapentaenoic acid. These results suggest that the activities of both the proteasome and TPPII are regulated in a parallel manner in cancer cachexia, and that both are induced by the same factor and probably have the same intracellular signalling pathways and transcription factors. © 2004 Elsevier Ireland Ltd. All rights reserved.

N-terminal His7-modification of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity

Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His7-modified analogue of GLP-1, N-pyroglutamyl-GLP-1 as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC50 values 32.9 and 6.7 nM, respectively) compared with native GLP-1 (IC50-37 nM). Similarly, both analogues stimulated cAMP production with EC50 values of 16.3 and 27 nM respectively compared with GLP-1 (EC50 4.7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5.6 mM glucose (P< 0.05 to P< 0.001) similar to native GLP-1. Both analogues (25 nM/kg body weight) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body weight) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biologically potent forms of GLP-1. The particularly powerful antihyperglycaemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes. © 2004 Society for Endocrinology.

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala8-substituted analogues of GLP-1, (Abu8)GLP-1 and (Val8)GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu8)GLP-1 and (Val8)GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu8)GLP-1 and (Val8)GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val8)GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu8 )GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val8)GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala8 in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val8)GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

Is the generation of neo-antigenic determinants by free radicals central to the development of autoimmune rheumatoid disease?

Biomolecules are susceptible to many different post-translational modifications that have important effects on their function and stability, including glycosylation, glycation, phosphorylation and oxidation chemistries. Specific conversion of aspartic acid to its isoaspartyl derivative or arginine to citrulline leads to autoantibody production in models of rheumatoid disease, and ensuing autoantibodies cross-react with native antigens. Autoimmune conditions associate with increased activation of immune effector cells and production of free radical species via NADPH oxidases and nitric oxide synthases. Generation of neo-antigenic determinants by reactive oxygen and nitrogen species ROS and RNS) may contribute to epitope spreading in autoimmunity. The oxidation of amino acids by peroxynitrite, hypochlorous acid and other reactive oxygen species (ROS) increases the antigenicity of DNA, LDL and IgG, generating ligands for which autoantibodies show higher avidity. This review focuses on the evidence for ROS and RNS in promoting the autoimmune responses observed in diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). It considers the evidence for ROS/RNS-induced antigenicity arising as a consequence of failure to remove or repair ROS/RNS damaged biomolecules and suggests that an associated defect, probably in T cell signal processing or/or antigen presentation, is required for the development of disease.

Future glucose-lowering drugs for type 2 diabetes

The multivariable and progressive natural history of type 2 diabetes limits the effectiveness of available glucose-lowering drugs. Constraints imposed by comorbidities (notably cardiovascular disease and renal impairment) and the need to avoid hypoglycaemia, weight gain, and drug interactions further complicate the treatment process. These challenges have prompted the development of new formulations and delivery methods for existing drugs alongside research into novel pharmacological entities. Advances in incretin-based therapies include a miniature implantable osmotic pump to give continuous delivery of a glucagon-like peptide-1 receptor agonist for 6-12 months and once-weekly tablets of dipeptidyl peptidase-4 inhibitors. Hybrid molecules that combine the properties of selected incretins and other peptides are at early stages of development, and proof of concept has been shown for small non-peptide molecules to activate glucagon-like peptide-1 receptors. Additional sodium-glucose co-transporter inhibitors are progressing in development as well as possible new insulin-releasing biological agents and small-molecule inhibitors of glucagon action. Adiponectin receptor agonists, selective peroxisome proliferator-activated receptor modulators, cellular glucocorticoid inhibitors, and analogues of fibroblast growth factor 21 are being considered as potential new approaches to glucose lowering. Compounds that can enhance insulin receptor and post-receptor signalling cascades or directly promote selected pathways of glucose metabolism have suggested opportunities for future treatments. However, pharmacological interventions that are able to restore normal β-cell function and β-cell mass, normalise insulin action, and fully correct glucose homoeostasis are a distant vision.

Management of type 2 diabetes:new and future developments in treatment

The increasing prevalence, variable pathogenesis, progressive natural history, and complications of type 2 diabetes emphasise the urgent need for new treatment strategies. Longacting (eg, once weekly) agonists of the glucagon-like-peptide-1 receptor are advanced in development, and they improve prandial insulin secretion, reduce excess glucagon production, and promote satiety. Trials of inhibitors of dipeptidyl peptidase 4, which enhance the effect of endogenous incretin hormones, are also nearing completion. Novel approaches to glycaemic regulation include use of inhibitors of the sodium-glucose cotransporter 2, which increase renal glucose elimination, and inhibitors of 11ß-hydroxysteroid dehydrogenase 1, which reduce the glucocorticoid effects in liver and fat. Insulin-releasing glucokinase activators and pancreatic-G-protein-coupled fatty-acid-receptor agonists, glucagon-receptor antagonists, and metabolic inhibitors of hepatic glucose output are being assessed. Early proof of principle has been shown for compounds that enhance and partly mimic insulin action and replicate some effects of bariatric surgery.

Influence of environmental factors on zoospores of Saprolegnia diclina

The numbers of zoospores produced by a pathogenic strain of Saprolegnia diclina and their behaviour are markedly influenced by a variety of environmental variables including temperature, pH, oxygen tension and the presence of biocides. The use of the latter is not recommended, as fish readily succumb to equivalent concentrations of biocides. Analysis of the pattern of distribution of resulting zoospore cysts demonstrates that zoospores become dispersed by random movement even while in the proximity of the parent colony’s nutrient source. However, the presence of amino acids, in particular aspartic and glutamic acid, at concentrations which occur in fish tissue promotes the directed movement of zoospores towards the nutrient source thereby encouraging the colonization of fresh sites.

New therapies for diabesity

Many patients with type 2 diabetes are obese (diabesity), and the two conditions together impose a particularly complex therapeutic challenge. Several differently acting agents are often required at the same time, encouraging development of more single-tablet combinations. Longer-acting (once daily and once weekly) injected agonists of glucagon-like peptide-1 are due to provide additional options to stimulate insulin secretion with weight loss and minimal risk of hypoglycemia. Further, dipeptidyl peptidase-4 inhibitors ("weight-neutral" insulinotropic agents) are also expected. Sodium-glucose cotransporter 2 inhibitors offer a new option to reduce hyperglycemia and facilitate weight loss by increasing the elimination of glucose in the urine. Selective peroxisome proliferator-activated receptor modulators are being studied to produce compounds with desired effects. Many other agents with antidiabetic and antiobesity activity are progressing in clinical development.

Amino acid-modified spray-dried powders with enhanced aerosolisation properties for pulmonary drug delivery

In this study, the amino acids arginine, aspartic acid, leucine, phenylalanine and threonine were investigated as 'dispersibility enhancers' in spray-dried powders for inhalation. Parameters such as spray-dried yield, tapped density, and Carr's Index were not predictive of aerosolisation performance. In addition, whilst the majority of amino acid-modified powders displayed suitable particle size distribution for pulmonary administration and potentially favourable low moisture content, in vitro particle deposition was only enhanced for the leucine-modified powder. In summary, leucine can be used to enhance the dispersibility and aerosolisation properties of spray-dried powders for pulmonary drug delivery. © 2007 Elsevier B.V. All rights reserved.

The use of amino acids to enhance the aerosolisation of spray-dried powders for pulmonary gene therapy

Background Pulmonary delivery of gene therapy offers the potential for the treatment of a range of lung conditions, including cystic fibrosis, asthma and lung cancer. Spray-drying may be used to prepare dry powders for inhalation; however, aerosolisation of such powders is limited, resulting in poor lung deposition and biological functionality. In this study, we examine the use of amino acids (arginine, aspartic acid, threonine, phenylalanine) to enhance the aerosolisation of spray-dried powders containing model non-viral gene vectors. Methods Lipid/polycation/pDNA (LPD) vectors, in the presence or absence of amino acids, were dispersed in lactose solutions, and spray-dried to produce appropriately sized dry powders. Scanning electron microscopy and laser diffraction were used to determine particle morphology and diameter, respectively. Gel electrophoresis was used to examine the influence of amino acids on the structural integrity of the LPD complex. In vitro cell (A.549) transfection was used to determine the biological functionality of the dry powders, and the in vitro aerosolisation performance was assessed using a multistage liquid impinger (MSLI). Results Both gel electrophoresis and in vitro cell transfection indicated that certain amino acids (aspartic acid, threonine) can adversely affect the integrity and biological functionality of the LPD complex. All amino acids significantly increased the aerosolisation of the powder, with the arginine and phenylalanine powders showing optimal deposition in the lower stages of the MSLI. Conclusions Amino acids can be used to enhance the aerosolisation of spray-dried powders for respiratory gene delivery, allowing the development of stable and viable formulations for pulmonary gene therapy.

Fixed-dose combination therapy for type 2 diabetes:sitagliptin plus pioglitazone

Type 2 diabetes is typically associated with insulin resistance and dysfunction of insulin-secreting pancreatic beta-cells. Addressing these defects often requires therapy with a combination of differently acting antidiabetic agents. A potential novel combination in development brings together the dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin with the thiazolidinedione pioglitazone into a fixed-dose single-tablet combination. The former component acts mainly to increase prandial insulin secretion; the latter improves insulin sensitivity.

The challenge of managing coexistent type 2 diabetes and obesity

The presence of obesity with type 2 diabetes increases morbidity and mortality from each condition. Excess adiposity accentuates insulin resistance and complicates the treatment of type 2 diabetes. Glucagon-like peptide 1 receptor agonists promote weight loss, whereas metformin, dipeptidyl peptidase 4 inhibitors, and a glucosidase inhibitors are typically weight neutral. The anabolic effects of increased insulin secretion and action restrict the benefits of treatment in obese patients. New treatments should ideally reduce hyperglycaemia and excess adiposity. Potential new treatments include analogues of intestinal and adipocyte hormones, inhibitors of renal glucose reabsorption and cellular glucocorticoid activation, and activators of cellular energy production.

Histidine acts as a co-germinant with glycine and taurocholate for Clostridium difficile spores

Aims: It is well established that the bile salt sodium taurocholate acts as a germinant for Clostridium difficile spores and the amino acid glycine acts as a co-germinant. The aim of this study was to determine whether any other amino acids act as co-germinants. Methods and Results: Clostridium difficile spore suspensions were exposed to different germinant solutions comprising taurocholate, glycine and an additional amino acid for 1 h before heating shocking (to kill germinating cells) or chilling on ice. Samples were then re-germinated and cultured to recover remaining viable cells. Only five amino acids out of the 19 common amino acids tested (valine, aspartic acid, arginine, histidine and serine) demonstrated co-germination activity with taurocholate and glycine. Of these, only histidine produced high levels of germination (97·9–99·9%) consistently in four strains of Cl. difficile spores. Some variation in the level of germination produced was observed between different PCR ribotypes, and the optimum concentration of amino acids with taurocholate for the germination of Cl. difficile NCTC 11204 spores was 10–100 mmol l-1. Conclusions: Histidine was found to be a co-germinant for Cl. difficile spores when combined with glycine and taurocholate. Significance and Impact of the Study: The findings of this study enhance current knowledge regarding agents required for germination of Cl. difficile spores which may be utilized in the development of novel applications to prevent the spread of Cl. difficile infection.