Effect of branched-chain amino acids on muscle atrophy in cancer cachexia

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2α (eukaryotic initiation factor 2α) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G-eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70S6k (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70S6k, caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G-eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation. © The Authors.

Do little embryos make big decisions? How maternal dietary protein restriction can permanently change an embryo's potential, affecting adult health

Periconceptional environment may influence embryo development, ultimately affecting adult health. Here, we review the rodent model of maternal low-protein diet specifically during the preimplantation period (Emb-LPD) with normal nutrition during subsequent gestation and postnatally. This model, studied mainly in the mouse, leads to cardiovascular, metabolic and behavioural disease in adult offspring, with females more susceptible. We evaluate the sequence of events from diet administration that may lead to adult disease. Emb-LPD changes maternal serum and/or uterine fluid metabolite composition, notably with reduced insulin and branched-chain amino acids. This is sensed by blastocysts through reduced mammalian target of rapamycin complex 1 signalling. Embryos respond by permanently changing the pattern of development of their extra-embryonic lineages, trophectoderm and primitive endoderm, to enhance maternal nutrient retrieval during subsequent gestation. These compensatory changes include stimulation in proliferation, endocytosis and cellular motility, and epigenetic mechanisms underlying them are being identified. Collectively, these responses act to protect fetal growth and likely contribute to offspring competitive fitness. However, the resulting growth adversely affects long-term health because perinatal weight positively correlates with adult disease risk. We argue that periconception environmental responses reflect developmental plasticity and 'decisions' made by embryos to optimise their own development, but with lasting consequences.

Effect of eicosapentaenoic acid, protein and amino acids on protein synthesis and degradation in skeletal muscle of cachectic mice

Atrophy of skeletal muscle reduces both the quality and quantity of life of patients with cancer cachexia. Loss of muscle mass is thought to arise from a reduction in protein synthesis combined with an enhanced rate of protein degradation, and few treatments are available to counteract this process. Eicosapentaenoic acid (EPA) has been shown to attenuate the enhanced protein degradation, but to have no effect on protein synthesis. This study examines the effect of EPA combined with a protein and amino-acid supplementation on protein synthesis and degradation in gastrocnemius muscle of mice bearing the cachexia-inducing MAC16 tumour. Muscles from cachectic mice showed an 80% reduction in protein synthesis and about a 50-fold increase in protein degradation compared with muscles from nontumour-bearing mice of the same age and weight. Treatment with EPA (1 g kg-1) daily reduced protein degradation by 88%, but had no effect on protein synthesis. Combination of EPA with casein (5.35 g kg-1) also had no effect on protein synthesis, but when combined with the amino acids leucine, arginine and methionine there was almost a doubling of protein synthesis. The addition of carbohydrate (10.7 g kg-1) to stimulate insulin release had no additional effect. The combination involving the amino acids produced almost a doubling of the ratio of protein synthesis to protein degradation in gastrocnemius muscle over that of EPA alone. No treatment had a significant effect on tumour growth rate, but the inclusion of amino acids had a more significant effect on weight loss induced by the MAC16 tumour than that of EPA alone. The results suggest that combination therapy of cancer cachexia involving both inhibition of the enhanced protein degradation and stimulation of the reduced protein synthesis may be more effective than either treatment alone. © 2004 Cancer Research UK.

Formulation and characterisation of lyophilised rapid disintegrating tablets using amino acids as matrix forming agents

Despite recent advances in the formulation of lyophilised rapid disintegrating tablets (RDTs), the inclusion of matrix supporting/disintegration enhancing agents has been limited to the use of saccharides and polyols. In this study, the feasibility of using amino acids as matrix forming agents in lyophilised RDTs was investigated. Twelve amino acids were chosen (alanine, arginine, threonine, glycine, cysteine, serine, histidine, lysine, valine, asparagine, glutamine and proline), and the suitability for freeze drying, mechanical properties and disintegration time after inclusion of the amino acids at varied concentration were studied. In addition, the porosity of the RDTs and wettability profile of the amino acids were investigated to understand the mechanisms of disintegration. The results suggest the suitability of these amino acids for the lyophilisation regime, as they displayed satisfactory safety margin between the glass transition and shelf temperature (-40 degrees C), except proline-based formulations. Moreover, the crystallisation behavior of alanine, glycine, cysteine and serine at high concentration increased the stability of the formulation. The characterisation of the RDTs suggests that high concentration of the amino acids is required to enhance the mechanical properties, whereas only optimum concentrations promote the disintegration. Moreover, wetting time of the amino acid and porosity of the tablet are the two factors that control the disintegration of RDTs.

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.

Use of amino acids as counterions improves the solubility of the BCS II model drug, indomethacin

The number of new chemical entities (NCE) is increasing every day after the introduction of combinatorial chemistry and high throughput screening to the drug discovery cycle. One third of these new compounds have aqueous solubility less than 20µg/mL [1]. Therefore, a great deal of interest has been forwarded to the salt formation technique to overcome solubility limitations. This study aims to improve the drug solubility of a Biopharmaceutical Classification System class II (BCS II) model drug (Indomethacin; IND) using basic amino acids (L-arginine, L-lysine and L-histidine) as counterions. Three new salts were prepared using freeze drying method and characterised by FT-IR spectroscopy, proton nuclear magnetic resonance ((1)HNMR), Differential Scanning Calorimetry (DSC) and Thermogravimetric analysis (TGA). The effect of pH on IND solubility was also investigated using pH-solubility profile. Both arginine and lysine formed novel salts with IND, while histidine failed to dissociate the free acid and in turn no salt was formed. Arginine and lysine increased IND solubility by 10,000 and 2296 fold, respectively. An increase in dissolution rate was also observed for the novel salts. Since these new salts have improved IND solubility to that similar to BCS class I drugs, IND salts could be considered for possible waivers of bioequivalence.

Multiple roles of charged amino acids in cytoplasmic loop 7 for expression and function of the multidrug and organic anion transporter MRP1 (ABCC1)

Multidrug resistance protein MRP1 mediates the ATP-dependent efflux of many chemotherapeutic agents and organic anions. MRP1 has two nucleotide binding sites (NBSs) and three membrane spanning domains (MSDs) containing 17 transmembrane helices linked by extracellular and cytoplasmic loops (CL). Homology models suggest that CL7 (amino acids 1141-1195) is in a position where it could participate in signaling between the MSDs and NBSs during the transport process. We have individually replaced eight charged residues in CL7 with Ala, and in some cases, an amino acid with the same charge, and then investigated the effects on MRP1 expression, transport activity, and nucleotide and substrate interactions. A triple mutant in which Glu(1169), Glu(1170), and Glu(1172) were all replaced with Ala was also examined. The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, (1169)AAQA). Same charge mutant D1183E was also not expressed, whereas expression and activity of R1166K were similar to wild-type MRP1. The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and (1169)AAQA were accompanied by changes in orthovanadate-induced trapping of [alpha-(32)P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Together, our results demonstrate the extreme sensitivity of CL7 to mutation, consistent with its critical and complex dual role in both the proper folding and transport activity of MRP1.

Amino acids as cryoprotectants for liposomal delivery systems

Liposomes provide an efficient delivery system for solubilisation and delivery of both small and macro molecules. However, they suffer from the disadvantage of instability when stored as aqueous dispersions. Cryoprotection of the liposomal systems provides an effective approach to overcome poor stability whilst maintaining formulation characteristics, although, the formulation of a freeze-dried product requires the consideration of not only the selection of an appropriate cryoprotectant, but also needs careful consideration of the processing parameters including pre-freezing conditions, primary and secondary drying protocols along with optimisation of cryoprotectant concentration. This current work investigates the application of amino acids as potential cryoprotectants for the stabilisation of liposomes, and results indicate that amino acids show biphasic nature of stabilisation with 4 mol of cryoprotectant per mole of the lipid exhibiting optimum cryoprotection. The investigations of process parameters showed that the pre-freezing temperatures below the glass transition of the amino acids followed by drying for over 6 h resulted in stable formulations. Studies investigating the efficiency of drug retention showed that the cryoprotection offered by lysine was similar to that shown by trehalose, suggesting that amino acids act as effective stabilisers. ESEM analysis was carried out to monitor morphology of the rehydrated liposomes. © 2007 Elsevier B.V. All rights reserved.

Preparation and characterisation of amino acids based trimethoprim salts

Trimethoprim (TMP) is a dihydrofolate reductase (DHFR) inhibitor which prevents the conversion of dihydrofolic acid into tetrahydrofolic acid, resulting in the depletion of the latter and leading to bacterial death. Oral bioavailability of TMP is hindered by both its low solubility and low permeability. This study aims to prepare novel salts of TMP using anionic amino acids; aspartic and glutamic acid as counter ions in order to improve solubility and dissolution. TMP salts were prepared by lyophilisation and characterized using FT-IR spectroscopy, proton nuclear magnetic resonance (1HNMR), Differential Scanning Calorimetry (DSC) and Thermogravimetric analysis (TGA). Both the amino acids formed salts with TMP in a 1:1 molar ratio and showed a 280 fold improvement in solubility. Investigation of the microbiological activity of the prepared salts against TMP sensitive Escherichia coli showed that the new salts not only retained antibacterial activity but also exhibited higher zone of inhibition which was attributed to improved physicochemical characters such as higher solubility and dissolution. The results are an important finding that could potentially impact on faster onset of antibacterial activity and reduced therapeutic dose when administered to patients. Studies are underway investigating the effect of ion-pairing TMP with amino acids on the permeability profile of the drug.

Amino acids in oral drug delivery:salts, ion-pairs and transcriptomics

Oral drug delivery is considered the most popular route of delivery because of the ease of administration, availability of a wide range of dosage forms and the large surface area for drug absorption via the intestinal membrane. However, besides the unfavourable biopharmaceutical properties of the therapeutic agents, efflux transporters such as Pglycoprotein (P-gp) and multiple resistance proteins (MRP) decrease the overall drug uptake by extruding the drug from the cells. Although, prodrugs have been investigated to improve drug partitioning by masking the polar groups covalently with pre-moieties promoting increased uptake, they present significant challenges including reduced solubility and increased toxicity. The current work investigates the use of amino acids as ion-pairs for three model drugs: indomethacin (weak acid), trimethoprim (weak base) and ciprofloxacin (zwitter ion) in an attempt to improve both solubility and uptake. Solubility was studied by salt formation while creating new routes for uptake across the membranes via amino acids transporter proteins or dipeptidyl transporters was the rationale to enhance absorption. New salts were prepared for the model drugs and the oppositely charged amino acids by freeze drying and they were characterised using FTIR, 1HNMR, DSC, SEM, pH solubility profile, solubility and dissolution. Permeability profiles were assessed using an in vitro cell based method; Caco-2 cells and the genetic changes occurring across the transporter genes and various pathways involved in the cellular activities were studied using DNA microarrays. Solubility data showed a significant increase in drug solubility upon preparing the new salts with the oppositely charged counter ions (ciprofloxacin glutamate salt exhibiting 2.9x103 fold enhancement when compared to the free drug). Moreover, permeability studies showed a 3 fold increase in trimethoprim and indomethacin permeabilities upon ion-pairing with amino acids and more than 10 fold when the zwitter ionic drug was paired with glutamic acid. Microarray data revealed that trimethoprim was absorbed actively via OCTN1 transporters while MRP7 is the main transporter gene that mediates its efflux. The absorption of trimethoprim from trimethoprim glutamic acid ion-paired formulations was affected by the ratio of glutamic acid in the formulation which was inversely proportional to the degree of expression of OCTN1. Interestingly, ciprofloxacin glutamic acid ion-pairs were found to decrease the up-regulation of ciprofloxacin efflux proteins (P-gp and MRP4) and over-express two solute carrier transporters; (PEPT2 and SLCO1A2) suggesting that a high aqueous binding constant (K11aq) enables the ion-paired formulations to be absorbed as one entity. In conclusion, formation of ion-pairs with amino acids can influence in a positive way solubility, transfer and gene expression effects of drugs.

Tumour associated proteolysis and protein metabolism

The effect of cancer cachexia on protein metabolism has been studied in mice transplanted with the MAC16 adenocarcinoma. The progressive cachexia induced by the MAC16 tumour was characterised by a reduction in carcass nitrogen between 16-30% weight loss and a reciprocal increase in tumour nitrogen content. Carcass nitrogen loss was accompanied by a concomitant decrease in gastrocnemius muscle weight and nitrogen content and also by a decrease in liver nitrogen content. The loss of gastrocnemius muscle throughout the progression of cachexia was attributable to a 60% decrease in the rate of protein synthesis and a 240% increase in the rate of protein degradation. The loss of skeletal muscle protein that may be partially mediated by an increased rate of protein degradation has been correlated with a circulatory catabolic factor present only in cachectic tumour-bearing animals, that degrades host muscle in vitro. The proteolysis-inducing factor was found to be heat stable, not a serine protease and was inhibited by indomethacin and eicosapentaenoic acid (EPA) in a dose-related manner. The proteolytic factor induced prostaglandin E2 formation in the gastrocnemius muscle of non tumour-bearing animals and this effect was inhibited by indomethacin and EPA. In vivo studies show EPA (2.0g/kg-1 by gavage) to effectively reverse the decrease in body weight in animals bearing the MAC16 tumour with a concomitant reduction in tumour growth. Muscle from animals treated with EPA showed a decrease (60%) in protein degradation without an effect on protein synthesis. In vivo studies show branched chain amino acid treatment to be ineffective in moderating the cachectic effect of the MAC16 tumour. The action of the factor was largely mimicked by triarachidonin and trilinoleia. The increased serum levels of arachidonic acid in cachectic tumour-bearing animals may thus be responsible for increased protein degradation through prostanoid metabolism. The understanding of protein metabolism and catabolic factors in the cachectic animal may provide future avenues for the reversal of cachexia and the treatment of cancer.metabolism and catabolicmetabolism and cat

Metabolic induction and early responses of mouse blastocyst developmental programming following maternal low protein diet affecting life-long health

Previously, we have shown that a maternal low protein diet, fed exclusively during the preimplantation period of mouse development (Emb-LPD), is sufficient to induce by the blastocyst stage a compensatory growth phenotype in late gestation and postnatally, correlating with increased risk of adult onset cardiovascular disease and behavioural dysfunction. Here, we examine mechanisms of induction of maternal Emb-LPD programming and early compensatory responses by the embryo. Emb-LPD induced changes in maternal serum metabolites at the time of blastocyst formation (E3.5), notably reduced insulin and increased glucose, together with reduced levels of free amino acids (AAs) including branched chain AAs leucine, isoleucine and valine. Emb-LPD also caused reduction in the branched chain AAs within uterine fluid at the blastocyst stage. These maternal changes coincided with an altered content of blastocyst AAs and reduced mTORC1 signalling within blastocysts evident in reduced phosphorylation of effector S6 ribosomal protein and its ratio to total S6 protein but no change in effector 4E-BP1 phosphorylated and total pools. These changes were accompanied by increased proliferation of blastocyst trophectoderm and total cells and subsequent increased spreading of trophoblast cells in blastocyst outgrowths. We propose that induction of metabolic programming following Emb-LPD is achieved through mTORC1signalling which acts as a sensor for preimplantation embryos to detect maternal nutrient levels via branched chain AAs and/or insulin availability. Moreover, this induction step associates with changes in extra-embryonic trophectoderm behaviour occurring as early compensatory responses leading to later nutrient recovery. © 2012 Fleming et al.

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.

Peptide conjugate hydrogelators

Molecular gelators are currently receiving a great deal of attention. These are small molecules which, under the appropriate conditions, assemble in solution to, in the majority of cases, give long fibrillar structures which entangle to form a three-dimensional network. This immobilises the solvent, resulting in a gel. Such gelators have potential application in a number of important areas from drug delivery to tissue engineering. Recently, the use of peptide-conjugates has become prevalent with oligopeptides (from as short as two amino acids in length) conjugated to a polymer, alkyl chain or aromatic group such as naphthalene or fluorenylmethoxycarbonyl (Fmoc) being shown to be effective molecular gelators. The field of gelation is extremely large; here we focus our attention on the use of these peptide-conjugates as molecular hydrogelators.

Peptide binding prediction for the human class II MHC allele HLA-DP2:a molecular docking approach

MHC class II proteins bind oligopeptide fragments derived from proteolysis of pathogen antigens, presenting them at the cell surface for recognition by CD4+ T cells. Human MHC class II alleles are grouped into three loci: HLA-DP, HLA-DQ and HLA-DR. In contrast to HLA-DR and HLA-DQ, HLA-DP proteins have not been studied extensively, as they have been viewed as less important in immune responses than DRs and DQs. However, it is now known that HLA-DP alleles are associated with many autoimmune diseases. Quite recently, the X-ray structure of the HLA-DP2 molecule (DPA*0103, DPB1*0201) in complex with a self-peptide derived from the HLA-DR a-chain has been determined. In the present study, we applied a validated molecular docking protocol to a library of 247 modelled peptide-DP2 complexes, seeking to assess the contribution made by each of the 20 naturally occurred amino acids at each of the nine binding core peptide positions and the four flanking residues (two on both sides).