The physicochemical properties of geraniin, a potential antihyperglycemic agent.

CONTEXT: Natural products play a vital role in the discovery of leads for novel pharmacologically active drugs. Geraniin (GE) was identified as the major compound in the rind of Nephelium lappaceum L. (Sapindaceae), while ellagic and gallic acids have been shown to be its main metabolites. GE and its metabolites possess a range of bioactive properties including being an anti-infective, anticarcinogenic, antihyperglycemic, and antihypertensive. OBJECTIVE: GE and its metabolites were investigated to establish its gastrointestinal absorption and physicochemical properties. MATERIALS AND METHODS: GE was purified from N. lappaceum rind extract using reverse-phase C18 column chromatography. Lipophilicity (log P) was determined using the 1-octanol/water shake-flask method. Equilibrium solubility of GE and its metabolites (20 mg) was determined in water and four biorelevant media: simulated gastric, simulated intestinal, fasted state-simulated intestinal, and fed state-simulated intestinal after 72 h. RESULTS AND DISCUSSION: The purification yield was 10.8%; where a 97-99% pure GE was obtained. Log P values for GE, ellagic, and gallic acids were established as -0.73 ± 0.17, 0.11 ± 0.06, and 0.71 ± 0.21, respectively, establishing them as polar compounds. All three compounds were found to exhibit poor solubility in gastric (0.61-8.10 mg/mL) but good solubility in intestinal fluids (3.59-14.32 mg/mL). CONCLUSION: The above results indicate that the compounds have limited gastrointestinal absorption due to its polarities. To consider these compounds as oral drug candidates, formulation strategies are being developed.

The significance of acid/base properties in drug discovery.

While drug discovery scientists take heed of various guidelines concerning drug-like character, the influence of acid/base properties often remains under-scrutinised. Ionisation constants (pK(a) values) are fundamental to the variability of the biopharmaceutical characteristics of drugs and to underlying parameters such as logD and solubility. pK(a) values affect physicochemical properties such as aqueous solubility, which in turn influences drug formulation approaches. More importantly, absorption, distribution, metabolism, excretion and toxicity (ADMET) are profoundly affected by the charge state of compounds under varying pH conditions. Consideration of pK(a) values in conjunction with other molecular properties is of great significance and has the potential to be used to further improve the efficiency of drug discovery. Given the recent low annual output of new drugs from pharmaceutical companies, this review will provide a timely reminder of an important molecular property that influences clinical success.

Stability and sterility of diluted enoxaparin under three different storage conditions.

AIM: This study assessed whether enoxaparin sodium diluted to a concentration of 20 mg/mL for clinical use with 0.9% sodium chloride remained stable and sterile for up to 43 days under three different storage conditions. METHODS: Enoxaparin dilutions in polypropylene syringes were stored under three different controlled conditions of temperature and light: (i) room temperature (22-26°C) under natural light; (ii) room temperature (22-26°C) in the dark; and (iii) controlled refrigeration (2-8°C) in the dark. A weekly assay of anti-Xa and anti-IIa activity was undertaken to determine if the diluted enoxaparin preparations retained anticoagulant activity, thus remaining suitable for clinical application. RESULTS: Our findings indicate that diluted enoxaparin, when stored under the tested varied conditions of light and temperature, retained greater than or equal to 90% of baseline anticoagulant activity for anti-Xa and anti-IIa effect for up to 43 days. CONCLUSIONS: The study results are significant for families, in that they suggest that at least a month's supply of enoxaparin could be dispensed at a time, reducing the frequency of patients/families returning for supply and providing a more convenient service for paediatric patients.

Stability of peroxide antimalarials in the presence of human hemoglobin.

Peroxide antimalarials, including artemisinin, are important for the treatment of multidrug-resistant malaria. These peroxides are known to react with iron or heme to produce reactive intermediates that are thought to be responsible for their antimalarial activities. This study investigated the potential interaction of selected peroxide antimalarials with oxyhemoglobin, the most abundant form of iron in the human body. The observed stability of artemisinin derivatives and 1,2,4-trioxolanes in the presence of oxyhemoglobin was in contrast to previous reports in the literature. Spectroscopic analysis of hemoglobin found it to be unstable under the conditions used for previous studies, and it appears likely that the artemisinin reactivity reported in these studies may be attributed to free heme released by protein denaturation. The stability of peroxide antimalarials with intact oxyhemoglobin, and reactivity with free heme, may explain the selective toxicity of these antimalarials toward infected, but not healthy, erythrocytes.

Temperature stability of oxytocin ampoules labelled for storage at 2°C-8°C and below 25°C: an observational assessment under controlled accelerated and temperature cycling conditions.

INTRODUCTION: Oxytocin, administered via injection, is recommended by WHO for the prevention and treatment of postpartum haemorrhage. However, the susceptibility of oxytocin injection to thermal degradation has led WHO and UNICEF to recommend cold-chain storage of all oxytocin products. Nevertheless, some oxytocin products supplied to the global market are labelled for storage at ≤25°C, often with a shorter shelf-life relative to products labelled for refrigeration. Differences in labelled storage requirements can lead to uncertainties among stakeholders around the relative stability of oxytocin products and specifically whether ≤25°C products are more resistant to degradation. Such confusion can potentially influence policies associated with procurement, distribution, storage and the use of oxytocin in resource-poor settings. OBJECTIVES: To compare the stability of oxytocin injection ampoules formulated for storage at ≤25°C with those labelled for refrigerated storage. DESIGN: Accelerated and temperature cycling stability studies were performed with oxytocin ampoules procured by the United Nations Population Fund (UNFPA) from four manufacturers. METHOD: Using oxytocin ampoules procured by UNFPA, accelerated stability (up to 120 days) and temperature cycling (up to 135 days between elevated and refrigerated temperatures) studies were performed at 30°C, 40°C and 50°C. Oxytocin content was quantified using a validated HPLC-UV method. RESULTS: All ampoules evaluated exhibited similar stability profiles under accelerated degradation conditions with the exception of one product formulated for ≤25°C storage, where the rate of degradation increased at 50°C relative to other formulations. Similar degradation trends at elevated temperatures were observed during temperature cycling, while no significant degradation was observed during refrigerated periods of the study. CONCLUSION: Oxytocin ampoules formulated for non-refrigerated storage demonstrated comparable stability to those labelled for refrigerated storage and should not be interpreted by stakeholders as offering a more stable alternative. Furthermore, these products should not be procured for use in territories with high ambient temperatures, where all oxytocin injection products should be supplied and stored under refrigerated conditions.

Probing the flexibility of the DsbA oxidoreductase from Vibrio cholerae--a 15N - 1H heteronuclear NMR relaxation analysis of oxidized and reduced forms of DsbA.

We have determined the structure of the reduced form of the DsbA oxidoreductase from Vibrio cholerae. The reduced structure shows a high level of similarity to the crystal structure of the oxidized form and is typical of this class of enzyme containing a thioredoxin domain with an inserted alpha-helical domain. Proteolytic and thermal stability measurements show that the reduced form of DsbA is considerably more stable than the oxidized form. NMR relaxation data have been collected and analyzed using a model-free approach to probe the dynamics of the reduced and oxidized states of DsbA. Akaike's information criteria have been applied both in the selection of the model-free models and the diffusion tensors that describe the global motions of each redox form. Analysis of the dynamics reveals that the oxidized protein shows increased disorder on the pico- to nanosecond and micro- to millisecond timescale. Many significant changes in dynamics are located either close to the active site or at the insertion points between the domains. In addition, analysis of the diffusion data shows there is a clear difference in the degree of interdomain movement between oxidized and reduced DsbA with the oxidized form being the more rigid. Principal components analysis has been employed to indicate possible concerted movements in the DsbA structure, which suggests that the modeled interdomain motions affect the catalytic cleft of the enzyme. Taken together, these data provide compelling evidence of a role for dynamics in the catalytic cycle of DsbA.

Preparation and in vitro evaluation of novel lipopeptide transfection agents for efficient gene delivery.

Gene therapy by delivery of nonviral expression vectors is highly desirable, due to their safety, stability, and suitability for production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factor in application on nonviral gene delivery. Despite recent advances in the field, there are still major obstacles to overcome. In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series of novel lipopeptide transfection agents, consisting of an alkyl chain, one cysteine, 1 to 4 histidine and 1 to 3 lysine residues. The lipopeptides were designed to facilitate dimerization (by way of the cysteine residues), DNA binding at neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidine residues). DNA/lipopeptide complexes were evaluated for their biophysical properties and transfection efficiencies. The number and identity of amino acids incorporated in the lipopeptide construct affected their DNA/lipopeptide complex forming capacity. As the number of lysine residues in the lipopeptide increased, the DNA complexes formed became more stable, had higher zeta potential (particle surface charge), and produced smaller mean particle sizes (typically 110 nm at a charge ratio of 5.0 and 240 nm at a charge ratio of 1.0). The effect of inclusion of histidines in the lipopeptide moiety had the opposite effect on complex formation to lysine, but was necessary for high transfection efficiency. In vitro transfection studies in COS-7 cells revealed that the efficiency of gene delivery of the luciferase encoding plasmid, pCMV-Luc, mediated by all the lipopeptides, was much higher than poly(L-lysine) (PLL), which has no endosomal escape system, and in two cases was slightly higher than that of branched polyethylenimine (PEI). Lipopeptides with at least two lysine residues and at least one histidine residue produced spontaneous transfection complexes with plasmid DNA, indicating that endosomal escape was achieved by incorporation of histidine residues. These low molecular weight peptides can be readily synthesized and purified and offer new insights into the mechanism of action of transfection complexes.

A protocol for the combined sub-fractionation and delipidation of lipid binding proteins using hydrophobic interaction chromatography.

Cellular lipids frequently co-purify with lipid binding proteins isolated from tissue extracts or heterologous host systems and as such hinder in vitro ligand binding approaches for which the apo-protein is a prerequisite. Here we present a technique for the complete removal of unesterified fatty acids, phospholipids, steroids and other lipophilic ligands bound to soluble proteins, without protein denaturation. Peroxisome proliferator activated receptor gamma ligand binding domain and intracellular fatty acid binding proteins were expressed in an Escherichia coli host and completely delipidated by hydrophobic interaction chromatography using phenyl sepharose. The delipidation procedure operates at room temperature with complete removal of bound lipids in a single step, as ascertained by mass spectrometry analysis of organic solvent extracts from purified protein samples. The speed and capacity of this method makes it amenable to scale-up and high-throughput applications. The method can also easily be adapted for other lipid binding proteins that require delipidation under native conditions.

Iron-mediated degradation kinetics of substituted dispiro-1,2,4-trioxolane antimalarials.

The iron-mediated reactivity of various dispiro-1,2,4-trioxolanes was determined by automated kinetic analysis under standard reaction conditions. The active antimalarial compounds underwent peroxide bond cleavage by Fe(II) resulting in products indicative of carbon-centered radical formation. The rate of reaction was heavily influenced by the presence of spiro-substituted adamantane and cyclohexane rings, and was also significantly affected by cyclohexane ring substitution. Steric hindrance around the peroxide oxygen atoms appeared to be the major determinant of reaction rate, however polar substituents also affected reactivity by an independent mechanism. A wide range of reaction rates was observed within this class of peroxide antimalarials, however iron-mediated reactivity did not directly correlate with in vitro antimalarial activity.

Precise control of drug loading and release of an NSAID-polymer conjugate for long term osteoarthritis intra-articular drug delivery.

A facile synthesis method of polymer diclofenac conjugates (PDCs) based on biocompatible polyurethane chemistry that provides a high drug loading and offers a high degree of control over diclofenac (DCF) release kinetics is described. DCF incorporating monomer was reacted with ethyl-l-lysine diisocyanate (ELDI) and different amounts of polyethylene glycol (PEG) in a one-step synthesis to yield polymers with pendent diclofenac distributed along the backbone. By adjusting the co-monomers feed ratio, the drug loading could be tailored accordingly to give DCF loading of up to 38 w/w%. The release rate could also be controlled easily by changing the amount of PEG in the backbone. Above 10 w/w% of PEG, the in vitro DCF release studies in physiological conditions showed an apparent zero-order profile without an initial burst effect for up to 120 days. The PDCs described may be suitable for long-term intra-articular (IA) delivery for the treatment of osteoarthritis (OA).

The binding interaction of synthetic ozonide antimalarials with natural and modified beta-cyclodextrins.

The current studies were undertaken to explore the potential basis for a significant difference in the pharmacokinetic parameters after intravenous administration of a synthetic ozonide (OZ) antimalarial drug candidate (1) to rats when formulated in either Captisol (a sulfobutylether substituted beta-cyclodextrin derivative ((SBE)(7)-beta-CD)) or a buffered aqueous vehicle. It was suspected that the differences may have been due to failure of 1 to rapidly dissociate from the cyclodextrin complex in vivo, perhaps due to an unusually tight binding within the cyclodextrin cavity. To address this hypothesis, the binding of representative synthetic OZ antimalarial drug candidates (including 1) with beta-cyclodextrin and (SBE)(7)-beta-CD was investigated by isothermal titration calorimetry and phase solubility analysis. It was found that each of the OZ compounds exhibited an exceptionally high binding constant ( approximately 10(6)/M) with both Cyclodextrins (CD). The nature of the complexation was investigated by molecular dynamics simulations and NMR to explore the mechanisms, which generated such high binding constants. The data suggested that the most probable cause of the unusually high binding constants was a very close fit within the cyclodextrin cavity that resulted in more favourable changes in both the enthalpy and entropy of the binding interaction, compared to published data for other drugs.

Alteration of the intravenous pharmacokinetics of a synthetic ozonide antimalarial in the presence of a modified cyclodextrin.

The pharmacokinetic profile and renal clearance of a novel synthetic ozonide antimalarial (1) was found to be significantly altered when intravenously administered to rats as a cyclodextrin-based formulation (0.1 M Captisol, a sulfobutylether beta-cyclodextrin derivative (SBE(7)-beta-CD)) compared to a cyclodextrin-free isotonic buffered glucose formulation. There was an 8.5-fold decrease in the steady-state blood volume of distribution, a 6.6-fold decrease in the mean residence time and a greater than 200-fold increase in renal clearance of 1 when administered in the cyclodextrin formulation. Analysis of the whole blood and plasma concentration profiles revealed an essentially constant blood to plasma ratio when 1 was administered in the cyclodextrin-free formulation, whereas this ratio changed as a function of time when administered in the presence of the cyclodextrin derivative. It is postulated that the observed differences were due to a very strong complexation interaction between 1 and the cyclodextrin, resulting in a slow dissociation of the complex in vivo, and altered distribution and excretion profiles. Preliminary studies using isothermal titration calorimetry (ITC) indicated that the association constant for the 1/Captisol complex was approximately two orders of magnitude higher than reported for typical drug/cyclodextrin complexes.

Chemical kinetics and aqueous degradation pathways of a new class of synthetic ozonide antimalarials.

Chemical stability of a new class of ozonide (1,2,4 trioxolanes) antimalarial compounds was investigated. The effects of pH, ionic strength, dielectric constant and cyclodextrin-complexation on the chemical stability and degradation product formation of selected compounds were examined. The mechanism of degradation in aqueous solution was probed using (18)O-labelled water and kinetic solvent isotope effect studies. The effect of stereochemistry was investigated using selected pairs of stereoisomers. The degradation of the ozonides in aqueous solution followed apparent first-order kinetics, with no effect of ionic strength and no indication of any direct involvement of water in the degradation mechanism. All major degradation products were identified and mass balance was confirmed. Stereochemistry had a significant effect on degradation rate; trans isomers degrading approximately four-fold faster than the corresponding cis isomers. The degradation rates were essentially independent of pH above pH 2; however, an additional specific acid catalysed pathway was dominant below pH 2. Solvent dielectric constant had a significant effect on the degradation rate. It is proposed that the degradation observed in aqueous solution occurred through a concerted heterolytic scission of the central ozonide ring, with chemical substituents on the cyclohexyl ring having only a minor influence on degradation rate.

The Hydrolysis of Diclofenac Esters: Synthetic Prodrug Building Blocks for Biodegradable Drug-Polymer Conjugates.

Degradation reactions on diclofenac-monoglycerides (3a,b), diclofenac-(p-hydroxybenzoate)-2-monoglyceride (3c), diclofenac (1), and diclofenac lactam (4) were performed at 37 °C in isotonic buffer solutions (apparent pH range 1-8) containing varying concentrations of acetonitrile (ACN). The concentration remaining of each analyte was measured versus time. Diclofenac-monoglycerides and diclofenac-(p-hydroxybenzoate)-2-monoglyceride (3c) were both found to undergo facile and complete hydrolysis in pH 7.4 isotonic phosphate buffer/10% ACN. Under mildly acidic, neutral or alkaline conditions, diclofenac-(p-hydroxybenzoate)-2-monoglyceride (3c) had the fastest hydrolysis rate (t1/2 = 3.23 h at pH 7.4), with simultaneous formation of diclofenac lactam (4) and diclofenac (1). Diclofenac-monoglycerides (3a,b) hydrolyzed more slowly under the same conditions, to again yield both diclofenac (1) and diclofenac lactam (4). There was also transesterification of diclofenac-2-monoglyceride (3b) to its regioisomer, diclofenac-1-monoglyceride (3a) across the pH range. Diclofenac was shown to be stable in neutral or alkaline conditions but cyclized to form the lactam (4) in acidic conditions. Conversely, the lactam (4) was stable under acidic conditions but was converted to an unknown species under alkaline or neutral conditions.

Kinetics of iron-mediated artemisinin degradation: effect of solvent composition and iron salt.

The antimalarial endoperoxides, such as artemisinin, are postulated to exert their potent parasiticidal activity via the formation of reactive intermediates in the iron-rich infected erythrocyte. The in vitro chemical reaction profile of putative endoperoxide antimalarials and ferrous iron is often qualitatively used to assess their potential antimalarial activity and to develop a structure-reactivity relationship. This study utilized LCMS to monitor the kinetics of artemisinin degradation and product formation in the presence of iron. A second order degradation reaction (k = 18 M(-1) h(-1)) was observed from the reaction of artemisinin with ferrous sulphate in aqueous acetonitrile to produce a number of stable isomeric rearrangement products. A systematic study of the effect of a number of solvent systems and different iron salts showed pronounced changes in reaction rate and product distribution. The significant effects observed in the current study highlight the need to carefully control reaction conditions when studying peroxide antimalarial stability or attempting to develop in vitro/in vivo correlations of endoperoxide antimalarials and their reactivity with iron.

Interaction of model aryl- and alkyl-boronic acids and 1,2-diols in aqueous solution.

The goal of this work was to quantitate ester formation between alkyl and aryl boronic acids and vicinal-diols or 1,2-diols in aqueous solution. As used here, 1,2-diols includes polyols with one or more 1,2-diol pairs. Multiple techniques were used including apparent pKa shifts of the boronic acids using UV spectrophotometry (for aryl acids) and titration (for aryl and alkyl acids). Isothermal microcalorimetry was also used, with all reactions being enthalpically favored. For all the acids and 1,2-diols and the conditions studied, evidence only supported 1:1 ester formation. All the esters formed were found to be significantly more acidic, as Lewis acids, by 3-3.5 pKa units than the corresponding nonesterified boronic acid. The equilibrium constants for ester formation increased with increasing number of 1,2-diol pairs but stereochemistry may also play a role as sorbitol with five possible 1,2-diol pairs and five isomers (taking into account the stereochemistry of the alcohol groups) was twice as efficient at ester formation compared with mannitol, also with five possible 1,2-diol pairs but only three isomers. Alkyl boronic acids formed esters to a greater extent than aryl acids. Although some quantitative differences were seen between the various techniques used, rank ordering of the structure/reactivity was consistent. Formulation implications of ester formation between boronic acids and 1,2-diols are discussed.

Evaluation of the impact of sulfobutylether7 -ß-cyclodextrin on the liquid chromatography-mass spectrometry analysis of biological samples arising from in vivo pharmacokinetic studies.

The utility of cyclodextrin (CD) complexation in improving apparent solubility of drugs in parenteral formulations is well established. Administration of these formulations delivers CD directly into the systemic circulation, and it may be necessary to demonstrate unaltered in vivo disposition of a drug coadministered with a CD. Crucial to the undertaking of such a study is the need for bioanalytical assays in which CD presence does not impact drug quantitation. This is of particular importance when assessing the potential impact of in vivo CD complexation on the urinary excretion of a drug, as CDs are predominantly eliminated via glomerular filtration, and hence are present in urine at significantly higher concentration than would be present in blood and plasma. Of 23 publications (in the past 30 years) describing preclinical and clinical assessment of drug pharmacokinetics after i.v. administration of CD-enabled formulations, only two reports clearly stated that the presence of CD had no impact on assay performance. In this work, we describe the simple process involved in (1) predicting the maximum concentrations of a modified CD, sulfobutylether7 -ß-CD (SBE7 -ß-CD), in plasma and urine samples from preclinical studies, and (2) evaluating the impact of SBE7 -ß-CD on the quantitative liquid chromatography-mass spectrometry analysis of rimantadine.

The effect of intravenous sulfobutylether7 -ß-cyclodextrin on the pharmacokinetics of a series of adamantane-containing compounds.

Intravenously administered (i.v.) drug-cyclodextrin (CD) inclusion complexes are generally expected to dissociate rapidly and completely, such that the i.v. pharmacokinetic profile of a drug is unchanged in the presence of CD. The altered pharmacokinetics of a synthetic ozonide in rats has been attributed to an unusually high-binding affinity (2.3 × 10(6) M(-1) ) between the drug and sulfobutylether7 -ß-cyclodextrin (SBE7 -ß-CD) with further studies suggesting a significant binding contribution from the adamantane ring. This work investigated the binding affinity of three adamantane derivatives [amantadine (AMA), memantine (MEM) and rimantadine (RIM)] to SBE7 -ß-CD and the impact of complexation on their i.v. pharmacokinetics. In vitro studies defined the plasma protein binding, as well as the impact of SBE7 -ß-CD on erythrocyte partitioning of each compound. SBE7 -ß-CD binding constants for the compounds were within the typical range for drug-like molecules (10(2) -10(4) M(-1) ). The pharmacokinetics of AMA and MEM were unchanged; however, significant alteration of RIM plasma and urinary pharmacokinetics was observed when formulated with CD. In vitro studies suggested two factors contributing to the altered pharmacokinetics: (1) low plasma protein binding of RIM, and (2) decreased erythrocyte partitioning in the presence of high SBE7 -ß-CD concentrations. This work demonstrated the potential for typical drug-cyclodextrin interactions to alter drug plasma pharmacokinetics.

Partitioning of halofantrine hydrochloride between water, micellar solutions, and soybean oil: Effects on its apparent ionization constant.

Recent studies in a conscious dog model demonstrated intestinal lymphatic transport to be a significant contributor to the bioavailability of the highly lipid-soluble free-base of halofantrine (Hf), and surprisingly, also the poorly lipid-soluble hydrochloride salt (Hf. HCl). Partial conversion of solubilized Hf. HCl to Hf base within the intestinal lumen prior to the lymphatic uptake seemed to be the most likely explanation for these results. This hypothesis was supported by studies exploring the partitioning behavior of Hf. HCl between soybean oil (SBO) and aqueous micellar solutions containing different ionic and nonionic surfactants. Mixed micelles prepared from sodium taurodeoxycholate (NaTC) and lecithin (PC) were chosen to represent fed-state intestinal fluids. The apparent ionization constants derived from the partitioning versus pH profiles showed marked shifts when compared with the likely aqueous pK(a) value. In the present paper, the apparent pK(a) values of Hf in aqueous micellar phases, without a coexisting oil phase, were investigated to further probe the mechanisms underlying the effect of micellar media on the apparent ionization equilibrium, and subsequently, on its partitioning behavior in the triphasic systems. Another aim of this study was to further evaluate the aqueous pK(a) value of Hf. The results indicate that the aqueous pK(a) of Hf is most probably in the range approximately 8-9, and that the ionization equilibrium is highly dependent on the solution environment. For example, marked pK(a) shifts of several units were observed for Hf in the presence of different micellar species and SBO. The apparent ionization equilibrium depends not only on interaction of Hf with the micelles, but also on its partitioning into the oil phase.