Characterization, bioactivity and pharmacokinetic study of a novel carbohydrate-peptide polymer: Glycol-split heparin-endostatin2 (GSHP-ES2).

Endostatin (ES) has attracted considerable attention for the treatment of anti-angiogenesis-related disorders. An 11-amino-acid peptide (ES2, IVRRADRAAVP) from the amino terminal of ES is of interest because it is the main active fragment of ES. However, both ES and ES2 have a poor stability and a short half-life, and other disadvantages need to be further resolved. Thus, we conjugated ES2 to glycol-split heparin derivatives (GSHPs) to yield the polymer-peptide conjugate, GSHP-ES2. This study showed that GSHP-ES2 exhibited increased stability, a wider pH activity range, better inhibition of endothelial cell proliferation, migration and tube formation in vitro, better anti-angiogenic activity and a longer half-life in vivo compared with ES2. These results also indicate that GSHP-ES2 has good potential for the treatment of angiogenesis-related diseases, either alone or in combination with other chemicals.

Tunable Nanocarrier Morphologies from Glycopolypeptide-Based Amphiphilic Biocompatible Star Copolymers and Their Carbohydrate Specific Intracellular Delivery.

Nanocarriers with carbohydrates on the surface represent a very interesting class of drug-delivery vehicles because carbohydrates are involved in biomolecular recognition events in vivo. We have synthesized biocompatible miktoarm star copolymers comprising glycopolypeptide and poly(ε-caprolactone) chains using ring-opening polymerization and "click chemistry". The amphiphilic copolymers were self-assembled in water into morphologies such as nanorods, polymersomes, and micelles with carbohydrates displayed on the surface. We demonstrate that the formation of nanostructure could be tuned by chain length of the blocks and was not affected by the type of sugar residue. These nanostructures were characterized in detail using a variety of techniques such as TEM, AFM, cryogenic electron microscopy, spectrally resolved fluorescence imaging, and dye encapsulation techniques. We show that it is possible to sequester both hydrophobic as well as hydrophilic dyes within the nanostructures. Finally, we show that these noncytotoxic mannosylated rods and polymersomes were selectively and efficiently taken up by MDA-MB-231 breast cancer cells, demonstrating their potential as nanocarriers for drug delivery.

Application of Polymeric Nanoparticles for CNS Targeted Zinc Delivery In Vivo.

A dyshomeostasis of zinc ions has been reported for many psychiatric and neurodegenerative disorders including schizophrenia, attention deficit hyperactivity disorder, depression, autism, Parkinson's and Alzheimer's disease. Furthermore, alterations in zinc-levels have been associated with seizures and traumatic brain injury. Thus, altering zinclevels within the brain is emerging as a new target for the prevention and treatment of psychiatric and neurological diseases. However, given the restriction of zinc uptake into the brain by the blood-brain barrier, methods for controlled regulation and manipulation of zinc concentrations within the brain are rare. Here, we performed in vivo studies investigating the possibility of brain targeted zinc delivery using zinc-loaded nanoparticles which are able to cross the blood-brain barrier. After injecting these nanoparticles, we analyzed the regional and time-dependent distribution of zinc and nanoparticles within the brain. Moreover, we evaluated whether the presence of zinc-loaded nanoparticles alters the expression of zinc sensitive genes and proteins such as metallothioneins and zinc transporters and quantified possible toxic effects. Our results show that zinc loaded g7 nanoparticles offer a promising approach as a novel non - invasive method to selectively enrich zinc in the brain within a small amount of time.

Intraperitoneal Administration of Muramyl Dipeptide ß-Heptylglycoside to Pregnant and Non-Pregnant Female Mice Modulates Production of Th1/Th2/Th17/Tr1 Cytokines by Splenocytes Ex Vivo.

Muramyl dipeptide ß-heptylglycoside (C7MDP) was administered to non-pregnant CBA female mice and pregnant mice after non-abortion-prone mating (CBA×BALB/c) and mating associated with a high rate of spontaneous abortion (CBA×DBA/2). In non-pregnant females, C7MDP increased the production of IL-2, IL-4, IL-5, IL-6, IL-17, IFNγ, TNFα, and GM-CSF at constant production of IL-1α and IL-10. C7MDP increased the production of IL-10 and IL-17 and suppressed the production of IFNγ on day 8 of gestation in non-abortion-prone mouse couples and stimulated the synthesis of IL-4 and IFNγ, reduced IL-5 production, and slightly increased IL-1α secretion after abortion-prone mating. On day 14 of gestation, C7MDP elevated the yield of IL-2, IL-4, IFNγ, TNFα, and GM-CSF in CBA×BALB/c and CBA×DBA/2 couples and IL-17 in the fi rst variant of mating.

Synthesis and molecular modelling studies of novel carbapeptide analogs for inhibition of HIV-1 protease.

Novel glycopeptides containing amino acids such as valine and alanine were designed, synthesized and tested for inhibition of the wild type C-SA HIV-1 protease enzyme. The incorporation of dipeptide sequences Val-Ala/Ala-Val to the sugar B-amino acid at two side chain positions resulted in a series of nine novel compounds. Compounds 3a, 3b, 3c, 3d, 4a, 4b and 5 displayed significant activities against the HIV protease enzyme. The glycopeptides are orders of magnitude less toxic to human MT-4 cells than lopinavir. Computational results were in good agreement with the experimental HIV-PR activity. The sugar hydroxyl group at the C(3) position interacts with the enzymatic Asp25/Asp25' residues. The docked position of the inhibitor is preserved during MD simulations and at least five hydrogen bond forms between the inhibitor and the enzymatic pocket. The results provide a platform for the progress of more effective carbohydrate supported inhibitors of HIV-1 and other aspartic proteases.

The role of neutral endopeptidase in caerulein-induced acute pancreatitis.

Substance P (SP) is well known to promote inflammation in acute pancreatitis (AP) by interacting with neurokinin-1 receptor. However, mechanisms that terminate SP-mediated responses are unclear. Neutral endopeptidase (NEP) is a cell-surface enzyme that degrades SP in the extracellular fluid. In this study, we examined the expression and the role of NEP in caerulein-induced AP. Male BALB/c mice (20-25 g) subjected to 3-10 hourly injections of caerulein (50 µg/kg) exhibited reduced NEP activity and protein expression in the pancreas and lungs. Additionally, caerulein (10(-7) M) also downregulated NEP activity and mRNA expression in isolated pancreatic acinar cells. The role of NEP in AP was examined in two opposite ways: inhibition of NEP (phosphoramidon [5 mg/kg] or thiorphan [10 mg/kg]) followed by 6 hourly caerulein injections) or supplementation with exogenous NEP (10 hourly caerulein injections, treatment of recombinant mouse NEP [1 mg/kg] during second caerulein injection). Inhibition of NEP raised SP levels and exacerbated inflammatory conditions in mice. Meanwhile, the severity of AP, determined by histological examination, tissue water content, myeloperoxidase activity, and plasma amylase activity, was markedly better in mice that received exogenous NEP treatment. Our results suggest that NEP is anti-inflammatory in caerulein-induced AP. Acute inhibition of NEP contributes to increased SP levels in caerulein-induced AP, which leads to augmented inflammatory responses in the pancreas and associated lung injury.

Dependence of DNA sequence selectivity and cell cytotoxicity on azinomycin A and B epoxyamide stereochemistry.

Evaluation of the importance of C18/C19 stereochemistry of azinomycin A/B epoxyamide partial structures with respect to DNA alkylation sequence selectivity is reported using a unique assay with a DNA oligomer containing imbedded normal (5'-GGC-3'/3'-CCG-5') and inverted (5'-CGG-3'/3'-GCC-5') azinomycin consensus cross-linking sequences. Both species were found to have unique selectivity profiles and alkylate DNA in a manner distinct from azinomycin B. Computational docking experiments support altered binding modes for the enantiomers.

Novel histidine-conjugated galactosylated cationic liposomes for efficient hepatocyte-selective gene transfer in human hepatoma HepG2 cells.

To enhance gene transfection to hepatocytes by cationic liposomes, it is necessary to overcome a number of barriers existing in the process from administration to gene expression. Recently we and other group have demonstrated that the escape of plasmid DNA (pDNA)/cationic liposome complexes (lipoplexes) from the endosome to cytoplasm was rate limiting. In this study, to enhance transfection efficiency by promoting the release of lipoplexes from the endosome to cytoplasm, we proposed utilizing the "proton sponge effect". Here, we synthesized a novel pH-sensitive histidine-modified galactosylated cholesterol derivative (Gal-His-C4-Chol), for a more efficient gene delivery to hepatocytes. Liposomes containing Gal-His-C4-Chol showed much greater transfection activity than conventional Gal-C4-Chol liposomes based on a receptor-mediated mechanism in HepG2 cells. Hence, this finding should contribute to the development of gene therapy using cationic liposomes toward their clinical application.

Audiologic monitoring for potential ototoxicity in a phase I clinical trial of a new glycopeptide antibiotic.

This study describes audiologic methodology and results for evaluating potential ototoxicity in a phase I clinical trial of a new glycopeptide. This study was conducted under good clinical practices, which are regulated by the US Food and Drug Administration (FDA) (21 Code of Federal Regulations), and input from the FDA was sought prior to study implementation. Healthy, normal volunteers underwent extensive medical and audiologic assessments as part of this phase I dose- escalation study of dalbavancin, a new glycopeptide, to assess potential side effects. Audiologic monitoring included air-conduction thresholds in the conventional (0.25-8 kHz) and high-frequency (10-16 kHz) ranges. At baseline, subjects were also tested using word recognition, bone conduction testing if indicated, and tympanometry. Full testing was to be repeated if any subject met the American Speech-Language-Hearing Association (ASHA) 1994 criteria for ototoxic change. However, no subjects demonstrated ototoxic change after receiving dalbavancin, nor were any false-positive results obtained.

Toxicological profile of a low molecular weight spleen peptide formulation used in supportive cancer therapy.

The toxicity of Polyerga (GP-1), a mixture of low molecular weight spleen peptides, extracted from porcine spleen was examined in in vitro and in vivo experiments. The following endpoints were examined: single dose toxicity, repeated dose toxicity, reproduction toxicity, mutagenic potential, local tolerance, immunotoxicity and general pharmacology. The standard therapeutic dose is 3 x 1 ml GP-1/week. The dosage can be increased temporarily to 2 ml/d. 1 ml injection solution contains 30 micrograms oligopeptides. The LD50 was determined in rats as 3.76 ml/kg b.w. i.v. Following repeated intramuscular administration of GP-1 no toxicity appeared in rats up to a dose level of 2 ml/kg b.w./d during the 13-week treatment period. In dogs the no-effect level of GP-1 was 0.32 ml/kg b.w./d i.m. (marginal body weight reduction at 0.80 ml/kg b.w./d i.m.). In an embryotoxicity study in rats, GP-1 possessed no teratogenic properties tested at dose levels exceeding the human therapeutic dose by a factor of 1000. No mutagenic potential was observed for GP-1 in the AMES test. No local irritations were observed at the intended injection sites and at injection sites made in error. None of the in vitro and in vivo pharmacological studies revealed any effect on the parameters tested, at doses up to 500 times the clinical dose. Hence, under the present test conditions, based on a daily therapeutic dose of 1 ml GP-1/patient (approximately 0.014 ml/kg b.w./d), the therapeutic ratio is at least 50 for the most sensitive endpoint 'repeated dose toxicity' reflecting the wide margin of safety for GP-1.

Epithelial autotoxicity of nitric oxide: role in the respiratory cytopathology of pertussis.

Bordetella pertussis releases a specific peptidoglycan fragment known as tracheal cytotoxin (TCT) that reproduces the respiratory epithelial cytopathology of whooping cough (pertussis). In vitro, TCT inhibits DNA synthesis in hamster trachea epithelial cells and causes specific destruction of ciliated cells in explants of human and hamster respiratory epithelium. We have recently demonstrated that TCT triggers production of intracellular interleukin 1 by respiratory epithelial cells, and this cytokine may act as an intermediate signal in the generation of TCT toxicity. Here we report the identification of a subsequent critical step in this pathway: induction of nitric oxide synthesis in the respiratory epithelium. The toxic effects of nitric oxide are consistent with spectroscopic evidence of the formation of iron-dinitrosyl-dithiolate complexes in TCT-treated cells. Aconitase, with its iron-sulfur center, is one expected target of nitric oxide, and TCT inhibited 80% of the activity of this enzyme in respiratory epithelial cells. The deleterious effects of TCT and interleukin 1 were dramatically attenuated by the nitric oxide synthase inhibitors NG-monomethyl-L-arginine and aminoguanidine. These results indicate that nitric oxide mediates the toxicity of TCT for the respiratory epithelium, thus implicating a central role for nitric oxide in the pathogenesis of pertussis.

A structure-activity relationship for induction of meningeal inflammation by muramyl peptides.

Components of bacterial peptidoglycans have potent biological activities, including adjuvant effects, cytotoxicity, and induction of sleep. Mixtures of peptidoglycan components also induce inflammation in the lung, subarachnoid space, and joint, but the structural requirements for activity are unknown. Using a rabbit model for meningitis, we determined the biological activities of 14 individual muramyl peptides constituting > 90% of the peptidoglycan of the gram-negative pediatric pathogen Haemophilus influenzae. Upon intracisternal inoculation, most of the muropeptides induced leukocytosis in cerebrospinal fluid (CSF), influx of protein into CSF, or brain edema, alone or in combination. The disaccharide-tetrapeptide, the major component of all gram-negative peptidoglycans, induced CSF leukocytosis and protein influx at doses as low as 0.4 microgram (0.42 nM). Modification of the N-acetyl muramic acid or substitution of the alanine at position four in the peptide side chain decreased leukocytosis but enhanced brain edema. As the size of the muropeptide increased, the inflammatory activity decreased. Muropeptide carrying the diaminopimelyl-diaminopimelic acid cross-link specifically induced cytotoxic brain edema. These findings significantly expand the spectrum of biological activities of natural muramyl peptides and provide the basis for a structure-activity relationship for the inflammatory properties of bacterial muropeptides.

[The anticoagulant action of a factor from the nondialyzed fraction of the ammoniacal extract of the lungwort, Pulmonaria mollissima]. / Protivosvertyvaiushchee deistvie antikoagulianta nedializuemoi fraktsii ammiachnogo ékstrakta iz travy medunitsy miagchaishei.

It has been established that a non-dialyzed fraction of ammoniacal extract from P. molissima contains an anticoagulant-glycopeptide. In in vivo experiments, it produces stable hypocoagulemia, primarily blocking fibrin self-assemblage, and prevents the animal's death from thromboplastinemia. It has been found that the aforesaid action does not exert any adverse effect on the blood contents, arterial pressure, respiration or urinary renal function.

[A comparative study of the immunological properties of peptidoglycans of different origins]. / Sravnitel'noe izuchenie immunologicheskikh svoistv peptidoglikanov razlichnogo proiskhozhdeniia.

The action of peptidoglycans (PG) of different origin has been experimentally studied in vivo. In these experiments PG of bacterial origin, such as blastolysin (BL), and synthetic PG, viz. muramyldipeptide (MDP) and its analog glucosaminylmuramyldipeptide (GMDP) have been used. Their toxicity, allergenic action, their effect on the phagocytic activity of peritoneal exudate macrophages (PEM), the accumulation of antibody-producing cells in the spleen, antibody titer in the blood serum and delayed hypersensitivity to nonbacterial antigens have been determined. As revealed in this study, BL does not differ from MDP in its toxicity and allergenic action. The phagocytic activity of PEM under the influence of BL only insignificantly differs from their activity under the influence of MDP, but is lower than under the influence of GMDP. The adjuvant action of BL is somewhat higher than that of synthetic PG.

A comparative efficacy and safety study of teicoplanin plus aztreonam versus gentamicin plus piperacillin in haematology oncology patients with clinically diagnosed septicaemia.

Infections due to Gram-positive bacteria, especially coagulase-negative staphylococci, have been increasing in immunocompromised patients during the last 5 years because of an increased use of Hickman catheters and oral gut decontamination with quinolones. Teicoplanin, a new glycopeptide antibiotic, has a long plasma half-life which allows once-a-day bolus administration, making it a 'user friendly' agent. A randomized comparative evaluation of teicoplanin plus aztreonam versus gentamicin plus piperacillin in leukaemic patients with a clinical diagnosis of septicaemia was undertaken. The objectives of this study were (1) to evaluate the efficacy and safety of teicoplanin and aztreonam in comparison to a 'standard antibiotic' regimen and (2) to assess the local and systemic tolerance of these drugs. Results of the study in more than 70 patients to date are presented, and the role of anti-Gram-positive antibiotics in the management of severe sepsis in immunocompromised patients is discussed.

[Experimental study on chemotherapeutic efficacy, acute toxic action and nephrotoxicity of combinations of eremomycin with tobramycin]. / Eksperimental'noe izuchenie khimioterapevticheskoi éffektivnosti, ostrogo toksicheskogo deistviia i nefrotoksichnosti kombinatsii éremomitsina s tobramitsinom.

Chemotherapeutic efficacy of eremomycin in combination with tobramycin was investigated on a model of experimental sepsis of albino mice caused by Staphylococcus aureus cultures resistant to methicillin. Eremomycin is a novel original antibiotic of the glycopeptide structure isolated in the USSR and tobramycin is an aminoglycoside. Acute toxicity of the combination with a wide range of the dose fixed proportions was studied on mice and the nephrotoxic action of the antibiotics and their combinations administered intravenously for 5 days was studied on albino rats. The experiments showed that the chemotherapeutic effect of eremomycin in combination with tobramycin was of synergistic nature. Acute toxicity of the combined drugs mainly summed up and somewhat increased when the proportion of tobramycin and eremomycin was 1:2.4 or 1:3.6. Eremomycin had a dose-depended nephrotoxicity. Summing up of the nephrotoxic action of the drugs on their combined use was observed.

[Preclinical toxicological study of the new antibiotic eremomycin. Chronic toxicity and effect on intrauterine development of rats]. / Doklinicheskoe toksikologicheskoe izuchenie novogo antibiotika éremomitsina. Khronicheskaia toksichnost', vliianie na vnutriutrobnoe razvitie krys.

Toxicity of eremomycin was studied after its multiple parenteral administration to albino rats, guinea pigs and dogs in doses equivalent by the body surface to the daily doses for humans i. e. 1 and 3 g. The antibiotic was administered for 1 to 6 months. Tolerance of the antibiotic by the dogs after intravenous and intramuscular administration was satisfactory. In some animals there were observed an insignificant increase in the activity of alanine aminotransferase and a rise in the level of urea in blood serum. Pathomorphological examination of the internal organs of the albino rats and dogs showed that in high doses the antibiotic could have a damaging effect on the kidneys and epithelium of the gastrointestinal tract. The level of the damages depended on the dose of the antibiotic and duration of its use. The damages induced by eremomycin were reversible. It had no marked effect on the peripheral blood count, coagulation system and erythrocyte resistance. In the tested doses the antibiotic had no unfavourable effect on the hearing function in the experiments with guinea pigs. Studies with rats revealed that eremomycin had no teratogenic effect. A slightly pronounced embryotoxic action was observed only after using the antibiotic in doses exceeding more than 12 times the approximate therapeutic dose.

The intraocular penetration and retinal toxicity of teicoplanin.

We investigated the intraocular penetration and retinal toxicity of teicoplanin, a relatively new glycopeptide antibiotic with activity similar to vancomycin when used to inhibit staphylococci and other gram positive organisms, particularly Streptococcus faecalia. Topically administered teicoplanin penetrated poorly into the aqueous and vitreous in rabbit eyes. Subconjunctival injection of the drug yielded aqueous levels above the minimum inhibiting concentration (3.1 micrograms/ml) only at one hour after injection. In the vitreous, drug levels were above the mean inhibitory concentration at 30 minutes after the subconjunctival injection, but rapidly declined thereafter. The maximum nontoxic, single-dose, intravitreal injection was 750 micrograms/0.1 ml. Rabbits received 8 micrograms/ml of teicoplanin in an intravitreal infusion solution without demonstrable retinal toxicity.

[Preclinical toxicological study of the new antibiotic eremomycin. Its acute toxicity for laboratory animals]. / Doklinicheskoe toksikologicheskoe izuchenie novogo antibiotika éremomitsina. Ostraia toksichnost' dlia laboratornykh zhivotnykh.

Eremomycin is relatively low toxic. LD50 of eremomycin on its intravenous administration to albino mice amounted to 1760 (1460-2130) mg/kg. It is 2.6, 3.5 and 6 times less toxic than ristomycin, vancomycin and teicoplanin, respectively. The rate of intravenous administration had no significant effect on eremomycin toxicity. Sensitivity of adult and preadolescent mice to eremomycin was almost the same. Eremomycin toxicity for male mice was somewhat higher than that for female mice. The use of 5 per cent glucose solution instead of distilled water as a solvent lowered 1.3-fold the toxicity of eremomycin in albino mice when it was administered intravenously. The toxic effect of eremomycin on the renal function played a significant role in the mechanism of the animal death due to the antibiotic. In experiments with guinea pigs eremomycin showed no allergenic effect. Unlike the other representatives of glycopeptide antibiotics, eremomycin had practically no local irritating effect which provided its recommendation for clinical trials not only as an intravenous but also intramuscular antibiotic.

Teicoplanin: renal tolerance and pharmacokinetics in rats.

The nephrotoxicity and pharmacokinetics of teicoplanin were studied in an experimental rat model. Nephrotoxicity was assessed by measuring urinary excretion of tubular cells and malate dehydrogenase. The experiments revealed that 1 mg/kg or more of teicoplanin daily resulted in an increase of excretion rates of tubular cells. Similarly to vancomycin, teicoplanin is accumulated in the kidneys. Nephrotoxicity induced by teicoplanin can be reduced by coadministration of fosfomycin and D-glucaro-1.5-lactam, and enhanced by administration of tobramycin. We conclude that renal function should be monitored closely during teicoplanin therapy.