KINETIC STUDIES OF THE DEGRADATION OF OXYCARBONYLOXYMETHYL PRODRUG OF ADEFOVIR AND TENOFOVIR IN SOLUTION

The decomposition kinetics of bis-POC PMEA and bis-POC PMPA followed pseudo-first order kinetics with the corresponding mono-POC ester detected as the only observable degradation product for all the pH values studied.The rates of hydrolysis of bis-POC PMEA over the pH range studied was described by $$ The ¹⁸O incorporation studies revealed that hydrolysis of bis-POC PMEA at pH 7.0 primarily proceeds via P-O cleavage with an additional minor pathway involving C?O bond cleavage. Hydrolysis of bis-POC PMPA was found to be about 2 fold slower than bis-POC PMEA at pH values above 6.0.     

Acyclic phosphonate nucleotides and human adenylate kinases: impact of a borano group on alpha-P position

Adenylate kinases are involved in the activation of antiviral drugs such as the acyclic phosphonates analogs PMEA and (R)PMPA. We examine the in vitro phosphorylation of PMEA and PMPA bearing a borano- or a H- group on the phosphorus atom. The alpha-borano or alpha-H on PMEA and PMPA were detrimental to the activity of recombinant human AMP kinases 1 and 2. Docking PMEA to the active site of AMP kinase 1 indicated that the borano group may prevent two conserved critical Arg interactions with the alpha-phosphate, resulting in substrate bad positioning.     

Some new acyclic nucleotide analogues as antiviral prodrugs: synthesis and bioactivities in vitro

A series of ester analogues of acyclic nucleotide PMPA and PMEA were synthesized as potent antiviral agents. The antiviral evaluation results indicated that bis benzyl ester prodrug of PMPA 5f and bis allyl ester prodrug of PMEA 5g exhibited potent antiviral activities. The IC(50) of 5f for HBV was 2.15 microM, and the IC(50) of 5g for HIV-1 was 1.61 microM.     

Hydrolysis kinetics of trisaccharides consisting of glucose, galactose, and fructose residues in subcritical water

The hydrolysis kinetics of trisaccharides consisting of glucose, galactose, and fructose residues with different glycosidic bonds, 1-kestose, d-melezitose, d-raffinose, and lactosucrose, in subcritical water were conducted over the temperature range of 150-230 degrees C and at a constant pressure of 10 MPa. The hydrolysis of trisaccharides in subcritical water proceeded consecutively, i.e., one cleavage of the two bonds antedated the other. The preceding cleavage was not expressed by the first-order kinetics, but by the kinetics considering the concentration of the acidic compounds, which were produced by the degradation of the constituent monosaccharides. The hydrolysis of the constituent disaccharides, except sucrose composed of the alpha-Glc-(1-->2)-beta-Fru bond, obeyed first-order kinetics. All of the rate constants of the hydrolytic kinetics were determined, and the values were found to depend on the type of bond.     

Antiinflammatory effects of phosphonomethoxyethyl analogues of adenine in a model of adjuvant arthritis.

The 9-(2-phosphonomethoxyethyl)adenine (PMEA) and its more bioavailable bis(pivaloyloxymethyl) ester, (bis-POM-PMEA), applied s.c. at doses of 5-50 mg/kg, profoundly suppress symptoms of rat adjuvant arthritis, such as paw swelling, sple-nomegaly, fibroadhesive perisplenitis and systemic NO levels. The 9-(2-phosphonomethoxypropyl)adenine, PMPA and bis-POC-PMPA are ineffective. The antiarthritic effect does not depend on the influence of the drugs on macrophage NO production.     

Synthesis and evaluation of novel amidate prodrugs of PMEA and PMPA

Some novel amidate prodrugs of PMEA and PMPA have been synthesised and tested in vitro for their biological activity. Compound 5 in particular showed greatly enhanced antiviral potency compared with the parent nucleotide analogue. In vitro enzymatic studies and structure-activity relationships indicate that the degradation mechanism of such prodrugs may be the same as that described for the phosphoramidate triesters of nucleotide analogues.     

HPLC method for determination of in vitro delivery through and into porcine skin of adefovir (PMEA)

A simple HPLC/UV method for the determination of the transdermal permeation and dermal penetration of a broad-spectrum antiviral drug adefovir (PMEA) was developed. The separation was achieved on a C18 column with the mobile phase composed of 10 mM KH2PO4 and 2 mM Bu4NHSO4 at pH 6.0 and 7% acetonitrile. The method was validated with respect to selectivity, linearity (0.1-50 microg/ml), precision, accuracy, and stability. Transdermal permeation of 2% PMEA was studied in vitro using the Franz diffusion cell and porcine skin. The flux values were 1.8, 3.0, and 0.6 microg/cm2/h from aqueous donor samples at pH 3.4 and 7.4, and isopropyl myristate, respectively. The respective skin concentrations at 48 h were 294, 263, and 971 microg/g from these vehicles. These results will serve as a lead for further studies on transdermal and topical delivery of antivirals from the group of acyclic nucleoside phosphonates.     

Anti-inflammatory Effects of Phosphonomethoxyethyl Analogues of Adenine in a Model of Adjuvant Arthritis

Abstract

The 9-(2-phosphonomethoxyethyl)adenine (PMEA) and its more bioavailable bis(pivaloyloxymethyl) ester, (bis-POM-PMEA), applied s.c. at doses of 5–50 mg/kg, profoundly suppress symptoms of rat adjuvant arthritis, such as paw swelling, sple-nomegaly, fibroadhesive perisplenitis and systemic NO levels. The 9-(2-phosphono-methoxypropyl)adenine, PMPA and bis-POC-PMPA are ineffective. The antiarthritic effect does not depend on the influence of the drugs on macrophage NO production.     

Raman spectroscopy study of acid-base and structural properties of 9-[2-(phosphonomethoxy)ethyl]adenine in aqueous solutions

The acid-base properties of the acyclic antiviral nucleotide analogue 9- [2-(phosphonomethoxy)ethyl] adenine (PMEA) in aqueous solutions are studied by means of Raman spectroscopy in a pH range of 1-11 and compared with the properties of its common adenosine monophosphate counterparts (5'-AMP, 3'-AMP, and 2'-AMP). Factor analysis is used to separate the spectra of pure ionic species (PMEA2-, HPMEA-, H2PMEA, H3PMEA+) in order to determine their abundance, sites of protonation, and corresponding spectroscopic pK(a) values. The characteristic Raman features of the neutral adenine moiety in PMEA2- and HPMEA- species resemble those of neutral adenine in the AMPs, whereas significant differences are observed between the Raman spectra of the N1-protonated adenine of the solute zwitterionic H2PMEA and its N1-protonated AMP counterparts. On the contrary, the spectrum of crystalline H2PMEA, adopting an "anti-like" conformation, is found to be similar to the N1-protonated AMPs in solution. To explain peculiar Raman features a "syn-like" conformation is suggested for N1-protonated PMEA species in aqueous solutions instead of an anti-like one adopted by H2PMEA in crystals or by common AMPs in aqueous solutions. A physical mechanism of the anti-like to syn-like conformational transition of the solute PMEA that is due to adenine protonation and the flexibility of the (phosphonomethoxy)ethyl group is proposed and discussed.     

Dependence of pepsin conformational states on pH and temperature]

A conformational behaviour of pepsin depending on pH and temperature was studied by circular dichroism, differential UV-spectroscopy, calorimetry and enzymatic hydrolysis kinetics. A subtile conformational transition of the enzyme accompanied by changes in the physico-chemical and enzymatic properties of the protein was observed within the temperature interval of 15--40 degrees and within the pH range of 1,1--5,6. The range of pepsin heat denaturation was studied. A diagram of pepsin conformational states under different values of pH and temperature was built.     

Hydrolysis of cellobiose using β-glucosidase from Penicillium funiculosum. Kinetic analysis

The kinetics of cellobiose hydrolysis was studied using β-glucosidase from Penicillium funiculosum, both free and immobilized on nylon powder, at different temperatures, pH values, enzymatic activities and initial cellobiose and glucose concentrations. The experimental results were fitted to a kinetic model by considering the substrate and product inhibitions as well as the thermal deactivation of β-glucosidase with a mean deviation of less than 10%. The immobilization of β-glucosidase led to an increase in the stability of the enzyme against changes in the pH value.     

New cycloAmb-nucleoside phosphonate prodrugs

cycloSal- and cycloAmb-nucleoside phosphonate prodrugs of PMEA were synthesized and characterized. Each of these compounds showed different disadvantages in hydrolysis. Thus, a new series of cycloAminobenzyl(cycloAmb)-PMEA prodrugs was synthezised and studied with regard to their hydrolysis properties and biological activity.     

Kinetics of beta-casein hydrolysis by wild-type and engineered trypsin

Apparent rate constants of tryptic hydrolysis of amide bonds containing Arg and Lys residues in beta-casein were determined by the analysis of kinetics of accumulation of 17 major peptide components revealed by high performance liquid chromatography. When studying pH influence on Arg/Lys bond cleavage preference, averaged rate constants over several Arg&bond;X and Lys&bond;X bonds were used for analysis of kinetics of wild-type trypsin, K188H, K188F, K188Y, K188W, and of K188D/D189K mutants. The pK(a1) value of 6.5 was found for all studied trypsins. For wild-type trypsin and its K188D/D189K mutant, pK(a2) was found to be 10. The lowest among studied engineered trypsins pK(a2) = 9.3 was determined for K188Y mutant. Considerable preference for the cleavage of Arg over Lys containing peptide bonds was demonstrated for all trypsins with engineered S2 site except for K188H and K188F. The comparison of individual rate constants for various bonds showed that during the hydrolysis by wild-type trypsin, the probabilities of splitting depend on secondary specificity and local hydrophobicity of amino acid residues, which are nearest to the hydrolyzed peptide bond (P2 site). The improvement of prediction of hydrolysis rates performed by the used program was achieved after considering the presence of hydrophobic neighborhood of Lys48--Ile49 and Arg202--Gly203 bonds.     

Properties of urease immobilized on the functional organic silica surface

The paper deals with kinetics of the urea hydrolysis by microbial-origin urease dissolved and immobilized on the organic silica surface. It is shown that hydrolysis kinetics for soluble urease is described by the Michaelis-Menten equation until the concentration of urea reaches 1 M. Two fractions differing in the Michaelis constant are revealed for silochrome immobilized urease. The rate of urea hydrolysis by native and immobilized urease was studied depending on the pH value in presence of the substrate in the 1 M and 5 mM concentration. The hydrolysis rate of 1 M urea in the buffer-free solution by silochrome-immobilized urease is practically independent of pH within 4.5-6.5. Application of a 2.5 mM phosphate-citrate buffer as a solvent causes an increase in the hydrolysis rate within this pH range. For a soluble urease the 1 M urea hydrolysis rate dependence on pH is ordinary at pH 5.8-6.0. If the substrate concentration is 5 mM, the pH-dependences for the rate of the urea hydrolysis by silochrome- and aerosil-immobilized urease are close and at pH above 6.0 coincide with those for a soluble enzyme. The found differences in the properties of soluble and immobilized ureases are explained by the substrate and reaction products diffusion.     

Phosphodiesterolytic activity of lanthanide (III) complexes with α-amino acids

Kinetics of the hydrolysis of BNPP (bis(4-nitrophenyl)phosphate) mediated by lanthanide - samarium (III) and ytterbium (III) - alone and in the presence of various alfa amino acids has been systematically studied at 37.0 °C and I = 0.15 M in NaClO₄, in the pH interval of 7-9. The rate of BNPP cleavage is sensitive to metal ion concentration, pH, and ligand to metal molar ratio. Hydrolysis follows Michaelis-Menten-type saturation kinetics. For both metals, high pH values markedly increase the observed activity. Besides, potentiometric titrations of all these systems under identical conditions allowed us to identify the active coordination compounds towards hydrolysis. The results show that complexes with phosphodiesterolytic activity are monomeric cationic species such as [Ln(aa)₃(OH)]²⁺ or [Ln(aa)₂(OH)₂]⁺. Since phosphodiesterolytic activity is evident above pH 7 and it is increased with increasing pH, hydrolytic reactions of the metals are competitive processes that could lead to their precipitation as Ln(OH)₃(s). In this sense, ligand excess (for example, ligand to metal molar ratio equal to 30) was employed. Furthermore, due to its more extended hydrolysis, ytterbium shows, in general, less activity than samarium under the studied conditions. In general, a good phosphodiesterolytic activity is observed for these complexes under similar conditions to the physiological ones. Amino acids could be easily derivatized without changing their coordinating ability, leading to lanthanide complexes possibly capable of efficiently hydrolyzing the phosphodiester linkages of nucleic acids.     

Phosphodiesterolytic activity of samarium(III) mixed ligand complexes containing crown ethers and α-amino acids

Phosphodiesterolytic activity of samarium complexes containing crown ethers and amino acids was systematically studied. Formation constants of mixed ligand Sm–crown ethers–amino acids complexes (crown ethers = 18-crown-6, 15-crown-5 and 12-crown-4 and amino acids = Gly and Arg) were determined at 37.0 °C and 0.50 M NMe₄Cl. Kinetics of the hydrolysis of BNPP (bis(4-nitrophenyl)phosphate) mediated by lanthanide(III)-mixed ligands complexes was studied under the same experimental conditions. The rate of BNPP cleavage is sensitive to metal ion concentration, pH, and ligand to metal molar ratio. Hydrolysis follows Michaelis–Menten-type saturation kinetics. High pH values markedly increase the observed activity. Potentiometric titrations results together with kinetic data of all these systems, under identical conditions, allowed us to identify the active species towards hydrolysis. Complexes with phosphodiesterolytic activity are monomeric hydroxylated cationic species. In general, a good phosphodiesterolytic activity is observed for these complexes under similar conditions to the physiological ones.     

Kinetics and time-temperature equivalence of polymer degradation

We studied the hydrolysis kinetics of amorphous polylactide. It was found the hydrolysis rate had a slow-to-fast transition at a certain molecular weight (Mn). This transition was not correlated with the mass loss and water uptake of samples, nor the pH values of testing media. We speculated that this transition was due to the slow diffusion of polymer chain ends. The chain ends did not significantly promote the hydrolysis of samples until their concentrations (approximately 1/Mn) reached a critical value. The degradation tests were also conducted over a temperature range from 37 to 90 degrees C. A time-temperature equivalent relationship of degradation processes was established and a master curve spanning a time range equivalent to 3-5 years at 37 degrees C was constructed. This master curve can be used to predict polymer degradation processes based on accelerated tests. The functional time and disappearance time of degradable polymers were also discussed.     

Phe5(4-nitro)-bradykinin: a chromogenic substrate for assay and kinetics of the metalloendopeptidase meprin

Phe5(4-nitro)-bradykinin has been identified as a good synthetic substrate to study the kinetics and mechanism of action of the metalloendopeptidase meprin. No convenient substrate for kinetic analysis of the enzyme had been previously described. HPLC analyses indicated that meprin cleaved bradykinin and nitrobradykinin between Phe5 (or Phe5(NO2)) and Ser6. Reaction rates for bradykinin were determined by quantitative HPLC analyses, whereas rates for nitrobradykinin were measured by continuous monitoring of the spectral change that occurs at 310 nm when the Phe(NO2)-Ser bond is hydrolyzed. For nitrobradykinin and unmodified bradykinin, respectively, Km values were 281 and 425 microM, kcat values were 28 and 22 s-1, and kcat/Km values were 9.7 x 10(4) and 5.1 x 10(4)M-1. The two products of bradykinin hydrolysis were not substrates for the enzyme, but they were inhibitors. The initial rates of hydrolysis of nitrobradykinin increased linearly with enzyme concentration (0.09-2.2 micrograms/ml), and increased linearly with temperature in the range from 15 to 55 degrees C. Hydrolysis of the substrate was optimal at alkaline pH values. The cysteine endopeptidases papain and cathepsin L and the metalloproteases thermolysin, angiotensin-converting enzyme, and neutral endopeptidase (EC 3.4.24.11) also cleaved nitrobradykinin, but at different peptide bonds than meprin. The single cleavage of nitrobradykinin at the Phe(NO2)-Ser bond and the concomitant spectral shift that occurs at alkaline pH makes this a particularly suitable substrate for meprin.