Kinetics of solvolysis and muscarinic actions of an N-methyl-N-(2-bromoethyl)amino analogue of oxotremorine

An N-methyl-N-(2-bromoethyl)amino analogue (2) of oxotremorine cyclized in phosphate buffer to an aziridinium ion (3). The first-order rate constants (k1) for the cyclization reaction at 22 and 37 degrees C (pH 7.3) were 0.14 and 0.85 min-1, respectively. Determination of k1 as a function of pH gave a pKa value of 5.6 for 2. The rate constants (k2) for the hydrolysis of 3 at 22 and 37 degrees C (pH 7.3) were 0.0083 and 0.040 min-1, respectively. Compound 3 was 3-fold more active than oxotremorine as a muscarinic agonist on the guinea pig ileum, whereas its nicotinic actions, as estimated on the frog rectus, were quite weak. Because of its greater rate of cyclization and the higher peak concentrations of the aziridinium ion which ensue, 2 may offer advantages over its (2-chloroethyl)amino analogue (1) as an alkylating ligand for muscarinic receptors.     

31P NMR and chloride ion kinetics of alkylating monoester phosphoramidates

31P NMR spectroscopy was used to study the solvolysis kinetics of a novel series of alkylating monoester phosphoramidates (4a-d) under model physiologic conditions. Halide ion kinetics were used to determine the rate of aziridinium ion formation. The solvolysis rates showed the expected dependence upon substitution at the reactive nitrogen; comparison of 4a with phosphoramide mustard (1a) indicated that replacement of the amino group by alkoxy decreased the solvolysis rate by approximately 10-fold. The rate of conversion of starting compound (4a-d) to solvolysis product was essentially equal to the rate of halide ion release, suggesting that the aziridinium ion is a short-lived intermediate. 1H NMR and 31P NMR kinetics experiments performed in the absence and presence of trapping agent (dimethyldithiocarbamate) confirmed that the aziridinium ion was too short-lived to be observed via NMR. These compounds were also tested for cytotoxicity against L1210 leukemia and B16 melanoma cells in vitro; the monoalkylators 4c and 4d showed no activity, 4a was weakly cytotoxic, and 4b was comparable in activity to phosphoramide mustard.     

Kinetics of phosphoramide mustard hydrolysis in aqueous solution

Hydrolysis of phosphoramide mustard was investigated using HPLC, 31P NMR, and GC-MS with specific deuterium labels. The hydrolysis of phosphoramide mustard in sodium phosphate buffers was found to follow apparent first-order kinetics. The rate of hydrolysis was temperature and pH dependent, being slower under acidic conditions. The hydrolysis was not catalyzed by hydroxyl ion, and its pH dependence appeared to be the result of a change in the mechanism of hydrolysis at different pH values. At a pH value approximately above the pKa of the phosphoramide mustard nitrogen, the major hydrolytic pathway of phosphoramide mustard was via the formation of the aziridinium ion, followed by nucleophilic attack. At pH values below its pKa, cleavage of the P-N bond predominated. At pH 7.4, the formation of an aziridinium ion was followed by a rapid hydrolysis to yield the monohydroxy and, subsequently, the dihydroxy products. The hydrolysis at this pH was adequately described by consecutive first-order kinetics. Seven species in the hydrolytic mixture have been identified as intact phosphoramide mustard, N-(2-chloroethyl)-N-(2-hydroxyethyl)phosphorodiamidic acid, N,N-bis-(2-hydroxyethyl)phosphorodiamidic acid, phosphoramidic acid, phosphoric acid, N,N-bis-(2-chloroethyl)amine, and N-(2-chloroethyl)-N-(2-hydroxyethyl)amine by GC-MS with the aid of deuterium labels. Phosphoramide mustard was found to be stabilized by chloride ion. The stabilization was linearly related to the chloride ion concentration, and the mechanism was found to be via the formation of phosphoramide mustard from the aziridinium and chloride ions. Phosphoramide mustard was significantly more stable in human plasma and in 5% human serum albumin as compared to aqueous buffers, an observation that may be important in vivo.     

Kinetics of degradation of 4-imidazolidinone prodrug types obtained from reacting prilocaine with formaldehyde and acetaldehyde.

The kinetics of decomposition of 4-imidazolidinone prodrug types obtained by reacting prilocaine (I) with formaldehyde and acetaldehyde has been studied in aqueous solution in the pH range 1-7.4 at 60 and 37 degrees C, respectively. At pH<5 the hydrolysis of the derivative derived from formaldehyde (II) to yield I obeyed apparent first-order kinetics. At higher pH, the decomposition reactions proceeded to an equilibrium and the reactions could be described by first- and second-order reversible kinetics. A plot of the logarithm of the apparent first-order rate constants for hydrolysis of II against pH resulted in a sigmoidal-shaped pH-rate profile characteristic for the hydrolysis of many N-Mannich bases. A half-life at pH 7.4 (60 degrees C) of 6.9h for compound II was calculated. Compared to II the 4-imidazolidinone derived from acetaldehyde (III) exhibited enhanced instability in aqueous buffer solutions. The decomposition was followed at 37 degrees C monitoring the decrease in concentration of intact (III). At acidic pH the reactions displayed strict first-order kinetics and the disappearance of III was accompanied by a concomitant formation of I. At pH 7.4, the rate data also applied reasonably well to first-order kinetics despite the observation that small amounts of III was formed at pH 7.4 from a solution containing equimolar concentrations of acetaldehyde and prilocaine (10(-4)M). In case of III, a bell-shaped pH-rate profile was obtained by plotting the logarithm of the pseudo-first-order rate constants against pH indicating the involvement of a kinetically significant intermediate in the reaction pathway and a change of the rate-limiting step in the overall reaction with pH. For the stability studies performed at pH 6.9 and 7.4 product analysis revealed that parallel to formation of (I) an unknown compound (X) emerged. Compared to III, compound X is hydrolysed to give I at a slower rate (t(50%)=30 h at 37 degrees C). Based on LC-MS data it is suggested that (X) is an isomeric form of III, which may exist in four diastereomeric forms. Thus, at physiological pH an initial relatively fast regeneration of I from III is to be expected followed by a slower drug activation resulting from hydrolysis of the isomeric form of III.     

Influence of pH and temperature on kinetics of ceftiofur degradation in aqueous solutions.

The objective of this study was to evaluate the stability of ceftiofur (1 mg mL(-1)) in aqueous solutions at various pH (1, 3, 5, 7.4 and 10) and temperature (0, 8, 25, 37 and 60 degrees C) conditions. The ionic strength of all these solutions was maintained at 0.5 M. Ceftiofur solutions at pH 5 and 7.4 and in distilled water (pH = 6.8) were tested at all the above temperatures. All other solutions were tested at 60 degrees C. Over a period of 84 h, the stability was evaluated by quantifying ceftiofur and its degradation product, desfuroylceftiofur, in the incubation solutions. HPLC was used to analyse these compounds. At 60 degrees C, the rate of degradation was significantly higher at pH 7.4 compared with pH 1, 3, 5 and distilled water. At both 60 degrees C and 25 degrees C, degradation in pH 10 buffer was rapid, with no detectable ceftiofur levels present at the end of 10 min incubation. Degradation rate constants of ceftiofur were 0.79+/-0.21, 0.61+/-0.03, 0.44+/-0.05, 1.27+/-0.04 and 0.39+/-0.01 day(-1) at pH 1, 3, 5, 74 and in distilled water, respectively. Formation of desfuroylceftiofur was the highest (65%) at pH 10. The rate of degradation increased in all aqueous solutions with an increase in the incubation temperature. At pH 7.4 the degradation rate constants were 0.06+/-0.01, 0.06+/-0.01, 0.65+/-0.17, and 1.27+/-0.05 day(-1) at 0, 8, 25, 37 and 67 degrees C, respectively. The energy of activation for ceftiofur degradation was 25, 42 and 28 kcal mol(-1) at pH 5, 7.4 and in distilled water, respectively. Desfurylceftiofur formation was the greatest at alkaline pH compared with acidic pH. Ceftiofur degradation accelerated the most at pH 7.4 and was most rapid at pH 10. The results of this study are consistent with rapid clearance of ceftiofur at physiological pH.     

Macromolecular prodrugs: X. Kinetics of fenoprofen release from PHEA-fenoprofen conjugate

The kinetics of fenoprofen release from poly[alpha,beta-(N-2-hydroxyethyl-DL-aspartamide)]-fenoprofen conjugate (PHEA-Fen) in aqueous buffer solutions (pH 10 and 1.1), simulated gastric (SGF) and intestinal fluids (SIF) was studied. In borate buffer pH 10, the following rate constants were obtained: k=0.2659 (t=60 degrees C) and k=0.0177 h(-1) (t=37 degrees C) and in glycine buffer solution pH 1.1 k=0.0036 h(-1). In SGF and SIF fenoprofen release did not occur in significant extend within 12 h. The hydrolysis of the ester bond between the polymeric carrier and fenoprofen followed the pseudo first-order kinetics, with activation energy indicative for the breakage of a sigma bond (E(a)=100.6 kJ mol(-1)). The concentration of the released fenoprofen was determined by high performance liquid chromatography (HPLC).     

Pathways of decomposition of propylbenzilylcholine mustard in neutral and alkaline solution

High-voltage electrophoresis has been used to follow the decomposition of propylbenzilylcholine mustard (PrBCM) in aqueous solution. Dilute solutions of PrBCM in 10 mM phosphate buffer, pH 7.5, or Krebs-Henseleit solution allowed to stand for 1 h at room temperature (22-24 degrees C) contain mainly the aziridinium ion derivative. At pH 7.5 the concentration of this ion declines slowly, giving rise first to the N-hydroxyethyl derivative and then ultimately, following hydrolysis of the ester bond, to NN-bis(2-hydroxyethyl)propylamine and benzilic acid. In contrast, in 5 mM NaOH the ester bond undergoes rapid hydrolysis, so that the major species present after 15 min at room temperature is the N-hydroxyethylaziridinium ion. This ion then undergoes slow reaction with hydroxy ion to yield the same final decomposition product, NN-bis(2-hydroxyethyl)propylamine, as is observed at pH 7.5.     

Degradation kinetics and mechanisms of a new cephalosporin, cefixime, in aqueous solution

Hydrolysis of cefixime in buffer solutions (pH 1-9) at 25 degrees C and a constant ionic strength of 0.3 was investigated using ion-pair reversed-phase HPLC. Hydrolysis rates followed pseudo first-order kinetics; the rate of hydrolysis of cefixime was very slow at pH 4-7, slightly faster at lower pH, and quite rapid at higher pH. In the early stages of hydrolysis, six major degradation products were isolated and identified: a beta-lactam ring-opened product and a 7-epimer (basic conditions), three lactones derived from intramolecular cyclization between the 2-carboxyl and 3-vinyl groups (acidic conditions), and an aldehyde derivative involving a 7-acyl moiety (neutral conditions). Principal degradation pathways for cefixime were found to involve initial cleavage of the beta-lactam ring.     

Stability of batanopride hydrochloride in aqueous solutions.

The degradation of batanopride hydrochloride, an investigational antiemetic drug, was studied in aqueous buffer solutions (pH 2-10; ionic strength, 0.5; 56 degrees C) in an attempt to improve drug stability for parenteral administration. Degradation occurs by two different mechanisms depending on the pH of the solution. In acidic media (pH 2-6), the predominant reaction was intramolecular cyclization followed by dehydration to form a 2,3-dimethylbenzofuran. There was no kinetic or analytical (high-performance liquid chromatography) evidence for the formation of an intermediate; therefore, the rate of dehydration must have been very rapid compared with the rate of cyclization. In alkaline media (pH 8-10), the primary route of degradation was cleavage of the C-O alkyl ether bond. In the intermediate pH range (pH 6-8), both reactions contributed to the overall degradation. Both degradation reactions followed apparent first-order kinetics. The pH-rate profile suggests that batanopride hydrochloride attains its optimal stability at pH 4.5-5.5. Citrate buffer was catalytic at pH 3 and 5, and phosphate buffer was catalytic at pH 8. No catalytic effect was observed for the borate buffer at pH 9-10.     

Products of the reaction between a diazoate derivative of 2'-deoxycytidine and L-lysine and its implication for DNA-nucleoprotein cross-linking by NO or HNO(2)

Recently, we have reported that a stable diazoate intermediate (dCyd-diazoate) is produced upon the reaction of dCyd with nitrous acid and nitric oxide [Suzuki, T., Nakamura, T., Yamada, M., Ide, H., Kanaori, K., Tajima, K., Morii, T., and Makino, K. (1999) Biochemistry 38, 7151-7158]. In this work, the reaction of dCyd-diazoate with L-Lys was investigated. When 0.4 mM dCyd-diazoate was incubated with 10 mM L-Lys in sodium phosphate buffer (pH 7.4) at 37 degrees C, two unknown products were formed in addition to dUrd. By spectrometric measurements, the products were identified as dCyd-Lys adducts with C4(dCyd)-N(alpha)(Lys) and C4(dCyd)-N(epsilon)(Lys) linkages (abbreviated as dCyd-alphaLys and dCyd-epsilonLys, respectively). The yields at the reaction time of 72 h were 28.0% dCyd-alphaLys, 13.4% dCyd-epsilonLys, and 11.1% dUrd with 33.9% unreacted dCyd-diazoate. When 0.4 mM dCyd-diazoate was incubated with 22 mg/mL poly(L-Lys) at pH 7.4 and 37 degrees C for 24 h, 82% of the free dCyd-diazoate disappeared, indicating adduct formation with the polymer. At pH 7.4 and 37 degrees C, dCyd-alphaLys and dCyd-epsilonLys were fairly stable and gave rise to no product after incubation for 7 days. At pH 4.0 and 70 degrees C, both adducts disappeared with the same first-order rate constant of 1.7 x 10(-)(6) s(-)(1) (t(1/2) = 110 h), which was approximately (1)/(3) of that of dCyd. These results suggest that if dCyd-diazoate is formed in DNA in vivo, it may react with free L-Lys and the side chain of L-Lys in nucleoproteins, resulting in stable adducts and DNA-protein cross-links, respectively.     

Kinetics and mechanisms of degradation of the antileukemic agent 5-azacytidine in aqueous solutions.

The hydrolytic degradation of 5-azacytidine was studied spectrophotometrically as a function of pH, temperature, and buffer concentration. Loss of drug followed apparent first-order kinetics in the pH region below 3. At pH less than 1,5-azacytosine and 5-azauracil were detected; at higher pH values, drug was lost to products which were essentially nonchromophoric if examined in acidic solutions. The apparent first-order rate constants associated with formation of 5-azacytosine and 5-azauracil from 5-azacytidine are reported. Above pH 2.6, first-order plots for drug degradation are biphasic. Apparent first-order rate constants and coefficients for the biexponential equation are given as a function of pH and buffer concentration. A reaction mechanism consistent with the data is discussed together with problems associated with defining the stability of the drug in aqueous solutions. At 50 degrees, the drug exhibited maximum stability at pH 6.5 in dilute phosphate buffer. Similar solutions were stored at 30 degrees to estimate their useful shelflife. Within 80 min, 6 times 10(-4) M solutions of 5-azacytidine decreased to 90% of original potency based on assumptions related to the proposed mechanisms.     

A mechanistic and kinetic study of the beta-lactone hydrolysis of Salinosporamide A (NPI-0052), a novel proteasome inhibitor

The aim of the present study was to investigate the mechanism of aqueous degradation of Salinosporamide A (NPI-0052; 1), a potent proteasome inhibitor that is currently in Phase I clinical trials for the treatment of cancer and is characterized by a unique beta-lactone-gamma-lactam bicyclic ring structure. The degradation of 1 was monitored by HPLC and by both low- and high-resolution mass spectral analyses. Apparent first-order rate constants for the degradation at 25 degrees C were determined in aqueous buffer solutions (ionic strength 0.15 M adjusted with NaCl) at various pH values in the range of 1 to 9. Degradation kinetics in water and in deuterium oxide were compared as a mechanistic probe. The studies were performed at pH (pD) 4.5 at 25 degrees C. To further confirm the reaction mechanism, the degradation was also performed in (18)O-enriched water and the degradation products subjected to HPLC separation prior to mass spectral analysis. Solubility and stability in (SBE)(7m)-beta-cyclodextrin (Captisol) solutions were also determined. The hydrolytic degradation of 1, followed by both HPLC and LC/MS, showed that the drug in aqueous solutions gives a species with a molecular ion consistent with the beta-lactone hydrolysis product (NPI-2054; 2). This initial degradant further rearranges to a cyclic ether (NPI-2055; 3) via an intramolecular nucleophilic displacement reaction. The kinetic results showed that the degradation of 1 was moderately buffer catalyzed (general base) and the rate constants were pH independent in the range of 1-5 and base dependent above pH 6.5. No acid catalysis was observed. The kinetic deuterium solvent isotope effect (KSIE) was 3.1 (kH/kD) and a linear proton inventory plot showed that the rate-determining step involved only a single proton transfer. This suggested that a neighboring hydroxyl group (as opposed to a second water molecule) facilitated water attack at pD 4.5. Mass spectral analysis from the (18)O-labeling studies proved that the mechanism involves acyl-oxygen bond cleavage and not a carbonium ion mechanism. 1 is unstable in water (t(90%) 相似文献   

The role of nucleophilicity in the ratio of primary to secondary solute-derived products in the decomposition of (1-acetoxypropyl)propylnitrosamine

The reactions of 1-propanediazotic acid, generated in situ from the hydrolysis of (1-acetoxypropyl)propylnitrosamine (1-APPN), with inorganic halides (X- = Cl-,Br-,I-) were studied in pH 8.0 buffer at 37 degrees C with specific emphasis on the extent of structurally rearranged solute-derived product. There is a direct correlation between the nucleophilicity of the halide and increased total and individual isomeric propyl halide (PrX) products. However, the relative formation of rearranged secondary propyl halide (2-PrX) is inversely proportional to solute nucleophilicity. The extent of isomerization ranges from 28% for Cl- to 13% for I-. In contrast, 2-propanol accounts for 33% of the total solvent-derived propanol yield. It is also demonstrated that the ratio of 1-PrX/2-PrX is inversely related to [X-] for Br- and I-. The data suggest that a nucleophilicity-driven association of the solute with a propanediazonium ion in the transition state leads to both primary and secondary products. The results are also discussed in terms of understanding the solvolysis products and DNA adducts previously observed from the metabolism of dipropylnitrosamine and the hydrolysis of N-propyl-N-nitrosourea.     

Studies on the reactions between daptomycin and glyceraldehyde

The objectives of this project were to determine the reaction pathways of daptomycin in the presence of glyceraldehyde in acidic solutions, and to quantitate the kinetics of the major pathways. In the presence of glyceraldehyde (pH range 1-7 at 25 to 60 degrees C), daptomycin formed two major products separable by RP-HPLC. The products were identified using UV spectroscopy, fluorimetry, mass spectrometry, and 2D-1H NMR. The reaction scheme involved the reversible formation of imine and anilide derivatives. Carbinolamine was believed to be a common intermediate in formation pathways of both products. The carbinolamine intermediate underwent either acid catalyzed dehydration resulting in imine formation or intramolecular hydrogen bonding and bond cleavage giving rise to anilide formation. In mild acid conditions, both products reversed to daptomycin. The reaction between daptomycin and glyceraldehyde was first-order with respect to both reactants. In a pH range of 1-7, the imine formation rate was pH dependent with a maximum rate at approximate pH values of 3-4. The observed pH dependence was consistent with the pH dependence of typical amine-aldehyde reactions.     

Mechanism of decarboxylation of p-aminosalicylic acid

The rate of decarboxylation of p-aminosalicylic acid (1) in aqueous solutions was studied at 25 degrees C (mu = 0.5) as a function of pH and buffer concentration. A pH-rate profile was generated by using the rate constants extrapolated to zero buffer concentration. The profile was bell-shaped, with the maximum rate of decarboxylation near the isoelectric pH. The rate constants obtained in buffered solutions indicated general acid catalysis. Bronsted behavior appeared to be adhered to. The two ionization constants of 1 were determined spectrophotometrically at 25 degrees C and at an ionic strength of 0.5. An HPLC method was used to characterize the degradation products of the reaction. Kinetic solvent deuterium isotope effects were studied to further confirm the mechanism of decarboxylation. Below pH 7.0, the mechanism of 1 decarboxylation is the rate controlling proton attack on the carbon-alpha to the carboxylic acid group of 1 anion and the ampholyte, followed by the rapid decarboxylation of the formed intermediate.     

Chemical reactivity of Ro-26-9228, 1alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol in aqueous solution

The degradation of Ro-26-9228, 1alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol, 2, was studied in aqueous solution in the pH range of 1.17-10.56 and in alcohol solutions, at 25, 40, and 50 degrees C. The degradation of Ro-26-9228 was found to be acid catalyzed and to be independent of potassium acetate buffer concentration. Above pH 4, the reaction rate is independent of pH, with a T90 of 14.3 h at 25 degrees C in pH 7.75 buffer. 19F nuclear magnetic resonance was used to study the ratio of the vitamin (6-s-trans) to previtamin form in acetonitrile at 40 degrees C. The equilibrium percentage of previtamin and the rate of approach to equilibrium were 13.8% and 0.2 h(-1), respectively. Nuclear magnetic resonance was used to elucidate the structure of the degradation products. Novel products were formed from the elimination of the fluorine and addition of solvent to C9, with formation occurring through the previtamin form. Additional degradation products result from reaction of the side chain 25-hydroxyl and addition of solvent to C1.     

Furosemide 1-O-acyl glucuronide. In vitro biosynthesis and pH-dependent isomerization to beta-glucuronidase-resistant forms

A biosynthetic acyl-type glucuronic acid conjugate of furosemide was isolated from in vitro incubation of pregnenolone-16 alpha-carbonitrile-induced rat liver microsomes containing UDP-glucuronyltransferase activity, furosemide, and UDP-glucuronic acid. Furosemide 1-O-acyl glucuronide (FG) was specifically hydrolyzed by beta-glucuronidase (BG) and was also labile to alkaline hydrolysis. FG concentration decreased at an apparent first order rate when incubated at 37 degrees C in buffer solution of pH values greater than 6.0 with only moderate hydrolysis of the conjugate at pH values less than 8.5. Formation of rearrangement forms of FG that were resistant to BG but labile to alkaline hydrolysis accounted for most of the disappearance of FG at this pH range. Radiochemical labeling of the conjugate with either 14C-furosemide or 14C-UDP-glucuronic acid was detected in the BG-resistant isomerization products of FG as they were separated by HPLC. The structure of FG and its isomerization products was further verified by negative ion thermospray liquid chromatography/mass spectrometry. The abundant (M - 1)-ion at mass 505, the aglycone fragment at m/z 329, and the characteristic sugar fragment ion of mass 175 were found in the spectra of FG and three additional isomers. An ion at m/z 221 was noted only in the case of the parent conjugate and thus may prove to be a characteristic ion for 1-O-acyl-linked glucuronides under negative ion thermospray. In vivo as well as in vitro rearrangement of FG to BG-resistant forms might affect the results of furosemide disposition studies which use BG hydrolysis to determine FG formation.     

Degradation kinetics of phentolamine hydrochloride in solution

The degradation kinetics of phentolamine hydrochloride in aqueous solution over a pH range of 1.2 to 7.2 and its stability in propylene glycol- or polyethylene glycol 400-based solutions were investigated. The observed rate constants were shown to follow apparent first-order kinetics in all cases. The pKa determination for phentolamine hydrochloride was found to be 9.55 +/- 0.10 (n = 5) at 25 +/- 0.2 degrees C. This indicated the protonated form of phentolamine occurs in the pH range of this study. The pH-rate profile indicated a pH-independent region (pH 3.1-4.9) exists with a minimum rate around pH 2.1. The catalytic effect of acetate and phosphate buffer species is ordinary. The catalytic rate constants imposed by acetic acid, acetate ion, dihydrogen phosphate ion, and monohydrogen phosphate ion were determined to be 0.018, 0.362, 0.036, and 1.470 L mol-1 h-1, respectively. The salt effect in acetate and phosphate buffers followed the modified Debye-Huckel equation quite well. The ZAZB value obtained from the experiment closely predicts the charges of the reacting species. The apparent energy of activation was determined to be 19.72 kcal/mol for degradation of phentolamine hydrochloride in pH 3.1, 0.1 M acetate buffer solution at constant ionic strength (mu = 0.5). Irradiation with 254 nm UV light at 25 +/- 0.2 degrees C showed a ninefold increase in the degradation rate compared with the light-protected control. Propylene glycol had little or no effect on the degradation of phentolamine hydrochloride at 90 +/- 0.2 degrees C; however, polyethylene glycol 400 had a definite effect.     

Some differential effects of 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride on guinea-pig atria and ileum

4-Diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (I) cyclizes at neutral pH to form an aziridinium salt. The formation and breakdown of the salt depend on the temperature (in the range 25 to 37 degrees C). In solution at 30 degrees C, peak levels, corresponding to 60-80% conversion, are reached after around 60 min and the half-life exceeds 100 min. In the presence of 0.9% NaCl conversion was reduced to 45-60%. I blocks muscarinic receptors in guinea-pig ileum and atria irreversibly and it is possible to produce dose-ratios on ileum with 10 nM I which are about 100 times those on atria. After about 30 min exposure to solutions of I (prepared 15-20 min previously so that formation of aziridinium ions is well-established) the graph of log (dose-ratio) against time is linear and similar plots were obtained with two different agonists, carbachol and ethoxyethyltrimethylammonium. With results for the ileum, extrapolation of the line suggests that it does not start from zero (dose-ratio = 1): this is because of an initial relatively rapid reversible block. This early phase is similar to that seen on ileum with 10 nM 4DAMP methobromide, which is a competitive antagonist, so is probably caused by competitive block by the aziridinium ion, which closely resembles 4DAMP metho-salts. The subsequent irreversible phase should be caused by alkylation of the receptors. I is easy to make and should be a valuable tool for the study of muscarinic receptors.     

Long-term stability of aqueous solutions of luteinizing hormone-releasing hormone assessed by an in vitro bioassay and liquid chromatography

The stability of aqueous solutions of luteinizing hormone-releasing hormone (LHRH) after extended storage at various temperatures was investigated using a newly developed HPLC assay and an in vitro dispersed pituitary cell culture bioassay. Good correlations were obtained between the potency obtained by HPLC and bioassay in samples stored at 37 degrees C or subjected to different stress conditions. No significant decrease in activity of LHRH was observed in aqueous solutions stored at 37 degrees C for up to 10 weeks, at 4 degrees C for 2 years, or subjected to repeated freezing and thawing for 5 d. Heating to 60 degrees C in sterile pH 9.0 buffer up to 11 d and storage at ambient temperature in nonsterile solution for 4 months produced well-distinguished degradation products and a decrease in potency. It is concluded that sterile aqueous solutions of LHRH are stable for at least 10 weeks at 37 degrees C and, thus, could be reliably used for chronic administration when long-term stability at body temperature is important.