Thyrotropin-Releasing Hormone (TRH) Uptake in Intestinal Brush-Border Membrane Vesicles: Comparison with Proton-Coupled Dipeptide and Na+-Coupled Glucose Transport

The mechanism of thyrotropin-releasing hormone (pGlu-His-Pro-NH₂; TRH) uptake across the luminal membrane of intestinal enterocytes was investigated using brush-border membrane vesicles (BBMV) from rabbit duodenum and jejunum and rat upper small intestine. [¹⁴C]Glucose accumulated within the intestinal vesicles (at 10 sec), in the presence of an inwardly directed Na⁺ gradient, 7- to 14-fold higher than equilibrium values (65 min). The vesicles also accumulated the dipeptide [¹⁴C]Gly-Sar. Dipeptide uptake was greatest in the presence of both an inwardly directed proton gradient and an inside negative membrane potential. The H⁺-dependent Gly-Sar transport was not affected by the presence of an excess (46-fold) of cold TRH. In contrast to the observations with glucose and Gly-Sar, the uptake of [³H]TRH after 10 or 60 sec (in each of the vesicle preparations) was not enhanced by either Na⁺ or H⁺ gradient conditions. The absence of vesicular accumulation of TRH was not due to peptide hydrolysis. For example, after a 60-sec incubation with rabbit jejunal BBMV no degradation of the tripeptide was evident. After 65 min, 6% of [³H]TRH had undergone degradation, by deamidation, to form TRH-OH. These studies provide no evidence for the oral absorption of TRH by a Na⁺- or H⁺-dependent carrier system in the brush-border membrane. Previous observations of TRH absorption in vivo may be accounted for by passive absorption of the peptide combined with its relative resistance to luminal hydrolysis.     

A Novel Oligodeoxynucleotide Inhibitor of Thrombin. I. In VitroMetabolic Stability in Plasma and Serum

Purpose. To determine the degradation rates and pathways of GS-522, a potent oligodeoxynucleotide (GGTTGGTGTGGTTGG) inhibitor of thrombin, in serum and plasma. Methods. A stability-indicating, anion-exchange HPLC method was developed and used to determine concentrations of GS-522 and metabolites. Results. In monkey plasma at 2 µM or below, the degradation of GS-522 can be fit to a first-order exponential with a k^p _obs ~ 0.01 min^–1. At 3 µM and above the degradation process deviates from a monoexponential decay profile. An initial fast degradation process is followed by a slower phase with an observed rate constant equal to that observed at 2 µM and below. In monkey serum, the K_Mand V_maxare 8.4 µM and 0.87 µM min^–1, respectively. Conclusions. The kinetics are consistent with an equilibrium binding of GS-522 to prothrombin in plasma (K_d = 50 nM) which saturates at GS-522 concentrations >2 µM. Compared to a scrambled sequence (GGTGGTGGTTGTGGT), with no defined tertiary structure, GS-522 is 4-fold more stable in serum. The metabolic profile in plasma is consistent with a 3-exonuclease catalyzed hydrolysis of GS-522.     

Prodrugs of Peptides. 6. Bioreversible Derivatives of Thyrotropin-Releasing Hormone (TRH) with Increased Lipophilicity and Resistance to Cleavage by the TRH-Specific Serum Enzyme

Bioreversible derivatization of TRH (pGlu–His–Pro–NH₂) to protect the tripeptide against rapid enzymatic inactivation in the systemic circulation and to improve the lipophilicity of this highly hydrophilic peptide was performed by N-acylation of the imidazole group of the histidine residue with various chloroformates. Whereas TRH was rapidly hydrolyzed at its pGlu–His bond in human plasma by a TRH-specific pyroglutamyl aminopeptidase serum enzyme, the N-alkoxycarbonyl derivatives were resistant to cleavage by the enzyme. On the other hand, these derivatives are readily bioreversible as the parent TRH is formed quantitatively from the derivatives by spontaneous hydrolysis or by plasma esterase-catalyzed hydrolysis. In addition to protecting the parent TRH against rapid inactivation in the circulation and hence potentially prolonging the duration of action of TRH in vivo, the N-alkoxycarbonyl prodrug derivatives were much more lipophilic than TRH as assessed by octanol–buffer partitioning. This property may enhance prodrug penetration of the blood–brain barrier and various other biomembranes compared to the parent peptide.     

The rapid hydrolysis of chlordiazepoxide to demoxepam may affect the outcome of chronic osmotic minipump studies

Background

In chronic studies, the classical benzodiazepine chlordiazepoxide (CDP) is often the preferred drug because, unlike other benzodiazepines, it is soluble in water. However, rapid CDP hydrolysis in solution has been described. This would diminish plasma levels in chronic minipump studies and introduce the corelease of active compounds.

Methods

Therefore, the present study aimed to explore the putative hydrolysis of CDP in aqueous solution over time and to identify the hydrolysis products. Moreover, we aimed to characterize the hydrolysis products for their in vitro (³H-flunitrazepam binding and oocyte electrophysiology) and in vivo (stress-induced hyperthermia paradigm) GABA_A receptor potency.

Results

CDP in solution hydrolyzed to the ketone structure demoxepam which was confirmed using mass spectrometry. The hydrolysis was concentration dependent (first-order kinetics) and temperature dependent. CDP exerted greater potency compared to demoxepam in vitro (increased activity at GABA_A receptors containing α₁ subunits) and in vivo (stress-induced hyperthermia), although ³H-flunitrazepam binding was comparable.

Conclusions

The classical benzodiazepine CDP is rapidly hydrolyzed in solution to the active compound demoxepam which possesses a reduced activity at the GABA_A receptor. Chronic studies that use CDP in aqueous solution should thus be interpreted with caution. It is therefore important to consider drug stability in chronic minipump applications.     

Bis(carbamoyloxymethyl) Esters of 2′,3′-Dideoxyuridine 5′-monophosphate (ddUMP) as Potential ddUMP Prodrugs

No HeadingPurpose. We previously reported the synthesis of bis(pivaloyloxymethyl) 2,3-dideoxyuridine 5-monophosphate (POM₂-ddUMP) (1a) as a membrane-transport prodrug formulation of the free parent nucleotide, ddUMP. Although successful at delivering ddUMP into cells in culture, POM₂-ddUMP was rapidly degraded by plasma carboxylate esterases after intravenous administration to experimental animals, and therefore has limited therapeutic potential as a systemically administered prodrug. We now report the synthesis of bis(N,N-dimethylcarbamoyloxymethyl)- and bis(N-piperidinocarbamoyloxymethyl) 2,3-dideoxyuridine 5-m onophosphate [DM₂-ddUMP (1b) and DP₂-ddUMP (1c), respectively], analogues of POM₂-ddUMP that were designed to be more resistant to degradation by plasma esterases..Methods. After entering cell by passive diffusion, it was anticipated that loss of one of the carbamoyloxymethyl groups of 1b and 1c would occur by spontaneous chemical hydrolysis to give the intermediate phosphodiesters, 2b and 2c. Cleavage of the remaining carbamoyloxymethyl groups by cellular phosphodiesterase I would generate ddUMP. 1b and 1c were prepared by condensation of 2,3-dideoxyuridine (ddU) with the appropriate bis(N-alkylcarbamoyloxymethyl) phosphate in DMA in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent).Results. The half-lives of 1b and 1c when incubated at a concentration of 10^–4 M in human plasma at 37°C were 3.5 h and 3.7 h, respectively, similar to the half-lives observed under the same temperature conditions in 0.05 M aqueous phosphate buffer, pH 7.4. By contrast, the half-life of the POM₂ prodrug, 1a, in plasma was only 5 min. The initial products of degradation of 1b and 1c were the phosphodiesters 2b and 2c. The latter compounds gave rise to ddUMP when incubated with snake venom phosphodiesterase I.Conclusions. These findings support the premise inherent in the design of 1b and 1c, namely that the carbamate prodrugs are far more resistant to hydrolysis by plasma carboxylate esterases than their POM counterparts and can revert to the free parent 5-mononucletides by successive chemical and enzymatic hydrolysis. Further studies of 1b and 1c as membrane-permeable prodrugs of ddUMP are in progress.     

The Relationship of Diastereomer Hydrolysis Kinetics to Shelf-Life Predictions for Cefuroxime Axetil

Cefuroxime axetil, an ester prodrug of cefuroxime, is comprised of a 50:50 mixture of diastereomers A and B. The first-order hydrolysis kinetics of cefuroxime axetil were investigated as a function of pH, temperature, buffers, and ionic strength. Chromatographically identified hydrolysis products were cefuroxime, ²-cefuroxime axetil, and ,-sulfoxides. Buffer catalysis was observed in acetate and phosphate buffers. No significant kinetic effect was observed for ionic strength in the range µ = 0.1-1.0. The pH–rate profiles for hydrolysis of cefuroxime axetil isomeric mixture were obtained at 45, 35, and 25°C. The equation defining the cefuroxime axetil hydrolysis rate constant as a function of pH was k _obs = k _H(a _H) + k _s + k _OH(K _w/a _H), exhibiting maximal stability in the pH range 3.5 to 5.5. The predicted profile at 5°C was in excellent agreement with experimental data in the pH range 3.6 to 5.5. In the pH range 1 to 9, the maximum difference observed for individual hydrolysis constants of isomers was 27%. Shelf-life estimates based on the hydrolysis rate constants for cefuroxime axetil as an isomeric mixture were shown to be equivalent to those based on individual hydrolysis rate constants for isomers A and B.     

Kinetic studies on the intramolecular acyl migration of β-1-O-acyl glucuronides: Application to the glucuronides of (R)- and (S)-ketoprofen, (R)- and (S)-hydroxy-ketoprofen metabolites,and tolmetin by 1H-NMR spectroscopy

Conjugation of carboxylate drugs with D-glucuronic acid is of considerable interest because of the inherent reactivity of the resulting β-1-O-acyl glucuronides. These conjugates can degrade by spontaneous hydrolysis and internal acyl migration. β-1-O-acyl glucuronides and their acyl migration products can also react covalently with macromolecules with potential toxicological consequences. The spontaneous degradation of the diastereoisomeric β-1-O-acyl glucuronide metabolites of the racemic drug ketoprofen, two of its ring-hydroxylated metabolites and of tolmetin β-1-O-acyl glucuronide was investigated by ¹H-NMR spectroscopy in buffer solutions, at pH 7.4 and 37°C. A plot of the logarithm of the peak integrals against time revealed first-order kinetics. Degradation rates and half-lives were calculated for each glucuronide using first-order reaction equations. Tolmetin glucuronide had the fastest degradation rate, whilst all of the ketoprofen-related glucuronides had similar degradation rates. The degradation of the diastereoisomeric glucuronides was stereoselective, with the rate for the (S)-isomer always slower compared with the (R)-isomer by approximately a factor of 2.     

Solubilization and Stabilization of a Benzylpenicillin Chemical Delivery System by 2-Hydroxypropyl-β-cyclodextrin

A dihydropyridine pyridinium salt redox carrier-based chemical delivery system for benzylpenicillin (1) was complexed with 2-hydroxypropyl--cyclodextrin (HPCD). The solubility of the lipophilic 1, which is incompatible with aqueous formulations, was dramatically increased and showed a linear dependency on the HPCD concentration. The degree of incorporation was 20 mg of 1 per g of complex. The stability study of 1 in various pH buffers indicated the base-catalyzed hydrolysis of the acyloxyalkyl linkage and the hydration of the 5,6 double bond of the dihydropyridine as the main degradation processes. The overall loss of 1, which follows first-order kinetics, was not influenced by changes in ionic strength and elimination of oxygen from the reaction medium. The HPCD complex of 1, which has a stability constant of 720–940 M ^–1, stabilized the chemical delivery system. The influence of the temperature on the stability of 1 is also discussed.     

The pharmacokinetics of low-dose dexamethasone in congenital adrenal hyperplasia

Summary The pharmacokinetics of dexamethasone, given at low dose, were studied in 13 patients with congenital adrenal hyperplasia (CAH) to ascertain whether kinetics differed in this inherited disorder of cortisol metabolism from those seen in healthy individuals.Changes in plasma dexamethasone concentration after intravenous bolus, measured using a simple novel radioimmunoassay, were well described by a two-compartment open model with first-order kinetics. Values for ₂: 0.206 h^–1, t_1/2: 3.53 h, Vc: 24.41 and f: 0.64 were similar to those previously reported for normal subjects. There were considerable interindividual differences in parameter values and C_maxp.o. (range 22–67 nmol/l).As suppression of the hypothalamo-pituitary-adrenal axis correlates with plasma dexamethasone levels, this variability may partly explain the differing dose and dose schedule requirements necessary to achieve adequate therapeutic control in the clinical management of CAH.     

Utility of the carboxylesterase inhibitor bis-para-nitrophenylphosphate (BNPP) in the plasma unbound fraction determination for a hydrolytically unstable amide derivative and agonist of the TGR5 receptor

1. The potent, functional agonist of the bile acid Takeda G-protein-coupled receptor 5 (TGR5), (S)-1-(6-fluoro-2-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-(isoquinolin-5-yloxy)ethanone (3), represents a useful tool to probe in vivo TGR5 pharmacology. Rapid degradation of 3 in both rat and mouse plasma, however, hindered the conduct of in vivo pharmacokinetic/pharmacodynamic investigations (including plasma-free fraction (f_{u plasma}) determination) in rodent models of pharmacology. Studies were therefore initiated to understand the biochemical basis for plasma instability so that appropriate methodology could be implemented in in vivo pharmacology studies to prevent the breakdown of 3.

2. Compound 3 underwent amide bond cleavage in both rat and mouse plasma with half-lives (T_1/2) of 39?±?7 and 9.9?±?0.1?min. bis(p-nitrophenyl) phosphate (BNPP), a specific inhibitor of carboxylesterases, was found to inhibit hydrolytic cleavage in a time- and concentration-dependent manner, which suggested the involvement of carboxylesterases in the metabolism of 3. In contrast with the findings in rodents, 3 was resistant to hydrolytic cleavage in both dog and human plasma.

3. The instability of 3 was also observed in rat and mouse liver microsomes. β-Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-dependent metabolism of 3 occurred more rapidly (T_1/2 approximately 2.22–6.4?min) compared with the metabolic component observed in the absence of the co-factor (T_1/2 approximately 89–130?min). Oxidative metabolism dominated the NADPH-dependent decline of 3, whereas NADPH-independent metabolism of 3 proceeded via simple amide bond hydrolysis.

4. Compound 3 was highly bound (approximately 95%) to both dog and human plasmas. Rat and mouse plasma, pre-treated with BNPP to inhibit carboxylesterases activity, were used to determine the f_{u plasma} of 3. A BNPP concentration of 500 μM was determined to be optimal for these studies. Higher BNPP concentrations (1000 μM) appeared to displace 3 from its plasma protein-binding sites in preclinical species and human. Under the conditions of carboxylesterases-inhibited rat and mouse plasma, the level of protein binding displayed by 3 was similar to those observed in dog and human.

5. In conclusion, a novel system has been devised to measure f_{u plasma} for a plasma-labile compound. The BNPP methodology can be potentially applied to stabilize hydrolytic cleavage of structurally diverse carboxylesterase substrates in the plasma (and other tissue), thereby allowing the characterization of pharmacology studies on plasma-labile compounds if and when they emerge as hits in exploratory drug-discovery programmes.

     

Pharmacokinetics of Zopolrestat,a Carboxylic Acid Aldose Reductase Inhibitor,in Normal and Diabetic Rats

The pharmacokinetics of zopolrestat, a carboxylic acid aldose reductase inhibitor, were examined in normal male rats dosed intravenously at 2 mg/kg and in normal and streptozotocin-diabetic male rats after oral administration at 50 mg/kg. After oral dosing, C _max was 127 µg/ml for normal rats and 144 µg/ml for diabetic rats. AUC(0–), however, was lower for diabetic rats than for normal rats and plasma half-life was longer in normal rats (8.0 vs 6.6 hr). Half-lives of zopolrestat in nerve, kidney, and lens were longer than plasma half-life and were similar for both diabetic and normal rats. Less than 2% of the dose was excreted in the urine as unchanged zopolrestat during the 48-hr period following dosing by diabetic or normal rats. Protein binding of zopolrestat was less extensive in plasma from diabetic rats than in plasma from normal rats. Similar kinetics were observed in diabetic animals receiving-five daily doses of zopolrestat at 50 mg/kg/day. There was no plasma or liver accumulation of zopolrestat at steady state, consistent with the observed half-lives. However, zopolrestat did accumulate in nerve, kidney, and lens to varying degrees during multiple dosing, reflecting the longer half-lives of zopolrestat in these tissues.     

Effect of urinary pH on the disposition of methadone in man

Summary The influence of urinary pH on the acute disposition of methadone in man was studied in five healthy volunteers. A cross-over experiment was performed in each subject. In the first experiment the subjects were treated with ammonium chloride (urinary pH 5.2) and in the other the urine was made alkaline (pH 7.8) by treatment with sodium hydrogen carbonate. d, 1-Methadone-HCl 10 mg (M) was administered intramuscularly on each occasion and blood, saliva and urine levels of M were determined by mass fragmentography. Plasma half-lives, volumes of distribution and body clearances of M were calculated in both experiments. The plasma half-lives in the -phase were 19.5±3.6 h (acidic urine) and 42.1±8.8 h (alkaline urine), respectively (p<0.001). The volumes of distribution were increased when the pretreatment was changed from ammonium chloride to sodium bicarbonate, namely from 3.51±0.41 l/kg to 5.24±0.83 l/kg (p<0.01). The body clearance decreased from 134±21 ml/min (acidic) to 91.9±9.1 ml/min (alkaline urine) (p<0.01). The ration M_plasma/M_RBC was about 2.3 and the elimination of M from RBCs was in good agreement with the plasma kinetics of M under both experimental conditions. The salivary levels of M did not reflect the plasma kinetics and considerable variation was seen in the ratio M_saliva/M_plasma (0.26–2.98). Thus, the present experiments demonstrate that pretreatment either with ammonium chloride or bicarbonate had profound effects on both the distribution and elimination kinetics of methadone.     

Epimerization and Hydrolysis of Dalvastatin,a New Hydroxymethylglutaryl Coenzyme A (HMG-CoA) Reductase Inhibitor

In aqueous solutions, dalvastatin (1) undergoes epimerization as well as hydrolysis. The transformation of the drug was studied as a function of pH at 25°C in aqueous solutions containing 20% acetonitrile. At all pH values, first-order plots for the conversion are biphasic, indicating rapid equilibration of 1 with its epimer (2) and slower hydrolysis of 1 to the corresponding -hydroxy acid (3). Apparent first-order rate constants for the biexponential equation are given as a function of pH. The alkyl–oxygen cleavage of the lactone ring results in the epimerization of 1 to 2, whereas the acyl–oxygen cleavage results in the hydrolysis of 1 to 3. The epimerization is an S_N1 reaction reaching an equilibrium of [l] _eq/[2] _eq = 1.27. The epimerization rate is increased with an increase in the water content of the solvent. The hydrolysis of 1 to 3 is acid and base catalyzed. The hydrolysis is reversible in acidic media and irreversible in neutral and basic media. At pH values greater than 9, the hydrolysis reaction proceeds more rapidly than the epimerization.     

Prodrugs of Peptides. 9. Bioreversible N-α-Hydroxyalkylation of the Peptide Bond to Effect Protection Against Carboxypeptidase or Other Proteolytic Enzymes

Various N--hydroxyalkyl derivatives of N-acyl amino acids and di- and tripeptides were prepared by hydrolysis or aminolysis of N-acyl 5-oxazolidinones. The stability of these derivatives was studied in aqueous solution as a function of pH. The compounds were all degraded quantitatively to their parent N-acylated amino acid or peptide and aldehyde but with vastly different rates. At pH 7.4 and 37°C the half-lives of decomposition ranged from 4 min to 1500 hr. The structural factors influencing the stability included both steric and polar effects within the acyl and N--hydroxyalkyl moieties as well as within the amino acid attached to the N--hydroxyalkylated N-acyl amino acid. Whereas the N-benzyloxycarbonyl (Z) derivatives of the dipeptides Gly-L-Leu and Gly-L-Ala were readily hydrolyzed by carboxypeptidase A, the N-hydroxymethylated compounds, i.e., Z-Gly(CH₂OH)-Leu and Z-Gly(CH₂OH)-Ala, were resistant to cleavage by the enzyme as revealed by their similar rates of decomposition in the presence or absence of the enzyme at pH 7.4 and 37°C. The results suggest that N--hydroxyalkylation of a peptide bond protects not only this bond but also an adjacent peptide bond against proteolytic cleavage. Since the N--hydroxyalkyl derivatives are readily bioreversible, undergoing spontaneous hydrolysis at physiological pH, this prodrug approach promises to overcome the enzymatic barrier to absorption of various peptides.     

Parenteral formulation and thermal degradation pathways of a potent rebeccamycin based indolocarbazole topoisomerase I inhibitor

The development of a practical and pharmaceutically acceptable parenteral dosage form of 1 is described. A cosolvent formulation strategy was selected to achieve the necessary human dose of 1 for administration via intravenous infusion. The final market formulation of 1 chosen for commercial development and Phase II clinical supplies was the topoisomerase inhibitor dissolved in a 50% aqueous propylene glycol solution vehicle with 50 mM citrate buffered to pH 4. The thermal degradation pathways of 1 in this aqueous propylene glycol vehicle in the pH range of 3–5 were determined by relative kinetics and degradation product identification using LC/MS, LC/MS/MS, and NMR analysis. The primary mode of degradation of 1 in this aqueous cosolvent formulation is hydrolysis affording the anhydride 2 (in equilibrium with the dicarboxylic acid 3) and release of the hydrazine diol side chain 11. Subsequent oxidative degradation of 11 occurs in several chemical steps which yield a complicated mixture of secondary reaction products that have been structurally identified.     

Degradation Mechanisms of Polysorbate 20 Differentiated by <Superscript>18</Superscript>O-labeling and Mass Spectrometry

Purpose

To investigate the mechanisms of polysorbate (PS) degradation with the added objective of differentiating the hydrolysis and oxidation pathways.

Methods

Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was utilized to characterize all-laurate polysorbate 20 (PS20) and its degradants. ¹⁸O stable isotope labeling was implemented to produce ¹⁸O-labeled degradation products of all-laurate PS20 in H₂ ¹⁸O, with subsequent UPLC-MS analysis for location of the cleavage site on the fatty acid-containing side chain of PS20.

Results

The analysis reveals that hydrolysis of all-laurate PS20 leads to a breakdown of the ester linkage to liberate free lauric acid, showing a distinct dependence on pH. Using a hydrophilic free radical initiator, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) to study the oxidative degradation of all-laurate PS20, we demonstrate that free lauric acid and polyoxyethylene (POE) laurate are two major decomposition products. Measurement of ¹⁸O incorporation into free lauric acid indicated that hydrolysis primarily led to ¹⁸O incorporation into free lauric acid via “acyl-cleavage” of the fatty acid ester bond. In contrast, AAPH-exposure of all-laurate PS20 produced free lauric acid without ¹⁸O-incorporation.

Conclusions

The ¹⁸O-labeling technique and unique degradant patterns of all-laurate PS20 described here provide a direct approach to differentiate the types of PS degradation.

     

Preformulation Stability Studies of the New Dipeptide Angiotensin-Converting Enzyme Inhibitor RS-10029

The degradation kinetics, products, and mechanisms of RS-10029 (2), 2-[2-[(l-carboxylic acid)-3-phenylpropyl]amino-l-oxopropyl] 6,7-dimethoxy- 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (S,S,S), in aqueous solutions from pH 1 to pH 13 were studied at 50, 60, and 80°C. Pseudo-first-order kinetics were obtained throughout the entire pH range studied, and the log(rate)-pH profile reflected four kinetic processes (k _o, k_o, k_o, and k _OH) as well as the three pk _a's of 2. Excellent mass balance (>96%) was obtained for the four major products 3–6 throughout the entire pH range studied even though four other minor products can be detected by high-performance liquid chromatography (HPLC). At pH 8.0 and below, intramolecular aminolysis leading to diketopiperazine (DKP) 5 accounted for greater than 65% of the neutral or water-catalyzed (k _o and k_o) processes. Amide hydrolysis leading to products 3 and 4 and epimerization of DKP 5 to the (R,S,S) diastereomer 6 accounted for the remaining 35% of the neutral or water catalyzed processes. At pH values above 8.0, DKP 5 formation begins to decrease as the amide hydrolysis increases so that both mechanisms account for the neutral or water-catalyzed k_o process. Above pH 11.0 amide hydrolysis dominates and is responsible for the specific base-catalyzed (k _OH) process. The four minor products detected by HPLC are two diastereomers (7 and 8) of 2 and the two diastereomers (9 and 10) of the DKP 5. The stability results between 2 and its ester prodrug (1) are compared.     

Disposition kinetics in dogs of diethyldithiocarbamate,a metabolite of disulfiram

Following the intravenous infusion of sodium diethyldithiocarbamate to dogs, the disposition kinetics of diethyldithiocarbamate (DDC), a metabolite of disulfiram, were assessed. Approximately 27% of the administered dose was S-methylated, this process exhibiting a mean first-order rate constant of 0. 0569 min^–1 (t_1/2=12.2 min), while the remainder was eliminated by other routes having a rate constant of 0.148 min^–1 (t_1/2=4.68 min). The methyl diethyldithiocarbamate (MeDDC) formed from DDC showed an elimination rate constant of 0.0141 min^–1 (t_1/2=49.2 min). These observations are discussed in the light of previous investigations where the presence of MeDDC has rarely been sought or reported. A few comparisons with prior studies, in which DDC or disulfiram was administered, are made by retrospective kinetic evaluation of published data. The results are discussed in relation to the duration of action of disulfiram in man.Glossary A plasma concentration intercept at the cessation of infusion (mass/volume) - A _T simplifying constant (mass/volume/time) - AUC _M area under the plasma concentration-time curve for MeDDC (mass × time/volume) - b time variable; equalst during infusion, equalsT after the cessation of infusion - B plasma concentration intercept at the cessation of infusion (mass/volume) - B _T simplifying constant (mass/volume/time) - C _D plasma concentration of DDC at any timet (mass/volume) - C _M plasma concentration of MeDDC, expressed as DDC, at any timet (mass/volume) - C _T plasma concentration of total DDC, expressed as DDC, at any timet;C _T=C_D+C_M (mass/volume) - C _t plasma concentration of total DDC, expressed as DDC, at any timet (mass/volume) - Cl _D total body clearance of DDC (volume/time) - Cl _M total body clearance of MeDDC (volume/time) - DDC diethyldithiocarbamate - f fraction of DDC that is methylated;f=K _DM/K _D - K _A apparent first-order rate constant (reciprocal time) - K _B apparent first-order rate constant (reciprocal time) - K _D apparent first-order rate constant for the elimination of DDC by all routes (reciprocal time) - K _M apparent first-order rate constant for the elimination of MeDDC by all routes (reciprocal time) - K _DE apparent first-order rate constant for the elimination of DDC by all routes except methylation (reciprocal time) - K _DM apparent first-order rate constant for theS-methylation of DDC (reciprocal time) - MeDDC methyl diethyldithiocarbamate - NaDDC sodium diethyldithiocarbamate (trihydrate) - Q zero-order infusion rate constant (mass/time) - Q ₁ zero-order infusion rate constant for the faster of two consecutive infusions (mass/time) - Q ₂ zero-order infusion rate constant for the slower of two consecutive infusions (mass/time) - t elapsed time since dosing (e.g., infusion) commenced - t elapsed time since the cessation of infusion - T duration of infusion (time) - T ₁ duration of the faster of two consecutive infusions (time) - T ₂ total duration of infusion when two consecutive infusions are administered (time) - V _D apparent volume of distribution of DDC - V _M apparent volume of distribution of MeDDC This work was supported by the Atkinson Charitable Foundation (Toronto, Ontario, Canada) and the Non-Medical Use of Drugs Directorate, Health and Welfare Canada (Grant No. 1212-5-206).     

Chemical and in vitro enzymatic stability of newly synthesized celecoxib lipophilic and hydrophilic amides

Five celecoxib (CXB) acylamide sodium salts, MP-CXB, Cy-CXB, Bz-CXB, CBz-CXB and FBz-CXB were synthesized and characterized. Two simple, fast and validated RP-HPLC methods were developed for simultaneous quantitative determination of the amides and celecoxib in aqueous and biological samples and LOD and LOQ were ≤13.6 and ≤40 ng/mL, respectively. The solubility and log P_app of the amides, in relevant media, were determined. The chemical hydrolysis, at 60, 70 and 80 °C, of MP-CXB was studied at GIT-relevant pH (1.2, 6.8 and 7.4) and of CY-CXB was studied at skin relative pH (5.4 and 7.4). Significant hydrolysis was observed for MP-CXB at pH 1.2 only with half-lives 28.28, 11.64 and 3.53 h at 60, 70 and 80 °C, respectively, with extrapolated half-lives of 2060 and 443 h at 25 and 37 °C, respectively. The hydrolysis of all amides was studied in rat live homogenate and only Cy-CXB was hydrolyzed with half-life of 3.79 h. The hydrolysis of MP-CXB and Cy-CXB was studied in human plasma and neither was hydrolyzed. It is finally suggested that hydrophobic interactions plays a role in the binding of susceptible acylamides to the hepatic hydrolyzing enzyme since only amides with saturated hydrocarbon chains underwent hydrolysis.     

Pharmacokinetics of spironolactone in man

Summary Five healthy male volunteers received 500 mg Aldactone^® orally together with 100 Ci ³H-20-21-spironolactone; one elderly patient received 1 mCi ³H-spironolactone without additional cold drug. For 6 days the disposition kinetics of the drug were studied in plasma, urine and feces. The tritium concentrations in plasma reached a peak between 25–40 min after administration amounting to 2–3% of the dose/1. Up to the 12th h, they fell rapidly and showed a monoexponential decline (t _1/2 : 2.57±0.27 days) between the 36th and 96th h. Later, a striking increase in the speed of elimination of radioactivity from plasma (t _1/2 : 1.66±0.21 days) was observed. The biological half-life of labeled material in plasma was longer than that of fluorigenic compounds. 47–57% of the dose were excreted in urine and the remaining amount culd be detected in feces (total recovery 90%). The half-life of the urinary excretion rate was distinctly shorter (t _1/2 : 0.9±0.11 days) than that of total radioactivity in plasma. This, together with an observed increase of the polar fraction in urine from 35 up to 85%, which was accompanied by a decrease in plasma from 55 to 35%, suggests either tubular reabsorption or enterohepatic recirculation of lipophilic compounds. TLC-separation of the lipophilic fraction in urine revealed two previously unknown compounds of which the main congener was identified as 3-(3-oxo-7-methylsulfonyl-6, 17-dihydroxy-4-androsten-17-yl) propionic acid -lactone, as well as canrenone and the metabolites which have already been described (Karim and Brown, 1972; Karim et al., 1975). This metabolite represents the main lipophilic degradation product in urine within the first hours, whereas the 6-OH-7-methylsulfinylspirolactone leveled off and seemed to be an endexcretion product. For further characterisation, the polar fraction was subjected to acidic hydrolysis. The known metabolic pathways of spironolactone degradation are discussed.The paper includes parts of the thesis of G. Luszpinski