Studying of drug solubility in water and alcohols using drug-ammonium ionic liquid-compounds.

Synthesis of three mefenamic acid (MEF) derivatives - ionic liquid compounds composed of MEF in an anionic form and ammonium cation (choline, MEF1), or {di(2-hydroxyethyl)dimethyl ammonium (MEF2)}, or {tri(2-hydroxyethyl)methyl ammonium compound (MEF3)} is presented. The basic thermal properties of pure compounds i.e. fusion temperatures, and the enthalpy of fusion of these compounds have been measured with differential scanning microcalorimetry technique (DSC). Molar volumes have been calculated with the Barton group contribution method. The solubilities of MEF1, MEF2 and MEF3 using the dynamic method were measured at constant pH in a range of temperature from (290 to 370) K in three solvents: water, ethanol and 1-octanol. The experimental solubility data have been correlated by means of three commonly known GE equations: the Wilson, NRTL and UNIQUAC with the assumption that the systems studied here present simple eutectic behaviour. The activity coefficients of pharmaceuticals at saturated solutions in each binary mixture were calculated from the experimental data. The formation of MEF-ionic liquid compounds greatly increases the solubility in water in comparison with pure MEF or complexes with 2-hydroxypropyl-ß-cyclodextrin. The development of these compounds formulations will assist in medication taking into account oral solid or gel medicines.

Synthesis, Biological Evaluation and Pharmacokinetic Studies of Mefenamic Acid - N-Hydroxymethylsuccinimide Ester Prodrug as Safer NSAID.

BACKGROUND: Non steroidal anti-inflammatory drugs are the most widely prescribed drugs to manage pain and inflammatory conditions, but their long term use is associated with gastrointestinal toxicity. OBJECTIVES: The study aimed to synthesize an ester-based prodrug of a non steroidal anti-inflammatory agent, mefenamic acid in order to improve the therapeutic index vis a vis to overcome the side effects such as gastrointestinal irritation and bleeding associated with the use of mefenamic acid. METHODS: The ester prodrug (MA-NH) was prepared by condensing mefenamic acid with N-hydroxymethylsuccinimide in the presence of Phosphorus oxychloride. The pharmacokinetic profile, including stability and release of mefenamic acid and N-hydroxymethylsuccinimide from the ester prodrug (MA-NH) was studied by RP- HPLC in acidic medium (pH 1.2), basic medium (pH 7.4), 80 % v/v human plasma, 10 % w/v rat intestinal homogenate and 10 % w/v rat liver homogenate (pH 7.4). RESULTS: The chemical structure of the title compound was characterized by using modern spectroscopic techniques. The prodrug was found to be stable in acid medium, but it hydrolyzed and released sufficient quantities of the drug in alkaline medium. The prodrug produced lesser number of ulcers and showed improved analgesic and anti-inflammatory activity as compared to the parent drug. CONCLUSION: The results indicate that the synthesized prodrug (MA-NH) is better in terms of analgesic and antiinflammatory activities and with less GI toxicity than the parent drug.

Identification and disposition of novel mono-hydroxyl mefenamic acid and their potentially toxic 1-O-acyl-glucuronides in vivo.

Mefenamic acid (MEF) is a widely prescribed non-steroidal anti-inflammatory drug that has been found associated with rare but severe cases of hepatotoxicity, nephrotoxicity and gastrointestinal toxicity. The formation of protein-reactive acylating metabolites such as 1-O-acyl-MEF glucuronide (MEFG) and 3'-hydroxymethyl-MEF 1-O-acyl-glucuronide is one proposed cause. In addition to the well-reported 3'-hydroxymethyl-MEF, two mono-hydroxyl-MEF (OH-MEFs) were recently identified in vitro. However, in vivo evidence is lacking and whether these OH-MEFs would be further glucuronidated to the potentially reactive 1-O-acyl-glucuronides (OH-MEFGs) is unknown. Utilizing UPLC-Q-TOF/MS and LC-MS/MS, the current study identified, for the first time, four OH-MEFs and their corresponding OH-MEFGs from plasma after a single oral administration of MEF (40 mg/kg) to rats, including an OH-MEF that has not been reported previously. The systemic exposure of these identified metabolites was high, with metabolic to parent AUC0 → 24 h ratios reaching 23-52% (OH-MEFs) and 8-29% (OH-MEFGs). These metabolites also had a long systemic exposure time in both single and 5 day multiple oral MEF-treated rats, with elimination half-lives between 9 h and > 24 h. In addition to these novel metabolites, the previously reported MEFG was also identified and its systemic exposure was found to be doubled after multiple MEF administrations. These pharmacokinetic results suggest that systemic toxicities caused by the potentially reactive MEFG and OH-MEFGs could be considerable, especially after repeated MEF treatment. Nevertheless, MEFG and OH-MEFGs had negligible uptake in the brain, indicating a minimal risk of brain toxicities. Furthermore, an in situ intestinal perfusion study revealed that during MEF absorption, it was extensively metabolized to MEFG while < 5% was metabolized to OH-MEFs and OH-MEFGs.

The nonenzymatic reactivity of the acyl-linked metabolites of mefenamic acid toward amino and thiol functional group bionucleophiles.

Mefenamic acid (MFA), a carboxylic acid-containing nonsteroidal anti-inflammatory drug, is metabolized into the chemically-reactive MFA-1-O-acyl-glucuronide (MFA-1-O-G), MFA-acyl-adenylate (MFA-AMP), and the MFA-S-acyl-coenzyme A (MFA-CoA), all of which are electrophilic and capable of acylating nucleophilic sites on biomolecules. In this study, we investigate the nonenzymatic ability of each MFA acyl-linked metabolite to transacylate amino and thiol functional groups on the acceptor biomolecules Gly, Tau, l-glutathione (GSH), and N-acetylcysteine (NAC). In vitro incubations with each of the MFA acyl-linked metabolites (1 µM) in buffer under physiologic conditions with Gly, Tau, GSH, or NAC (10 mM) revealed that MFA-CoA was 11.5- and 19.5-fold more reactive than MFA-AMP toward the acylation of cysteine-sulfhydryl groups of GSH and NAC, respectively. However, MFA-AMP was more reactive toward both Gly and Tau, 17.5-fold more reactive toward the N-acyl-amidation of taurine than its corresponding CoA thioester, while MFA-CoA displayed little reactivity toward glycine. Additionally, mefenamic acid-S-acyl-glutathione (MFA-GSH) was 5.6- and 108-fold more reactive toward NAC than MFA-CoA and MFA-AMP, respectively. In comparison with MFA-AMP and MFA-CoA, MFA-1-O-G was not significantly reactive toward all four bionucleophiles. MFA-AMP, MFA-CoA, MFA-1-O-G, MFA-GSH, and mefenamic acid-taurine were also detected in rat in vitro hepatocyte MFA (100 µM) incubations, while mefenamic acid-glycine was not. These results demonstrate that MFA-AMP selectively reacts with the amino functional groups of glycine and lysine nonenzymatically, MFA-CoA selectively reacts nonenzymatically with the thiol functional groups of GSH and NAC, and MFA-GSH reacts with the thiol functional group of GSH nonenzymatically, all of which may potentially elicit an idiosyncratic toxicity in vivo.

The solution properties of mefenamic acid and a closely related analogue are indistinguishable in polar solvents but significantly different in nonpolar environments.

This study investigates the cosolute effects of mefenamic acid (XA) and flufenamic acid (FA). These compounds serve as model of a drug discovery lead compound and a structural analogue. The activity coefficients of XA and FA in different solvents were obtained from solubility measurements at 25°C. The effect of varying concentrations of FA on the solubility of XA in four different solvents, including toluene, cyclohexane, ethanol, and an ethanol-water mixture (80:20, v/v), was investigated. The magnitude of change in the activity coefficient of XA in the presence of FA in different solvents was used to elucidate the thermodynamic effect of FA on the solubility of XA. Nuclear magnetic resonance and Fourier-transform infrared spectroscopy were used to obtain molecular level information about the interactions of the compounds in solution. The presence of FA increases XA solubility in toluene and in cyclohexane as much as seven-fold. Conversely, in ethanol and the ethanol-water mixture, similar levels of FA have essentially no effect on the solubility of XA. The solution properties investigated show that despite the close structural similarity between XA and FA, the two compounds are strongly distinguishable in nonpolar solvents. Conversely, the solution properties of the same two solutes are indistinguishable in polar solvents. A solubilization model based on solute-cosolute interactions is presented.

Design, synthesis, biological evaluation, and comparative Cox1 and Cox2 docking of p-substituted benzylidenamino phenyl esters of ibuprofenic and mefenamic acids.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently associated with gastric mucosal and renal adverse reactions, related to inhibition of cyclooxygenase1 (Cox1) in tissues where prostaglandins exert physiological effects. This led us to develop a set of ibuprofenic acid and mefenamic acid esters, namely: 4-((4-substituted benzylidene)amino)phenyl 2-(4-isobutylphenyl)propanoate and 4-((4-substituted benzylidene)amino)phenyl 2-((2,4-dimethylphenyl)amino)benzoate analogs, which were synthesized by condensation of the corresponding acids with Schiff's bases [4-(4-substituted benzylideneamino)phenols] involving dicyclohexyl carbodiimmide (DCC) as mild dehydrating agent. The main objective is to reduce the GIT toxicity associated with acute and chronic NSAIDs use. Anti-inflammatory, analgesic as well as ulcerogenic activities of the prepared esters were evaluated in vivo and compared with that of ibuprofen as reference standard in all screenings, involving the carrageenan induced paw oedema model and hot plate method. Most of the synthesized esters showed remarkable analgesic and anti-inflammatory activities. Interestingly, all of the compounds were found to be non-ulcerogenic under the tested conditions. This evidence have suggested that modification of the carboxyl function of representative NSAIDs results in retained or enhanced anti-inflammatory and analgesic activities with reduced ulcerogenic potential. Additionally, a comparative AutoDock study into Cox 1 and Cox2 has been done involving both of rigid and flexible docking for potential selectivity of our compounds within different Cox enzymes and to find out the binding orientation of these novel esters into their binding site. Some of the newly prepared aforementioned compounds showed considerable more Cox2 over Cox1 binding affinities by flexible docking better than rigid one.

Tyrosine and glycine derivatives as potential prodrugs: design, synthesis, and pharmacological evaluation of amide derivatives of mefenamic acid.

This study deals with the synthesis, pharmacological activity, and kinetic studies of mefenamic acid (MA) prodrugs of tyrosine and glycine. The synthesis involved a series of protection and deprotection reactions. The hydrolysis of these prodrugs in the intestine was confirmed by hydrolysis kinetics studies in simulated gastric fluid, simulated intestinal fluid, and 80% plasma. The prodrugs were also evaluated for analgesic, anti-inflammatory, and ulcerogenic activities. The glycine prodrug showed maximum analgesic activity of 86%, and both tyrosine and glycine prodrugs showed better anti-inflammatory activity of 74% and 81%, respectively, when compared to the 40% of MA. Further, the prodrugs showed fewer gastric ulcers compared to MA; tyrosine and glycine prodrugs had an average ulcer index of 9.1 and 4.5, respectively, while an average ulcer index of 24.2 was observed with MA. These findings suggest that both prodrugs are better in action as compared to MA, and are advantageous in having fewer gastrointestinal side effects.

Application of diffusion-edited NMR spectroscopy for the structural characterization of drug metabolites in mixtures.

Diffusion-edited NMR spectroscopy is used to enable the structural characterization of low level metabolites in the presence of endogenous compounds, and organic solvents. We compared data from standard one-dimensional (1D) (1)H, 1D NOESY-presaturation, and 1D diffusion-edited experiments run on 20 microg and 100 microg samples of ethacrynic acid glutathione thioether (EASG) and a previously unreported metabolite of mefenamic acid, mefenamic acid glutathione thioester (MSG). The 1D NOESY-presaturation technique gave spectra with the best signal-to-noise (S/N) ratio, approximately three times that observed with the standard (1)H experiment, with respect to the metabolite signals. However, it was not selective for solvent signals as overlapping metabolite signals were also suppressed by this technique. In some cases, these signals were key to determining the site of glutathione attachment on the parent molecule. 1D NOESY-presaturation spectra also produced baseline distortions and inconsistent integration values. By comparison, 1D diffusion-edited experiments were found to selectively and simultaneously remove multiple solvent signals, resolve overlapping metabolite signals, and provide more uniform integration for metabolite signals overlapping with or proximal to solvent peaks, without producing baseline distortions. However, the diffusion-edited experiments caused significant signal attenuation of the metabolite signals when compared with a standard (1)H spectrum. Partially purified metabolites isolated from biological matrices were also characterized by using two-dimensional diffusion-ordered spectroscopy (DOSY). DOSY spectra acquired on a sample of EASG purified from rat bile proved useful in 'separating' the signals of EASG, from those of a co-eluting bile acid and parent drug ethacrynic acid (EA) in the diffusion-dimension in regions where there was no spectral overlap. In the low-field regions of high overlap, the DOSY experiment did not effectively separate the signals from the individual components. Diffusion based experiments provide a way to determine the total number of components that are present in a metabolite sample as well as an ability to identify them based on the chemical shift information, without the need for laborious chromatography on small samples.

Glycerolysis of acyl glucuronides as an artifact of in vitro drug metabolism incubations.

During an investigation of the in vitro glucuronidation of benoxaprofen by human liver S-9 fraction, an unusual drug-related entity possessing a protonated molecular ion that was 74 mass units greater than the parent drug was observed. It was identified as the glycerol ester of benoxaprofen. Formation of this entity required inclusion of uridine diphosphoglucuronic acid (UDPGA) in the incubation, suggesting the formation of benoxaprofen acyl glucuronide followed by transesterification with the glycerol present in the incubation due to its presence as a stabilizer for liver subcellular fractions. Formation occurred during the sample work-up procedure while the samples were subjected to evaporation in vacuo, which does not remove glycerol. Conversion of purified benoxaprofen acyl glucuronide to the glycerol ester was demonstrated in glycerol at 37 degrees C. Other drugs that are converted to acyl glucuronides in vitro (diclofenac, mefenamic acid, tolmetin, and naproxen) were also shown to form corresponding glycerol esters when incubated with human liver S-9 fraction and UDPGA. The potential formation of glycerol esters of carboxylic acid drugs undergoing acyl glucuronidation in vitro represents an experimental artifact to which drug metabolism scientists should be aware.

Experimental and computational studies of epithelial transport of mefenamic acid ester prodrugs.

PURPOSE: A series of ester derivatives of mefenamic acid were synthesized with the aim of suppressing local gastrointestinal toxicity of mefenamic acid. A computational method was used to assist the design of the prodrug and to gain insights into the structure relationship of these compounds as P-glycoprotein (P-gp) substrates. The prodrugs were studied for their enzymatic stability, bidirectional permeability across Caco-2 monolayer, and their potential as transporter modulators METHODS: Bidirectional transport studies were performed using Caco-2 cells. Compounds exhibiting an efflux ratio of > or =2 were further examined for their potential interaction with P-gp and multidrug resistance-associated protein (MRP) using verapamil and indomethacin. Calcein efflux inhibition studies were conducted to investigate the efflux mechanism of these compounds. Geometry optimization of the esters was performed, and the spatial separation of two electron donor groups of each prodrug was measured. RESULTS: Morpholinoethyl ester (3) and pyrrolidinoethyl ester (4) of mefenamic acid showed evidence of efflux mechanism. Inhibition by verapamil had a pronounced effect on the transport of 3 and 4. Indomethacin, however, completely inhibited the apical efflux of 3 but enhanced the efflux ratio of 4. Both compounds increased the ratio of cellular calcein accumulation by 3- to 5-fold over control. Consistent with the experimental data, the computational results suggest the involvement of P-gp or its interaction in 3 and 4 transport. CONCLUSIONS: Apical efflux of 3 is associated with P-gp and MRP, but the efflux of 4 involves P-gp and/or MRP. The computational approach used in this study provided the basis for P-gp substrates of compounds 3 and 4 from their electron donor subunits spatial separation.

Synthesis and analgesic activity of N-Arylhydrazone derivatives of mefenamic acid.

PURPOSE: A series of N-Arylhydrazone derivatives of mefenamic acid (a known non-steroidal anti-inflammatory drug) were synthesized in order to obtain new compounds with potential analgesic and anti-inflammatory activity. METHODS: The structures of all synthesized compounds were confirmed by means of infrared, proton magnetic resonance and mass spectroscopy. All compounds were evaluated for their analgesic and anti-inflammatory activities by abdominal constriction test (writhing test) and carrageenan-induced rat paw edema test respectively. RESULTS: Most of the synthesized compounds induced significant reduction in the writhing response when compared to control. Among them, compounds 11, 12, 15, 16, 19, 20, and 21 were significantly more potent than mefenamic acid in the writhing test. The anti-inflammatory activity of these 7 compounds were evaluated and compounds 11, 12, 16, 19 and 20 showed significant anti-inflammatory activity in comparison to control but their effect was weaker than mefenamic acid. CONCLUSIONS: The antinociceptive relative activity of some of these newly synthesized compounds is greater than mefenamic acid but they are not potent anti-inflammatory agents.

Synthesis and evaluation of some acyloxyethyl mefenamates as possible prodrugs.

Various acyloxyethyl mefanamates were synthesized and evaluated for potential application as prodrugs. Their kinetics of hydrolysis were examined in aqueous solutions of pH 1.0 and 7.4 and in human plasma at 37 degrees C. Among the synthesized compounds, the beta-carboxypropionylethyl mefenamate and the pivaloyloxyethyl mefenamate show high stability against enzymatic and non enzymatic hydrolysis. On the other hand the acetyloxyethyl mefenamate shows t1/2s of 1.4 h, 1.41 h and 3.61 h in human plasma, solutions of pH 7.4 and pH 1.0 respectively; However, its hydrolysis to mefenamic acid in plasma was not quantitative. Preliminary in vivo study shows that acetyloxyethyl mefenamate gave plasma concentration of mefenamic acid lower than that of control after oral administration. The calculated AUC0-inf for the acetyloxyethyl and control were 45 and 85 micrograms.h/ml respectively.

Reactivity of mefenamic acid 1-o-acyl glucuronide with proteins in vitro and ex vivo.

Mefenamic acid is a nonsteroidal anti-inflammatory drug commonly used in analgesia. The use of this drug has been implicated in several cases of nephrotoxicity including acute renal failure and tubulointerstitial nephritis. One theory of drug-induced tubulointerstitial nephritis is that the drug or a derivative of the drug becomes irreversibly bound to certain sites in renal tissue and an immune response is directed against the hapten-host conjugate. Previous studies have shown that in humans the nonsteroidal anti-inflammatory drug mefenamic acid is metabolized by both phase I enzymes and the phase II enzyme family UDP-glucuronosyltransferase. Indeed, three glucuronides were identified and isolated from human urine by semipreparative HPLC after oral administration of mefenamic acid. This study focuses on mefenamic acid glucuronide and further characterizes this acyl glucuronide in terms of stability and its ability to bind irreversibly to proteins. Stability studies of mefenamic acid glucuronide in aqueous buffer highlighted the relative stability of this acyl glucuronide at physiological pH. The half-life at 37 degrees C, pH 7.4, was 16.5 +/- 3.1 hr, which is considerably longer than those reported for many acyl glucuronides. The degradation of mefenamic acid glucuronide was accelerated under alkaline conditions, decreasing the half-life to 5 +/- 1.6 hr at pH 8.0. Mefenamic acid glucuronide, although extremely stable in buffer at physiological pH, was found to bind irreversibly to human serum albumin in vitro. Irreversible binding to cellular proteins in culture was also evident with the addition of mefenamic acid to the heterologous Chinese hamster lung fibroblast cell line V79 expressing the human UDP-glucuronosyltransferase isoenzyme UGT1*02. This binding was directly related to glucuronide formation, because irreversible binding was not evident in the untransfected cell line V79.

Bromoacetamido analogs of indomethacin and mefenamic acid as affinity-labeling agents and mechanistic probes for prostaglandin H2 synthase.

Affinity-labeling agents, 1-[4-(bromoacetamido)benzyl]-5-methoxy-2-methylindole-3-acetic acid (I) and 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (II), were synthesized on the basis of their respective nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and mefenamic acid [Askonas & Penning (1991) Biochemistry 30, 11553-11560]. Compounds I and II are now shown to inhibit homogeneous ram seminal vesicle prostaglandin H2 (PGH2) synthase by two kinetically distinct complexes. They are competitive inhibitors versus arachidonic acid via the formation of high-affinity E.I complexes, and they cause time-dependent inactivation of the holoenzyme via low-affinity E.I complexes. Compounds I and II, unlike classical NSAIDs, were found to inactivate both the cyclooxygenase and peroxidase reactions of the synthase in a parallel manner. Inactivation was accompanied by the incorporation of 2 mol of either radiolabeled I or II per synthase monomer. The covalent bonds that result were stable to boiling in SDS, indicating that I and II offer alternatives to aspirin in locating NSAID binding sites. Incubation of aspirin-treated PGH2 synthase with radiolabeled I reduced the stoichiometry of incorporation to 1.0, suggesting that one of the sites modified corresponds to the cyclooxygenase site. By saturating the cyclooxygenase site with mefenamic acid, I and II only abolished the peroxidase activity of the enzyme, suggesting that the second site of modification corresponds to the peroxidase site. When PGH2 synthase was incubated with mefenamic acid and I or II, only the peroxidase activity was inactivated. Subsequent removal of all drugs by dialysis gave a preparation of PGH2 synthase that could perform the cyclooxygenase reaction, but lacked the ability to cleave ethyl hydroperoxide to ethanol and water.(ABSTRACT TRUNCATED AT 250 WORDS)

Structures of mefenamic acid metabolites from human urine.

Three major metabolites of mefenamic acid were isolated from the urine of a normal adult man receiving mefenamic acid orally. The structures of those metabolites were determined as glucuronides of mefenamic acid, its hydroxymethyl derivative, and its carboxylic acid derivative on the basis of spectral data.

Oral antipyretic therapy: evaluation of the N-aryl-anthranilic acid derivatives mefenamic acid, tolfenamic acid and flufenamic acid.

The antipyretic activity of three N-aryl-anthranilic acid derivatives, mefenamic acid, tolfenamic acid and flufenamic acid, was compared and their optimal antipyretic dose determined in a trial in 87 children (aged 5 months to 15 years), who suffered from infections and fever exceeding 38.5 degrees C. Tolfenamic acid proved to be the most potent antipyretic agent of the three drugs; it was eight times more powerful than mefenamic acid and three times more powerful than flufenamic acid. The optimal antipyretic doses were: mefenamic acid 4 mg/kg, tolfenamic acid 0.5 mg/kg and flufenamic acid 1.5 mg/kg. It is evident that the antipyretic activity of these anthranilic acid derivatives is even greater than their antirheumatic effect, the difference being most noticeable in the case of tolfenamic acid.