Acridonacetic acid: pharmacological properties and clinical use

The summarized data on the biological activity of acridoneacetic acid, a low-molecular interferon inducer are presented: the amount and type of induced interferon, producer cells, interaction with viruses; the antiviral, antitumoral and anti-inflammatory activity of the preparation has been demonstrated. The specific features of interferon induction by virus (a biological inducer), in contrast to acridoneacetic acid (a chemical inducer) have been noted. The possible mechanisms of interferon induction by low-molecular interferonogen are considered. A number of interferonogens, salts of acridoneacetic acid, proposed as medicinal preparations are considered. The results of the clinical use of Cycloferon, a medicinal form of acridoneacetic acid, are presented.     

Some biochemical and pharmacological properties of an epoxide metabolite of alclofenac

The properties of an epoxide metabolite of alclofenac have been investigated in a number of in vitro and in vivo tests. Alclofenac epoxide was shown to inhibit the activity of yeast alcohol dehydrogenase and form a conjugate with cysteine. The epoxide, but not alclofenac itself, showed mutagenic effects on strains of Salmonella typhimurium sensitive to alkylating agents, but had no effect on strains sensitive to intercalating agents. In addition the epoxide was active in a cell transformation assay using as a target Syrian hamster cells. No acute toxic reactions were observed in mice treated with alclofenac epoxide and the compound was devoid of analgesic and acute antiinflammatory activity. Alclofenac epoxide was found to be a sensitising agent in the guinea-pig when administered either by injection with complete Freund's adjuvant or topically as an ethanolic solution. It is postulated that the formation of the epoxide may explain some of the therapeutic and toxicological properties of alclofenac in man.     

A novel eicosanoid is the major arachidonic acid metabolite of cultured human monocytes

Human peripheral blood monocyte-macrophages (M) generate a novel eicosanoid during in vitro culture. The metabolite is generated during incubation of the cells with ¹⁴C — arachidonic acid (AA). Lack of prior recognition of this metabolite probably results from the facts that: 1) on thin-layer chromatography (TLC) in two standard solvent systems, the novel metabolite co-chromatographed with either prostaglandin D₂ or thromboxane B₂, and 2) its generation, under the conditions studied, does not occur until between 90 and 180 minutes after culture initiaton which is a time period beyond that used for most leukocyte studies. The generation of the metabolite is inhibited by nordihydroguaiaretic acid (NDGA) but not by indomethacin. Base hydrolysis did not alter its migration on TLC. On both reversed phase and straight phase high pressure liquid chromatography (HPLC), the novel peak isolated by TLC elutes as a single major peak of radioactivity with a retention time different from the known leukotrienes, hydroxy acids, or their metabolites. Furthermore, the peak isolated on HPLC has a single ultraviolet absorption maximum at 270 nm. M cultured for 1 week prior to a 24 hour incubation with ¹⁴C-AA generated proportionally less of the novel eicosanoid (roughly 68% of total radiolabeled product) than did M cultured for 3 weeks prior to a similar incubation with ¹⁴C-AA (roughly 86% of total radiolabeled product). Under the conditions studied, the novel eicosanoid is the major AA metabolite generated from exogenous AA by cultured M and it appears to be generated in increasing quantity as the M differentiate.     

A novel eicosanoid is the major arachidonic acid metabolite of cultured human monocytes

Human peripheral blood monocyte-macrophages (M phi) generate a novel eicosanoid during in vitro culture. The metabolite is generated during incubation of the cells with 14C - arachidonic acid (AA). Lack of prior recognition of this metabolite probably results from the facts that: 1) on thin-layer chromatography (TLC) in two standard solvent systems, the novel metabolite co-chromatographed with either prostaglandin D2 or thromboxane B2, and 2) its generation, under the conditions studied, does not occur until between 90 and 180 minutes after culture initiation which is a time period beyond that used for most leukocyte studies. The generation of the metabolite is inhibited by nordihydroguaiaretic acid (NDGA) but not by indomethacin. Base hydrolysis did not alter its migration on TLC. On both reversed phase and straight phase high pressure liquid chromatography (HPLC), the novel peak isolated by TLC elutes as a single major peak of radioactivity with a retention time different from the known leukotrienes, hydroxy acids, or their metabolites. Furthermore, the peak isolated on HPLC has a single ultraviolet absorption maximum at 270 nm. M phi cultured for 1 week prior to a 24 hour incubation with 14C-AA generated proportionally less of the novel eicosanoid (roughly 68% of total radiolabeled product) than did M phi cultured for 3 weeks prior to a similar incubation with 14C-AA (roughly 86% of total radiolabeled product). Under the conditions studied, the novel eicosanoid is the major AA metabolite generated from exogenous AA by cultured M phi and it appears to be generated in increasing quantity as the M phi differentiate.     

Unaided trifluoroacetic acid pretreatment solubilizes polyglutamine peptides and retains their biophysical properties of aggregation

Understanding the biophysical mechanism of polyglutamine (polyGln) aggregation is important to unravel the role of aggregates in the pathology of polyGln repeat disorders. To achieve this, synthetic polyGln peptides are widely used. Their disaggregation and solubilization is essential because it plays a crucial role in reproducing biophysical experimental data under in vitro conditions. Pretreatment with trifluoroacetic acid (TFA) and hexafluoroisopropanol (HFIP) at a 1:1 ratio is currently the method of choice to achieve solubility of polyGln peptides. Here we report that the disaggregation and solubilization of polyGln peptides can be achieved by TFA alone. We tested TFA due to the close similarity of it with HFIP in the nature of H-bond breakage and formation, higher cost, and the problems faced by us in the availability of HFIP. Our results demonstrate that the TFA disaggregated polyGln sequences give similar solubilization yield, aggregation kinetics, thioflavin T (ThT) binding, and structural features in comparison with the TFA/HFIP method. Furthermore, we show by limited validation studies that the proposed TFA method can replace the existing TFA/HFIP disaggregation method of polyGln sequences.     

15(S)-hydroxyeicosatetraenoic acid is the major arachidonic acid metabolite in human bronchi: association with airway epithelium

15(S)-Hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) was by far the most abundant metabolite of arachidonic acid in chopped human bronchi, as identified by reverse phase HPLC, uv spectrometry, and GC/MS. The quantitation of monohydroxyeicosatetraenoic acids (mono-HETEs) was performed by the use of 16(S)-hydroxy-9(Z),12(Z),14(E)-heneicosatrienoic acid as internal standard. Thus, significant amounts of 15-HETE were obtained in incubations of bronchi in buffer alone, but the addition of exogenous arachidonic acid (3-100 microM), dose-dependently increased the formation, with maximal levels reached at around 10 min. In contrast, challenge with ionophore A23187 or anti-human IgE did not stimulate the production of 15-HETE in the bronchi. Nordihydroguaiaretic acid inhibited the production of 15-HETE, whereas indomethacin did not. Small amounts of 8,15-diHETEs were detected in incubations with exogenous 15H(P)ETE. Lipoxins were however not detected under any of the incubation conditions used. Furthermore, removal of the airway epithelium substantially diminished the production of 15-HETE in the bronchi. Finally, bronchi were obtained from three patients with asthma, and the amounts of 15-HETE in these specimens were significantly higher than those found in tissues from nonasthmatics. Also, in peripheral lung parenchyma and pulmonary blood vessels 15-HETE was the major mono-HETE after stimulation with arachidonic acid but the levels were about 10 times lower than in the bronchi. As another difference, challenge of the parenchyma with the ionophore A23187 made 5-HETE the predominant mono-HETE. Taken together, airway epithelium appears to be the major source of 15-HETE in the human lung and the findings in specimens of asthmatics raise the possibility that 15-HETE somehow is involved in airway inflammation.     

Raoultella planticola, a new strain degrading 2,4,5-trichlorophenoxyacetic acid

A new strain that degrades the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was isolated from soil, which was exposed to factors related to the petrochemical industry. According to its physiological, biochemical, cultural, and morphological traits, together with the sequence of the 16S rRNA gene, the strain was identified as Raoultella planticola 33-4ch. The strain could consume 2,4,5-T as a sole source of carbon and energy. The amount of 2,4,5-T in the culture medium decreased by 51% after five days of incubation. Raoultella planticola 33-4ch consumes 2,4,5-T to produce 4-chlorophenoxyacetic, phenoxyacetic, and 3-methyl-2,6-dioxo-4-hexenoic acids.     

Transformation of ferulic acid to 4-vinyl guaiacol as a major metabolite: a microbial approach

The majority of the flavours and fragrances used worldwide are produced by chemical synthesis at low price. However, consumers prefer natural compounds because of increasing health and nutrition awareness in routine life. Hence, biotransformation is an alternative process to produce natural aroma compounds. Microorganisms have been gradually used more to produce natural aroma compounds with various applications in food, agriculture and pharmaceutical industries. This paper reviews the role of microorganisms in the transformation of ferulic acid to 4-vinyl guaiacol. The microbial processes based on biocatalytic method are discussed in terms of their advantages over chemical synthesis, plant cell cultures and enzyme catalyzed reactions. Thus, the transformation of ferulic acid by microorganisms could have possible use in food, pharmaceutical industry and become an increasingly important platform for the production of natural aroma compounds.     

Ashwagandha leaf derived withanone protects normal human cells against the toxicity of methoxyacetic acid, a major industrial metabolite

The present day lifestyle heavily depends on industrial chemicals in the form of agriculture, cosmetics, textiles and medical products. Since the toxicity of the industrial chemicals has been a concern to human health, the need for alternative non-toxic natural products or adjuvants that serve as antidotes are in high demand. We have investigated the effects of Ayurvedic herb Ashwagandha (Withania somnifera) leaf extract on methoxyacetic acid (MAA) induced toxicity. MAA is a major metabolite of ester phthalates that are commonly used in industry as gelling, viscosity and stabilizer reagents. We report that the MAA cause premature senescence of normal human cells by mechanisms that involve ROS generation, DNA and mitochondrial damage. Withanone protects cells from MAA-induced toxicity by suppressing the ROS levels, DNA and mitochondrial damage, and induction of cell defense signaling pathways including Nrf2 and proteasomal degradation. These findings warrant further basic and clinical studies that may promote the use of withanone as a health adjuvant in a variety of consumer products where the toxicity has been a concern because of the use of ester phthalates.     

Rapid determination of chloroprocaine and its major metabolite, 2-chloroaminobenzoic acid, in plasma by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic method for determination of the 2-chloroprocaine, local anesthetic of ester type, and its major metabolite 2-chloroaminobenzoic acid, has been developed and validated. A single-step extraction procedure is employed followed by high-performance liquid chromatographic separation using reversed-phase column and analysis using variable length UV detection. Lidocaine was used as internal standard for 2-chloroprocaine measurement and p-aminobenzoic acid was used as internal standard for 2-chloroaminobenzoic acid analysis. The analysis of spiked plasma demonstrated good accuracy and precision of the method with limit of detection 0.1 μg/ml for 2-chloroprocaine and 0.5 μg/ml for 2-chloroaminobenzoic acid. The method has been used for pharmacokinetic studies in laboratory animals.     

Identification and quantitative determination of a major circulating metabolite of gambogic acid in human

Gambogic acid (GA), a promising anticancer candidate, is a polyprenylated xanthone abundant in the resin of Garcinia morella and Garcinia hanburyi. The major circulating metabolite of GA in human, 10-hydroxygambogic acid (10-OHGA), was identified by comparison of the retention time and mass spectra with those of reference standard using liquid chromatography–tandem mass spectrometry. The reference standard of 10-OHGA was isolated from bile samples of rats after intravenous injection of GA injection, and its structure was confirmed by NMR. Then, a selective and sensitive method was developed for the quantitative determination of this metabolite in human plasma. After liquid–liquid extraction by ethyl acetate, the analyte and the internal standard were separated on a Sepax HPC18 column (100 mm × 2.1 mm i.d., 3.0 μm) with a mobile phase of 10 mM ammonium acetate water solution containing 0.1% formic acid–acetonitrile (20:80, v/v). The detection was performed on a single quadrupole mass spectrometer equipped with electrospray ionization (ESI) source. The calibration curve was linear over the range of 3–2000 ng/mL for 10-OHGA. The developed quantification method can now be used for the pharmacokinetic and pharmacological studies of 10-OHGA after intravenous infusion of GA injection in human.     

Leukotriene C4: The major lipoxygenase metabolite of arachidonic acid in dog spleen

Slices of dog spleen converted [¹⁴C]-arachidonic acid (AA) to a polar material which conjugated with [³H]-glutatione. Nordihydroguaiaretic acid (NDGA) and 5,8,11,14, Eicosatetraynoic acid (ETYA) but not indomethacin, inhibited the conversion of [¹⁴C]-arachidonic acid by the spleen slices into the polar material indicating that it is derived through the lipoxygenase pathway. Physicochemical analysis of the polar metabolite of arachidonic acid after thin-layer chromatography and high pressure liquid chromatography revealed that it has chemical properties identical to authentic leukotriene C₄ standard (LTC₄). The biological activity of the purified material was found to be similar to the slow reacting substance of anaphylaxis (SRS-A), viz, it caused contraction of the guinea-pig ileum which was abolished by FPL-55172, a specific SRS-A receptor antagonist. These data suggest that dog spleen slices convert arachidonic acid through lipoxygenase pathway into a polar material that appears to be identical to LTC₄.     

Synthesis and stereochemical determination of diunsaturated valproic acid analogs including its major diunsaturated metabolite

Valproic acid, an antiepileptic drug, is transformed into diunsaturated metabolites in humans. Synthesis of the geometric isomers of 2-(1'-propenyl)-2-pentenoic acid and 2-(1'-propenyl)-3-pentenoic acid was attempted using known procedures. The final product, a mixture of isomers, was converted into tert-butyldimethylsilyl or ethyl derivatives. Capillary gas-liquid chromatography-mass spectrometry analysis of the derivatives showed at least three isomeric dienoic acids from synthesized products. Argentation thin-layer chromatography was effective in resolving the isomeric mixture into a single isomer or mixture of two isomers. Thin-layer chromatography and gas-liquid chromatography retention data, photochemical isomerization studies, and nuclear magnetic resonance spectrometry were used to characterize the dienoic acids. By comparison of the retention times of the diunsaturated metabolites with synthesized reference compounds, the structure assigned to the major diunsaturated metabolite is 2-[(E)-1'-propenyl](E)-2-pentenoic acid.     

Peptide binding to HLA-DR1: a peptide with most residues substituted to alanine retains MHC binding

Major histocompatibility complex (MHC) glycoproteins play an important role in the development of an effective immune response. An important MHC function is the ability to bind and present 'processed antigens' (peptides) to T cells. We show here that the purified human class II MHC molecule, HLA-DR1, binds peptides that have been shown to be immunogenic in vivo. Detergent-solubilized HLA-DR1 and a papain-cleaved form of the protein lacking the transmembrane and intracellular regions have similar peptide binding properties. A total of 39 single substitutions were made throughout an HLA-DR1 restricted hemagglutinin epitope and the results determine one amino acid in this peptide which is crucial to binding. Based on this analysis, a synthetic peptide was designed containing two residues from the original hemagglutinin epitope embedded in a chain of polyalanine. This peptide binds to HLA-DR1, indicating that the majority of peptide side chains are not required for high affinity peptide binding.     

DNA cleavage by phenylhydroquinone: the major metabolite of a fungicide o-phenylphenol

The interaction of o-phenylphenol (OPP) and its metabolites with DNA was examined. As a model system, the reactivities of OPP and its metabolites with DNA were studied by using pUC18 DNA. The major metabolite formed in vitro from OPP by mixed function oxidase was phenylhydroquinone (PHQ). This result corresponds to the findings that PHQ in the form of glucuronide conjugate was the main product detected in bladder of OPP fed rats in vivo. When supercoiled pUC18 DNA (form I) was incubated with PHQ at concentrations from 1 X 10(-6) M to 2 X 10(-1) M, DNA strand scission by PHQ was observed at a concentration as low as 1 X 10(-5) M and the amount of linear form (form III) increased with increasing PHQ concentration. PHQ causes DNA strand scission. The DNA cleavage by OPP and phenyl-p-benzoquinone (PBQ) was barely detectable. The DNA cleavage by PHQ was inhibited by superoxide dismutase (SOD), catalase and several oxygen radical scavengers such as polyethylene glycol, tert-butanol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine. The production of superoxide radical from PHQ was confirmed by cytochrome c reduction assay. These results indicate that the oxygen radicals such as superoxide, hydroxyl radicals and some others generated in the process of oxidation of PHQ in aqueous solution are responsible for the DNA cleavage. In order to identify the sites of cleavage of DNA by PHQ, a 5'-end 32P-labeled 206 base-pair EcoRI-BglI fragment of pUC18 DNA was incubated with PHQ. The DNA was then analyzed by sequencing gel electrophoresis followed by autoradiography. When the DNA was incubated with PHQ and further treated with piperidine, cleavage was detected relatively more frequently at guanine residues. The attack seemed to occur at guanine residues in general, but was not restricted to guanines with specific residues in the vicinity.     

Synthesis and analysis of conjugates of the major vitamin E metabolite,alpha-CEHC

Glucuronide and sulphate conjugates of 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), the major metabolite of alpha-tocopherol (vitamin E), have been synthesized and used for the first direct analysis of conjugated urinary vitamin E metabolites. The metabolites of vitamin E (alpha-tocopherol) could be useful as markers of the function(s) of vitamin E in vivo. A number of methods have been described for the analysis of urinary vitamin E metabolites but these have relied on either acid or enzymatic deconjugation of the metabolites prior to analysis by high performance liquid chromatography or gas chromatography/mass spectrometry. These methods have provided useful information about the amount and types of metabolites excreted in the urine but suffer from a number of disadvantages. Deconjugation has been shown to produce artifacts as a result of the conversion of alpha-CEHC to alpha-tocopheronolactone and the efficiency of deconjugation is also difficult to assess. Methods that allow the direct measurement of the conjugated metabolites would overcome these problems and would also substantially reduce the preparation and analysis time. Here we describe the use of conjugated standards to characterize alpha-CEHC conjugates in human urine by tandem mass spectrometry (MS-MS). The future use of MS-MS to measure urinary vitamin E metabolites is also discussed.