Digoxigenin Monodigitoxoside Research Articles

Phytochemicals, antioxidants, estimation of cardiac glycoside (Scillaren A) and detection of major metabolites using LC-MS from Drimia species

Drimia Jacq., a bulbous geophyte belonging to family Hyacinthaceae Batsch ex Borkh. is well known for its medicinal values. Leaves and bulbs of eight genotypes of Drimia were sequentially extracted with three solvents (DCM, MeOH and H2O) and analysed for their phytochemicals (TPC, TFC and TTC) and in vitro antioxidant activities (DPPH, ABTS radical scavenging activities and phosphomolybdenum assay). Bulbs of D. indica (triploid form) extracted with MeOH showed the highest TPC (22.19 ± 1.00 mg GAE/g DW). Higher TFC was recorded in MeOH extract of bulbs of D. raogibikei (58.65 ± 1.95 mg QE/g DW) whereas significantly maximum TTC (857.17 ± 38.62 mg GAE/g DW) was found in MeOH extract of D. razii. DPPH radical scavenging activity was highest in DCM bulb extract of D. coromandeliana (12.22%). DCM extract of D. polyantha showed greatest (64.26%) ABTS radical scavenging activity. Phosphomolybdenum reduction potential was maximum in MeOH leaf extract of D. indica (triploid form) (52.86 ± 0.20 mg AAE/g DW). RP-HPLC estimation of cardiac glycoside (Scillaren A) from the bulbs of eight genotypes of Drimia revealed the highest (5.54 ± 0.03 mg/g DW) quantity in DCM extracts of D. coromandeliana. Presence of a bufadienolide (Scillaren A) was confirmed by FTIR and HR-LCMS. Additionally, HR-LCMS analysis revealed the presence of three cardenolides (convallatoxin, digoxigenin monodigitoxoside and peruvoside) and 160 major metabolites in the bulbs of Drimia species. Present study is the first comparative account of phytochemicals, antioxidants, cardiac glycosides and LC-MS profiling of major metabolites from eight Indian Drimia genotypes.

Molecular docking and ADMET-based mining of terpenoids against targets of type-II diabetes

Despite the availability of various medicines for the treatment of diabetes, plant-based drugs draw special attention due to their low cost and lesser side effects. The rhizomes of plant Hedychium coronarium have been reported for its anti-diabetic activity previously. In the present study, docking and computational ADME parameters of a few terpenoid ligands isolated from the above-mentioned plant against two control molecules (Sitagliptin, Metformin) were compared. The retrieved docked images indicated the docking sites in the target protein. Out of the five compounds, Digoxigenin monodigitoxoside has shown the best docking result. The LD50 of this compound is very similar to control and lower than others. This in silico study confirmed that terpenoids present in the plant rhizome are a potent drug candidate for the treatment of type-II diabetes. We have identified a few best-fit terpenoid molecules against identified targets using in silico studies, which may give new drugs to combat type-II diabetes after wet-lab validation.

Properties of Novel Anti-digoxin Antisera in Radioimmunoassay Using Homologous and Site Heterologous Tritium-Labeled Antigens Involving a [3H]-Leucine Moiety

The specificities of antisera against digoxin C-3' or C-3'' hemisuccinate-bovine serum albumin (BSA) conjugate were assessed by cross-reactivity studies with digoxin metabolites by radioimmunoassay (RIA) using the homologous and the site heterologous tritium-labeled antigens. One of the tracers used was digoxin 3'-hemisuccinyl-[3H]-leucine; the other was digoxin 3''-hemisuccinyl-[3H]-leucine, which had been prepared from digoxin 3''-hemisuccinate. When the tracer with [3H]-leucine at the C-3' position was used, antisera (I-1, I-3) elicited by digoxin 3'-hemisuccinate-BSA conjugate showed the following cross-reactivity: digoxigenin bisdigitoxoside (0.34%, 76%), digoxigenin monodigitoxoside (0.11%, 65%), digoxigenin (0.02%, 26%) and dihydrodigoxin (9.4%, 1.2%). However, when using the homologous antigen, antiserum (I-1) was highly specific against the digitoxose chain. When the site heterologous antigen, digoxin 3''-hemisuccinyl-[3H]-leucine was combined, this antiserum showed high cross-reactivity to digoxin degradation products. This digoxin RIA using antiserum (I-1) with the homologous antigen measures unmetabolized digoxin. On the other hand, the RIA system using antiserum (I-3) with the homologous antigen had cross-reactivity with the metabolites in accordance with their relative cardio-activities, so this system would be useful in therapeutic drug monitoring of digoxin.

Preparation of specific antisera to digoxin by using digoxin C-3' and C-3" hemisuccinate-bovine serum albumin conjugates.

The preparation and antigenic properties of digoxin C-3' and C-3" hemisuccinate-bovine serum albumin (BSA) conjugates are described. The antisera were prepared by immunizing rabbits with each of the digoxin-BSA conjugates, and properties of the antisera were characterized by RIA with 3H-labeled digoxin. The antidigoxin antiserum from immunization with digoxin 3'-hemisuccinate-BSA conjugate possessed high specificity for digoxin, exhibiting fairly low cross-reactions with dihydrodigoxin (2.1%), digoxigenin monodigitoxoside (0.9%), digoxigenin bisdigitoxoside (0.6%), and digoxigenin (0.1%).

Metabolism of digoxin and digoxigenin digitoxosides in rat liver microsomes: involvement of cytochrome P4503A

1. The sequential metabolism of digoxin (Dg3) to digoxigenin bis-digitoxoside (Dg2), digoxigenin mono-digitoxoside (Dg1) and digoxigenin (Dg0) was investigated in rat liver microsomes. 2. Kinetic studies produced results consistent with a single enzyme mechanism describing the successive oxidative cleavages. Formation of Dg2 was catalysed with mean (SD) Km and Vmax of 125 22 muM and 362 37/pmol/min/mg/protein, respectively. The corresponding values for the formation of Dg1 were 61 5 muM and 7 1 pmol/min/mg/protein. Dg0 formation was catalysed with the apparent values of 30 9 muM and 310 30 pmol/min/mg/protein. 3. Chemical inhibition of cytochrome P450 (CYP) 3Asubfamily with ketoconazole and triacetyoleandomycin decreased the formation of Dg2 and Dg1 by up to 90%. Antibodies specific to rat CYP3A2 lowered the rate of oxidative cleavage of Dg3 and Dg2 by up to 85%. Inhibition of CYP2E1, CYP2C subfamily and CYP1A2 by chemical and immunoinhibition did not affect initial rates of metabolism of Dg3 and Dg2. In contrast, Dg1 metabolism was not affected by triacetyloleandomycin as well as by antibodies to CYP3A2, CYP2C11, CYP2E1, CYP2B1 2B2 and CYP1A2. It was however inhibited by 80% by gestodene and 17 alpha -ethynylestradiol (selective inhibitors of human CYP3A). 4. Collectively, these data support the involvement of CYP3A in the cleavage of Dg3 and Dg2 in rat liver microsomes. The enzyme-metabolizing Dg1 remains to be identified.

Specific and sensitive determination of digoxin and metabolites in human serum by high performance liquid chromatography with cyclodextrin solid-phase extraction and precolumn fluorescence derivatization

A precolumn fluorescence derivatization high performance liquid chromatographic method has been developed for the simultaneous determination of digoxin and its metabolites digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside digoxigenin, and dihydrodigoxin (20- R and 20- S epimers) in human serum. Digoxin and its metabolites were extracted from serum samples (containing digitoxin as internal standard) with a cyclodextrin solid-phase extraction (SPE) column. Fluorescent derivatives were formed by reaction of the analytes with 1-naphthoyl chloride in the presence of 4-dimethylaminopyridine under a nitrogen atmosphere in a glove box with controlled relative humidity (26% r.h. or less). The derivatives were isolated using cyclodextrin and Cl SPE columns sequentially, and determined by HPLC using silica column separation and fluorescence detection. Calibration curves were linear over the concentration range from 0.25 to 4.0 ng ml −1. Recoveries of digoxin and its metabolites from serum ranged from 62 to 86%, and coefficients of variation from repetitive analyses ranged from 6.9 to 20.9% and from 5.8 to 12.2% at 0.5 ng ml −1 and 2.0 ng ml −1, respectively. This method has been shown capable of specifically determining digoxin and its major metabolites in serum, and has been successfully used in the determination of digoxin and its metabolites in serum samples collected from patients undergoing digoxin therapy. This method thus permits the investigation of digoxin metabolism and pharmacokinetics after the administration of commercial dosage forms.

Regioselective schiff's base mediated deglycosidation of digitalis glycosides. New efficient synthesis of digoxigenin bis-digitoxoside and digoxigenin mono-digitoxoside

Dialdehydes derived from digoxin glycosides via sodium periodate oxidation were regioselectively deglycosylated by various amino acids in anhydrous methanolic solution to afford excellent yields of digoxigenin bis-digitoxoside and digoxigenin mono-digitoxoside in a single step.

Metabolism of digoxin, digoxigenin digitoxosides and digoxigenin in human hepatocytes and liver microsomes

In vitro metabolism of digoxin and its cleavage-related compounds was investigated using hepatocytes in primary culture and microsomal fractions both isolated from human livers. On these models, digoxin (DG3) and digoxigenin bisdigitoxoside (DG2) were not shown to be significantly metabolized in vitro in man. Therefore, it appeared that the stepwise cleavage of DG3 and DG2 sugars was not cytochrome P450 dependent. This enzymatic system probably plays a minor role in humans for this particular reaction. However, digoxigenin monodigitoxoside (DG1) and digoxigenin (DG0) which are known to be formed after intra-gastric hydrolysis of DG3, were extensively converted to polar compounds (mainly glucuronides). In addition, using human liver microsomes, a wide variability in UDP-glucuronyl transferase (UDPGT) activities responsible for DG1 glucuronidation was demonstrated. These results suggest that two main factors may contribute to the overall interindividual variability of digoxin biotransformation: 1), the individual intra-gastric pH which influences the sugar cleavage leading to DG1 and DG0; ii), a variability in the level of the hepatic UDPGT specific for digitalis compounds conjugation.

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Beta-methyldigoxin (β-MD) is a cardiac glycoside widely used to treat heart failure and arrhythmia but its pharmacokinetics is not well known. Pharmacokinetic characteristics of β-MD were studied in five healthy volunteers. After a single oral dose of β-MD (0.2mg), the serum glycoside concentrations and urinary glycoside excretions were measured over seven days. The concentrations of β-MD and its metabolites in serum and urine were deter mined by high performance liquid chromatography-fluorescence polarization immunoassay method.β-MD appeared in serum 15min after administration and reached the peak serum concentration (1.88±0.71ng/ml) at 0.70±0.33hr. Elimination half-life, volume of distribution, and renal clearance were found to be 46.5±14.1hr, 9.5±2.4l/kg, and 46.0±14.3ml/min, respectively. These kinetic parameters were different from data reported previously . Digoxin was the only metabolite observed in serum and its peak concentration was 0.31± 0.07ng/ml at 1.10±0.34hr. The percentages of the β-MD dose renally excreted by 156 hr as β-MD, digoxin, digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside, and digoxigenin were 29.55, 23.42, 1.28, 0.36, and 0.50%, respectively. From serum concentration-time curves, the presence of enterohepatic circulation of β-MD was suggested.

Reduction of digoxin to 20R-dihydrodigoxin by cultures of Eubacterium lentum.

The anaerobic bacterium Eubacterium lentum, a common constituent of the intestinal microflora, inactivates digoxin by reducing the unsaturated lactone ring. Reduction of the cardiac glycoside by growing cultures of E. lentum ATCC 25559 proceeded in a stereospecific manner, with the 20R-dihydrodigoxin constituting more than 99% of the product formed. This is in contrast to the 3:1 ratio of 20R and 20S epimers formed in the chemical catalytic hydrogenation. Formation of the reduced glycosides proceeded quantitatively when an overall concentration of 10 micrograms/ml was added to the cultures. E. lentum did not hydrolyze the digitoxose sugars from C-3 of the parent glycoside. However, the synthetically prepared sugar-hydrolyzed metabolites (digoxigenin, digoxigenin monodigitoxoside, and digoxigenin bisdigitoxoside) were reduced to the corresponding dihydro metabolites. Repetition of the experiments with a feces sample from a volunteer who was known to be a converter of digoxin to dihydrodigoxin gave results identical to those obtained with pure E. lentum cultures.

Digoxin and metabolites in urine and feces; a fluorescence derivatization—high-performance liquid chromatographic technique

A high-performance liquid chromatography method is described for the determination of digoxin and its metabolites digoxigenin, digoxigenin monodigitoxoside, digoxigenin bis-digitoxoside and dihydrodigoxin (20 S and 20 R) excreted in urine and feces. The urine sample or fecal supernatant is extracted with methylene chloride in the presence of digitoxigenin or digitoxin as internal standard. Pre-column derivatization is achieved using 1-naphthoyl chloride with subsequent separation of the derivatized compounds on either a normal- or reversed-phase system with fluorescence detection. Recoveries for digoxin and all metabolites from fecal samples were in the range 60–74%, which is comparable to that previously determined for urine samples. Standard curve data revealed linearity over a wide range of concentrations. Coefficients of variation for the analysis were less than 10% for all compounds over a range of 5–125 ng per ml urine and 10–250 ng per 200 mg feces. Peaks for digoxin and metabolites in urine and feces were obtained when human excreta were analyzed.

Distribution of digoxin, digitoxin and their cardioactive metabolites in human heart and kidney tissue. A postmortem study

A method was developed for the specific determination of digoxin and digitoxin, as well as their semisynthetic derivatives and dependent cardioactive metabolites, in autopsy samples of heart and kidney. A collective of six patients on long-term treatment with therapeutic doses of beta-acetyldigoxin had a mean myocardial digoxin content of 46.1 +/- 25.0 ng/g (SD); kidney: 50.3 +/- 30.3 ng/g. Digoxigenin bisdigitoxoside represented the second most important metabolite in heart and kidney; digoxigenin monodigitoxoside and digoxigenin follow, respectively. In a collective of seven patients on maintenance treatment with digitoxin, the mean tissue levels were higher but the metabolic pattern was similar (myocardial digitoxin content: 78.9 +/- 38.4 ng/g, renal content: 104.1 +/- 44.1 ng/g). The amount of digoxin formed by hydroxylation under long-term treatment with digitoxin in heart and kidney were approximately 10 ng/g. A case of digoxin intoxication differed both in the tissue content and in the metabolic distribution.

Variable cross-reactivity of digoxin metabolites in digoxin immunoassays.

The authors investigated the cross-reactivity of the major known digoxin metabolites--digoxigenin, digoxigenin monodigitoxoside, digoxigenin bisdigitoxoside, and dihydrodigoxin--and of digitoxin in three 125I-radioimmunoassays and one enzyme immunoassay for digoxin. Digitoxin and dihydrodigoxin exhibit low cross-reactivity and nonparallel dilution responses for these assays. The cross-reactivities of the other three substances are significant for all assays studied with digoxigenin and monodigitoxoside having nonparallel and enhanced tracer displacement compared with digoxin itself. The authors demonstrate that because of nonparallel tracer displacement estimates of cross-reactivity calculated by the 50% displacement method fail to adequately predict the error induced in digoxin assays by digitoxin. They conclude that digoxin metabolites in serum are measured to various extents as the parent digoxin compound by all of the immunoassays they studied. In view of the varying biologic activity of digoxin metabolites and the large patient to patient variations in digoxin metabolism, the cross-reactivities the authors observe may help to explain the discrepancies in correlation of clinical response to measured serum digoxin values reported in other studies.

Digoxin and metabolites in urine: A derivatization—high-performance liquid chromatographic method capable of quantitating individual epimers of dihydrodigoxin

A high-performance liquid chromatographic method is described for the determination of digoxigenin, digoxigenin monodigitoxoside, digoxigenin bis-digitoxoside, digoxin, and dihydrodigoxin as the 3,5-dinitrobenzoyl esters. The method is applied to a 10 ml urine sample by adding digitoxigenin as internal standard, extracting with methylene chloride, derivatizing with 3,5-dinitrobenzoyl chloride in pyridine, chromatographing with a normal- phase system and detecting at 254 nm. Derivatized digoxigenin, digoxigenin mono- and bis- digitoxoside, and digoxin each yielded one symmetrical peak with the limit of sensitivity of the method being approximately 100 ng/ml. Analysis of a commercially obtained sample of dihydrodigoxin resulted in two well-separated, symmetrical peaks that represent the two epimers of derivatized dihydrodigoxin. Data indicate rapid and complete esterification of all primary and secondary alcohol moieties in the various molecules and the derivatives are shown to be stable in chloroform for at least four days. The procedure appears to be suitable for metabolic investigations and as a prototype for future analytical developments.

Influence of 16β formylation on Na, K-ATPase inhibition by cardiac glycosides

The inhibitory effect of formylated cardiac steroids (gitaloxin and its derivatives) on guinea-pig heart Na, K-ATPase was compared to that of other cardiac steroids with various hydroxy substituents. The decreasing order of potency of aglycones at equilibrium was as follows: gitaloxigenin greater than digitoxigenin greater than ouabagenin greater than digoxigenin greater than gitoxigenin greater than diginatigenin. This sequence was different to the sequence of drugs hydrophobic character. The compounds with hydroxy groups in the vicinity of the lactone ring (gitoxigenin, diginatigenin) were less potent than the hydrophilic compound ouabagenin. We propose that intramolecular bounding between 16 beta-OH and the lactone ring contributes to the relatively low potency of gitoxigenin and diginatigenin. The formylation of 16 beta-OH increased the potency of gitoxigenin by a factor of 41. The formylated compound (gitaloxigenin) was 5-fold more potent than digitoxigenin. The 3 beta-glycosylation of digoxigenin lead to pseudo-irreversible inhibitors of Na, K-ATPase. The half-time to achieve the equilibrium (for 5 mumol/l) was equal to 54 s, 90 s and 108 s respectively for digoxigenin monodigitoxoside, digoxin and desacetyllanatoside C. However, at equilibrium the three glycosides were equipotent, suggesting the existence of steric effects at the sugar site of the receptor. The sequence of potency observed for monodigitoxosides, monodigitalosides and tridigitoxosides after 60 min incubation was similar to that observed for the corresponding aglycones. These results suggest that the strongly negative inductive group 16 beta-OCHO is tightly bound to Na, K-ATPase, possibly to the same receptor site than that which is thought forming hydrogen and ionic bonds with the lactone ring. They show that the high toxicity of gitaloxin in guinea-pig heart is likely due to its high potency as Na, K-ATPase inhibitor.

On the substrate specificity of the digitoxigenin monodigitoxoside conjugating UDP-glucuronyltransferase in rat liver

The aim of the present study was to investigate the specificity of the UDP-glucuronosyltransferase (EC 2.4.1.17) involved in the conjugation of digitoxigenin monodigitoxoside. By in vitro assays with detergent activated liver musomes it was found that (1) digitoxigenin monodigitoxoside is by far the best substrate of all cardenolides and cardenolide digitoxosides tested. (2) In the presence of saturating UDP-glucuronate concentrations an apparent K m of 5.8 μM was obtained from linear Lineweaver-Burk plots together with a V max of about 150 pmoles/mg musomal protein/min. (3) Neither phenobarbital nor polycyclic aromatic hydrocarbons caused a considerable induction of the enzyme without change of the apparent K m , but spironolactone did. (4) The conjugation of the substrate (4 μM) could only be inhibited by the 3'-epi-digitoxoside of digitoxigenin. (5) 25–50 μM substrate inhibited only the conjugation of the 3'-epimer and that of digoxigenin monodigitoxoside. It is suggested that there is a form of glucuronyltransferase which specifically conjugates digitoxigenin monodigitoxoside.

Cleavage of digoxigenin digitoxosides by rat liver microsomes.

Microsomal monoxygenases can oxidize the axial hydroxyl of the terminal digitoxosyl of digoxin (dg-3), digoxigenin bis-, and digoxigenin mono-digitoxoside (dg-2 and dg-1, respectively) to an oxo-group. The corresponding metabolites (15'-dehydro-dg-3, 9'-dehydro-dg-2, and 3'-dehydro-1, respectively) have been identified by chromatographic and chemical methods. Only after this oxibation the terminal sugar can be split off, presumably by beta-elimination. Therefore, for the degradation of dg-3 three successive cytochrome P450 catalyzed oxidations are necessary before digoxigenin (dg-0) can be obtained. The highest oxibation rate was observed with dg-1 (120-150 pmoles/mg microsomal protein/min) and by far the lowest with dg-2 (6-7 pmoles/min) as the substrate (concentration was 30/microM). The latter may contribute to the effect that dg-2 is the main dg-3 metabolite in vivo. Pretreatment of rats with canrenoate enhanced the microsomal oxidation of dg-3, dg-2, and dg-1 by a factor of 3.2, 2.3 and 1.3, respectively. In contrast, there was no increase after pretreatment with phenobarbital.

Reverse Phase Thin Layer Chromatographic Procedure for Identification of Digoxin and Related Fluorescing Substances

A one-step method was developed to replace the current USP monograph procedrues for identification of digoxin and determination of related fluorescing substances. The new method is much quicker than the previous tests, which required paper chromatography and fluorometric measurements, respectively. Using reverse phase silica gel plates (C18 bonded to silica gel), digoxin was separated from digitoxin, gitoxin, and digoxigenin mono-digitoxoside, and partially resolved from digoxigenin bis-digitoxoside. A digoxin reference standard is used to correlate Rf values for identification, and a dilute solution of gitoxin is also co-spotted to give a maximum intensity limit for related fluorescing substances.

Glucuronidation of digitalis glycosides by rat liver microsomes: Stimulation by spironolactone and pregnenolone-16α-carbonitrile

In the present studies, glucuronidation of [ 3H]digoxigenin monodigitoxoside and [ 3H]digitoxigenin monodigitoxoside has been investigated in vitro using rat liver microsomes. During a 40-min incubation period, the conjugation of digitoxigenin monodigitoxoside was five times greater than of digoxigenin monodigitoxoside at a substrate concentration of 5 μm and three times greater at a 20 μM substrate concentration. No difference in conjugation rates was observed between male and female rats, with either substrate. Pretreatment with spironolactone (100 mg/kg/day for 3 days) stimulated the conjugation of both substrates in females, but no significant changes were observed in males with either substrate. Pretreatment (75 mg/kg/day) with pregnenolone-16α-carbonitrile (PCN) caused enhanced glucuronidation of both substrates in both sexes. The conjugation rates in the PCN-pretreated rats were approximately twice those of the spironolactone-pretreated rats. The possible role of this increased conjugation in the protective effect of spironolactone and PCN against digitalis toxicity is discussed.

Digoxin Degradation in Acidic Dissolution Medium

The release of digoxin and its simultaneous conversion to digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside, and digoxigenin in a USP dissolution test medium were followed by high-pressure liquid chromatography. Two products, Tablets A and B, were manufactured by solvent deposition and simple blending methods, respectively. Tablet A released digoxin faster than Tablet B in distilled water and in artificial intestinal juice, and no decomposition was observed. In the USP dissolution test medium, the rate of hydrolysis to digoxigenin bisdigitoxoside was almost equal to that of hydrolysis to digoxigenin monodigitoxoside, and a comparatively large formation rate of digoxigenin was observed. Concentrations of digoxin and its decomposition products were described by differential equations that included dissolution rates of digoxin (rapidly dissolving digoxin and digoxin crystals) and an apparent hydrolysis rate. In the earlier stage of dissolution, hydrolysis was rate determining; in the later stage, dissolution became the rate-determining step for overall digoxin degradation. To suppress digoxin hydrolysis in the USP dissolution test medium, a developmental formulation study was performed. The incorporation of magnesium oxide and magnesium hydroxide-aluminum hydroxide in the tablet formulations inhibited digoxin hydrolysis by 15.3 and 14.5%, respectively, after dissolution for 30min without serious delay of drug release.