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Metabolism of halogenated hydroxyquinolines.

Introduction

Quinoline derivatives have been earlier used for a long time in the treatment of human amoebic dysentery and bacillary dysenteries (de Alencar and Sampaio, 1963; Heseltine and Campbell, 1960; Heseltine and Freeman, 1959). The Quinolin-8-ol nucleus provides the basis for a great variety of drugs. Several halogenated derivatives of the Quinolin-8-ol consist of iodine, chlorine or bromine substitution on the 5 and / or 7 position of the quinoline nucleus (fig 1).

The systemic use of halogenated derivatives of Quinolin-8-ol gained great worldwide acceptance, especially in those countries where amoebiasis was common. It was effective against mobile and cystic forms of disease and due to its relatively poor systemic absorption was not used for amebic infections of the organs, particularly liver abscess (Neldner, K. H., 1977). Many of the halogenated Quinolin8-ols have also been used for topical antifungal and antibacterial therapy. Halquinol is generally a mixture of 5-chloro-quinoline-8-ol (5-CHQ), 7-chloro-quinolin-8-ol (7-CHQ) and 5, 7-dichloro-quinolin-8-ol (DCHQ) in a specific ratio (BP vet). Halquinol has been used with brand names such as Halquivet and Quixalin for topical application. Halquinol has also been used as a feed additive with trade name such as Quixalud, Roxallin in poultry and swine feeds. (Readett, 1965, Robert, 1996).

Though halogenated quinolines have been used for a long time, information on their metabolism is scarce. The metabolism of some of the halogenated derivatives like 5-chloro-7-iodo-quinolin-8-ol (ICHQ), 5, 7-dibromo-quinolin-8-ol (DBHQ) and Halquinol (CHQ) has been investigated to some extent.

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Based on the available literature on the subject the Pharmacokinetic model of Halogenated Quinoline-8-ol derivatives is summarized in Figure 2. Up on the administration of the Halogenated derivative the main metabolites formed in vivo by the phase II enzymes are the conjugates of glucuronide and sulfate. A small fraction of the originally administered Halogenated Quinoline-8-ol (HOX) could still remain as free form. More than 90% of the Glucuronide and Sulfate conjugates as well as the free form of HOX are excreted into the urine and the bile. A negligible fraction could undergo dehalogenation in the Phase I stage of the metabolism pathway to form unknown metabolites. Available literature illustrates studies conducted in laboratory animals, radiolabelled metabolic studies in mice & calf and studies conducted in human being. This review would restrict to structure activity relationship of halogenated quinolinols with respect to in vivo metabolism.

[FIGURE 2 OMITTED]

Metabolism of halogenated hydroxyquinolines in Laboratory animals

ICHQ (known in trade as Chinoform), was widely used earlier as an antimicrobial in the intestinal lumen (Bories and Tuliez, 1972) and suspected to cause SMON (subacute myeloptico-neuropathy) as per a report from Japan (Oakley, 1973). The difference in the species for ICHQ (I) absorption, metabolism and excretion has been investigated (Hayashi et al., 1976). In this study separate method of unmetabolised I (free form) and its conjugated metabolites, i.e. glucuronide (I-G) and sulfate (I-S) in urine and bile were estimated across various species. The results for the species differences are listed in Table 1. The urinary excretion ration in rat, the order of conjugate was I-S>I-G, while in guinea-pig, I-G>I-S, and in man, I-G>I-S. In the order of biliary excretion ration in guinea pig was characteristically I-S>I-G differently from rat, in which it was I-G>I-S (Refer Fig 3). In all cases, unmetabolised I was of trace and negligible.

As seen, in the bile of guinea pig I-S was exclusively excreted. The total excreted ration was largely lowered owing to bile fistula. The results show that bile effect on absorption of I in guinea pig was much greater than rat. Furthermore, the biological stability of the conjugates of I was studied and the conversion of I-G [??] I-S was found to occur in rat. In addition to urinary and biliary excretion, as to blood concentration after oral administration, the complexed (conjugated form) absorbability of I was shown. In small animals (rat and guinea-pig), the order of the concentration of unmetabolised I and the conjugate was I-G and I-S > unmetabolised I. In the beagle dogs which was sensitive to the toxicity of I, unmetabolised I > I-G and I-S. This data has been compared by C.T.Chen et al., in which they have correlated that man and dogs that were sensitive to SMON disease had higher plasma concentration of unmetabolised I than I-G and I-S as shown in Fig 4.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

A comparative pharmacokinetic fate of quinolin-8-ol (OX) and ICHQ (I) was studied in rats by Kiwada et al., 1977. In this study 14 C- labeled OX and I was synthesized for the study. Both the test compounds were administered intraveinously. Although both OX and I are metabolized to glucuronide and sulfate there ware some difference obtained from the excretion data. After intravenous administration of OX, glucuronide was more excreted (about 65% of dose) than sulfate (about 25% of dose) in urine in contrast to the case of I. In bile, only 10% as glucuronide was excreted. The difference from I was considered due to the difference of molecular weight caused by the halogen substitution.

The in vivo and in vitro fates of OX, 5-CHQ, DCHQ, and ICHQ and also their conjugates were studied in rats (Sawada et al., 1978). Male albino rat of Donryu (260 - 280 g) were dosed with 3 mg of 5-CHQ or its conjugate (3 mg equivalent to 5CHQ), or 3 mg of DCHQ or its conjugate (3 mg equivalent to DCHQ) was administered intravenously to the rat. The intravenous dosage forms were aqueous solution for 5-CHQ CG, 5-CHQ CS and DCHQ CS, 0.0467 M NaHCO3 for DCHQ CG, 0.1 N HCl for MC and 0.1 N NaOH for DCHQ. During experimentation, rat was kept in restraint cage and allowed to take only water ad libitum, Urine and bile samples were collected at an appropriate time interval until 8 hr through polyethylene cannulae set into bladder and bile duct, respectively.

After the end of the trial, the conjugates were determined from the urine and bile samples. The enzymatic activity in terms of P-Glucuronidase and Arylsulfatase from the kidney and liver were determined as these enzymes are responsible for the conjugation reactions. Binding of the conjugates to the rat plasma protein and hydrolysis of conjugate by metal ion were also estimated. The results obtained from the above study were that the compounds were metabolized to glucuronides and sulfates after intravenous administration. Glucuronides were excreted in bile and urine, but sulfate were excreted exclusively in urine (Ref Table 2). Unmetabolised forms were almost not excreted. Glucuronides were more excreted in the bile and in the order of OX<5-CHQ<DCHQ<ICHQG, as it could be seen that the molecular weights increased by halogen susbstituents favors biliary excretion of glucuronide. Since protein binding considered one of controlling factor for drug transport, the binding of conjugates, i.e. glucuronide and sulfate to plasma protein was determined. The order of glucuronide tendency to plasma protein was again 8-OXG<5 CHQCG<DCHQG<ICHQG.

Although sulfates were well hydrolysed as glucuronides in the body, sulfates (especially, ICHQ sulfate) resisted enzymatic hydrolysis in vitro by mitochondrialysosomal and microsomal enzymes in both liver and kidney in contrast to glucuronides to them (Ref Table 3).

The conjugates were metabolized by [Cu.sup.2+] [Fe.sup.2+], [Zn.sup.2+], and [Mg.sup.2+] (Refer Table 4). Especially in the presence of [Cu.sup.2+] or [Fe.sup.2+] DCHQ sulfate and ICHQ sulfate which were extremely stable in the enzymatic hydrolysis were unstable. Accordingly, the effects of the metal ions as well as the hydrolytic enzymes were suggested for the hydrolysis of the conjugate in the body.

Recently there has been active interest of the antituberculosis activities of a monohalogenated 5-CHQ against clinical Mycobacterium tuberculosis and also against Alzheimer's disease (Bondiolottie, et al., 2006). There have been a few studies of the antituberculosis activity of quinolines. For example, clioquinol had good activity in guinea pigs but not in mice (Lewis et al., 1970, Tison., 1952). Halogenated quinolines were reported as a possible cause of subacute myelooptic neuropathy (SMON), an uncommon neurological syndrome that occurred primarily in Japan. The cause of the syndrome is, however, far from established, as environmental factors, such as B12 deficiency, are also likely to be important. Nevertheless, recently, the interest in 5-CHQ has been increased due to its favorable effects on Alzheimer's disease (Hongmanee et al., 2007, Doraiswamy et al., 2004, Finefrock et al., 2003).

In recent reports 5-CHQ have been demonstrated to act as Zinc ionophore and to induce apoptosis of human cancer cells. It has been found that (Haijun et al., 2009) 5CHQ targets zinc to lysosomes, and is associated with an increase in lysosomal permeability, release of lysosomal enzymes into the cytoplasm, and cleavage of the proapotic protein Bid (BH3 - interacting domain death agonist). Thus suggesting that zinc ionophores such as 5-CHQ may serve as anticancer agents.

Radiolabelled metabolic studies of Halogenated Hydroxy1q25uinolines

Early study was done on ICHQ with rats receiving 1 mg oral dose of [I.sup.125] labeled drug. The proportions of free and conjugated ICOQ in urine, feces and plasma have been determined (Liewendahl, 1968). The experiment showed iodine-125 tissue distribution in rats 8 hrs, 24 hr and 48 hr after a 1 mg oral administration of labeled ICHQ; deiodination of the molecule was weak, and was about 6%.

A radiolabelled metabolism study of Halquinol (mono and dichlorohydroxyquinolines-[Cl.sup.36]) was done in the rat and the calf (Bories and Tulliez, 1972). Preliminary balance study using colorimetric determination of chlorohydroxyquinolines enabled the recovery in the urine and feces of only 60% of the orally administered dose. Therefore, it was necessary to use a labeled molecule for establishing this balance, and for studying incorporation, distribution, and depletion in the tissues. 5-Chloro- and 5, 7- dichloro-8-hydroxyquinolines labeled with chlorine36 were synthesized. This labeling seemed appropriate, as there was little dehalogenation of the ICHQ and DBHQ in earlier reports (Hayashi et al., 1976 and Rodriguez and Close, 1968).

The synthesis was carried out by controlled chlorination of quinolin-8-ol with [Cl.sup.36]-sulfuryl chloride. It gave a mixture of 5-chloro-and 5, 7-dichloro-quinolin-8-ol having radiochemical purity higher than 99% as determined by radio chromatography. The radioactive composition determined by radio chromatography was 90 and l0%, respectively. The approximate specific activity of the mixture was 0.102 mCi/mM.

Administration of the drug in rats

For the balance study, three male Wistar rats, of the same age and weight (250 g), were put in metabolism cages and given feed and water ad libitum. In a first experiment, a single dose of 3.75 mg (1.85 [micro],Ci) of [Cl.sup.36]-CHQ (representing 250 ppm of the diet) dissolved in 1 ml of peanut oil was administered to the rats by stomach intubation. In a second experiment, the labeled drug was blended at the same level in the feed. Urine and feces were collected separately and stored at -25[degrees]C.

The experimental procedure used for determining long-term accumulation involved giving [Cl.sup.36]-CHQ at 80 ppm level in the diet (1.2 mg or 0.6 [micro]Ci per day) to six male Wistar rats having the same age and weight (150 8). The animals were sacrificed after 1, 3, 6, 9, 12, and 15 days, and the tissues analyzed for radioactivity. For the depletion study, six male Wistar rats of the same age and weight (150 g) were fed for 15 days with 100 ppm of unlabeled CHQ, then with 100 ppm of labeled CHQ (1.5 mg or 0.75 pCi per day) for 8 days. The animals were then sacrificed every 12 hr, and chlorine-36 tissue levels determined.

Administration of the drug in calf

A balance study was carried out on a 2-month-old French Frisian calf put in a metabolism cage and fed with 10 1itres of milk a day. 30 [micro]Ci of labeled CHQ in capsule form (about 60 mg), representing 50 ppm of the calf's diet, were intubated into the stomach. Urine and feces were collected separately for 15 days, homogenized, and samples stored at -25[degrees]C.

The CHQ residues in the calf tissues were studied. The same calf, were fed with 10 1itres of milk per day, received 40 ppm of unlabeled CHQ (about 50 mg) for 15 days, then for the next 8 days received 25 mg of nonradioactive CHQ in the morning and 25 mg of labeled CHQ (12 [micro]Ci) in the evening, both in capsule form. The calf was slaughtered on the morning of the ninth day, and the tissues were excised, ground in liquid nitrogen, and lyophilized.

Radioactivity assays was performed by liquid scintillation on a Nuclear-Chicago 6860 counter for urine, feces and tissue for their assay. The counting efficiency varied between 50% for the kidney and spleen samples and 92% for the urine and fat. Identification of [Cl.sup.36]- Labeled metabolites for the chlorinated derivatives was done by Thin-Layer Chromatography (TLC) and the radioactive spots were located by a Packard 7201 radio chromatogram scanner. Determination of elimination compounds were done by identification in calf urine and kidney. Evaluation of CHQ dechlorination were also done.

In the study, the radioactive product used had a slightly different composition than that of Halquinol (CHQ) as a result of perfected microsynthesis. Considering that the two chlorinated derivatives had a similar metabolism, overall consideration to the mixture of the two radioactive substances and expressed radioactive residues in tissues per million parts of CHQ were given. Tables 5 and 6 show the result of the balance studies in the rat and calf. Urinary and fecal withdrawal was very rapid, with more than 90 of the radioactivity being eliminated within 48 hr in the rat. These results are similar to those obtained in experiments carried out with ICHQ and DBHQ (Liewendahl, 1968; Ritter and Jermann, 1966; Rodriguez and Close, 1968). Urinary elimination is predominant in the calf and fecal elimination is predominant in the rat; these species differences have already been observed by earlier (Haskins and Luttermoser, 1953) with ICHQ.

CHQ was eliminated both in free and conjugated forms, and undergoes limited dechlorination. Radioactive distribution in calf urine was as follows: 54.3% free, 31.7% conjugated, and 12.1 % in the form of chlorides. The three types of hydrolysis applied to calf urine, from which the free radioactive fraction had been extracted, cause liberation of the same amount of radioactivity (about 30%). This suggested that conjugated metabolites were in the form of glucuronides. The radio chromatogram, and ultraviolet and infrared spectra gave the identity between urinary metabolites (free or conjugated) and 5-CHQ and DCHQ of the original product. The respective percentages of the two chlorinated derivatives revealed by radio chromatography and expressed in radioactivity were 16 and 84%, while the product administered had a composition of 10 and 90%. The lower elimination of 5, 7-dichloro derivative in the study may correspond either to its preferential storage or to a partial or total dechlorination.

The fact that part of the radioactivity (12.1 %) was in the form of chlorides in the urine, and that Quinolin-8-ol was not determined, lead to the fact that only partial dechlorination occurred. Only 5% of the radioactivity in calf and rat feces was extracted. Given the chelating properties of Quinolin-8-ol and the low solubility of resulting chelates (iron in particular), it may be hypothesized that most of the radioactivity eliminated in the feces may be a chelated form. Calf tissue, serum, and bile residue levels are given in Table 7. This shows that after long-term administration at a 40 ppm level, the quantities of radioactivity are very low, most being found in the liver and kidneys, where metabolism and elimination occur. Radioactivity level in the bile indicates considerable elimination by this route. Residues of 5-chloro-and 5, 7-dichloro-quinolin-8-ol were found in the kidneys in proportions similar to those of the administered product. Table 8 shows that [Cl.sup.36] balance in most rat tissues is established within 6 to 9 days, and that no accumulation occurs.

The depletion of radioactivity following withdrawal of drug is shown in Table 9. Molecules containing Chlorine-36 is rapidly eliminated from liver and kidneys, the residue levels reaching that of the other tissues after 36 hr.

To draw a overall conclusion from the balance study with chlorine-36 labeled mixture of 5-CHQ and DCHQ showed the following distribution of [Cl.sup.36] in the urine and feces of the rat and calf: rat, urine 39.3% and feces 60.4%; calf, urine 80.5% and feces 10.5 %.

Chlorine-36 accumulation and depletion studied in rat tissues, the tissue balance was established within 6 days, and 95 % depletion occurred in 3 days. Free or conjugated unchanged chloroquinolines were excreted in urine, and unchanged chloroquinolines were present in tissues. There was only 12% molecule dechlorination.

Metabolism of halogenated hydroxyquinolines in human beings

As several halogenated derivatives of the hydroxyquinoline were widely used as amoebicide and intestinal bactericides, some studies on their metabolism have been reported in humans for ICHQ and DBHQ. However data available are still scarce for other halogenative derivatives of OX.

In man receiving a single dose of 0.1 to 1 mg of ICHQ excreted 43% in his urine as glucuronide and sulfate conjugates within 10 to 18 days (Liewendahl and Lamberg, 1967).

In the study conducted on the metabolism of the DBHQ in man (Rodriguez and Close, 1968) receiving one single oral dose of 1.98 g of DBHQ, from whom urine samples were collected at 24 hrs interval until 161 hrs for assay of the metabolites. The urinary elimination in man for the single dose of DBHQ amounted to 36 molar % after 24 hrs and 48 molar % after 72 hrs. The main metabolite consisted of glucuronide of DBHQ, which represented about 98 per cent of urinary metabolite found. Unchanged DBHQ was excreted as a free compound but in very small quantities. Less than 1.3 molar % showed debromination. This confirms with other experiments conducted in other species for the dehalogenation of halogenated derivatives of quinolin-8-ol

Conclusion

From the various metabolic fate and pharmacokinetic analysis done in the various studies show that majority of HOX derivative either remain free or get conjugated (Glucuronide and Sulfate conjugate) HOX forms which get excreted in the urine/bile and there was limited dehalogenation of these compounds. Further studies need to be undertaken in farm end species like poultry and swine to validate these findings.

Abbreviations used:

Quinolin-8-ol (OX), Halogenated quinoline-8-ol (HOX), 5-chloro-quinolin-8-ol (5CHQ), 5, 7-dichloro-quinolin-8-ol (DCHQ), and 5-chloro-7-iodo-quinolin-8-ol (ICHQ), 5, 7-dibromo-quinolin-8-ol (DBHQ), Halquinol (CHQ)

References

[1] Bondiolotti, G.P., Pollera, C., Pirola, R. and Bareggi, S. R., 2006, "Determination of 5-chloro-7-iodo-8-quinolinol (clioquinol) in plasma and tissues of hamsters by high-performance liquid chromatography and electrochemical detection, " J. Chromatogr. B, 837(1-2), pp. 87-91.

[2] Bories, G. F., and Tulliez, J. E., 1972, "Metabolism of mono- and dichlorohydroxyquinolines-Cl 36 in the rat and calf, " J. Agric. Food Chem., 20(2), pp. 417-420.

[3] Chen, C.T., Samejima, K. and Tamura, Z., 1973, "Synthesis of 5-Chloro-7 iodo-8-quinolinol Sulfate, " Chem. Pharm. Bull (Tokyo), 21, pp. 911-913. [4] de Alencar, J., Magalhaes, V. B. and Sampaio, V. D., 1963, "Treatment of amebiasis with chlorohydroxyquinoline, ". Hospital(Rio J), 63, pp. 67-72.

[5] Doraiswamy, P. M. and Finefrock, A. E., 2004, "Metals in our minds: therapeutic implications for neurodegenerative disorders, " Lancet Neurol., 3, pp. 431-434.

[6] Finefrock, A. E., A. I. Bush, and P. M. Doraiswamy., 2003, "Current status of metals as therapeutic targets in Alzheimer's disease, " J. Am. Geriatr. Soc., 51, pp.1143-1148.

[7] Haijun, Y., Yunfeng, Z., Stuart, E.L., and Wei-Qun, D., 2009, "Clioquinol targets zinc to lysosomes in human cancer cells, " Biochem. J., 417, pp. 133139.

[8] Haskins, W.T., and Luttermoser, G.W., 1953, "Urinary excretions of vioform and diodoquin in rabbits, " J. Pharmaco.l Exp. Ther., 109(2), pp. 201- 205.

[9] Hayashi, M., Fuwa, T., Awazu, S. and Hanano, M., 1976, "Differences in species of iodochlorhydroxyquin absorption, metabolism, and excretion, " Chem. Pharm. Bull, 24, pp. 2589-2596.

[10] Heseltine, W. W., and Campbell, P. J., 1960, "Laboratory studies on chlorhydroxyquinoline, " J. Trop. Med. Hyg., 63, pp.163-165.

[11] Heseltine, W. W. and Freeman, F. M., 1959, "Some pharmacological and microbiological properties of chlorhydroxyquinoline and related compounds, " J. Pharm. Pharmacol, 11, pp. 169-174.

[12] Hongmanee, P., Rukseree, K., Buabut, B., Somsri, B., and Palittapongarnpim, P., 2007, "In vitro activities of cloxyquin (5-chloroquinolin-8-ol) against Mycobacterium tuberculosis, " Antimicrob. Agents Chemother., 51(3), pp. 1105 - 1106.

[13] Lewis, A., and Shepherd, R. G., 1970. Antimycobacterial agents, p. 449. In A. Burger (ed.), Medicinal chemistry, 3rd ed. John Wiley & Sons, New York, NY

[14] Liewendahl, K. and Lamberg, B.A., 1967, "Metabolism of 125 iodochloroxyquinoline in man. I. Absorption, binding and excretion, " Nucl. Med., 6, pp. 20-31.

[15] Liewendahl, K., 1968, "Iodochloroxyquinoline and the thyroid gland, " Acta Endocrinol, 59, S10- S80

[16] Neldner, K. H., 1977, "The halogenated 8-hydroxyquinolines, "Int. J. Dermatology, 16, pp. 267-273.

[17] Oakley, G. P. Jr., 1973, "The neurotoxicity of the halogenated hydroxyquinolines. A commentary. "JAMA, 225 (4), pp. 395-397.

[18] Readett, M. D., 1965, "Halquinol in dermatology, " Br.J. Dermatol., 77(11), pp. 593-595.

[19] Ritter, P. and Jermann, M., 1966, "Comparative studies on the resorption and metabolism of a therapeutically-used 8-hydroxyquinoline, " Arzneimittelforschung, 16(12), pp. 1647-1652.

[20] Robert, A. S., 1996, " Role of growth promotants in poultry and swine feed, "ASA Tech Bulletin, AN04.

[21] Rodriguez, L. A. M. and Close, J. A., 1968, "The metabolism of the 5-7dibromo-8-hydroxyquinoline (Broxyquinoline) in man, " Biochem. Pharmacol., 17(8), pp. 1647-1653.

[22] Sawada, Y., Hayashi, M., Awazu, S. and Hanano, M., 1978, "In vivo and in vitro fates of 8-hydroxyquinoline derivatives in rat, " Chem. Pharm. Bull, 26 (5), pp. 1357-1363.

[23] Tateishi, J., Kuroda, S., Saito, A. and Otsuki. L., 1972, "Strain-differences in dogs for neurotoxicity of clioquinol, " The Lancet, 299(7763), pp. 1289-1290.

[24] Tison, F., 1952, "Treatment of experimental tuberculosis in guinea pig by iodochloroxy quinoline, "Ann. Inst. Pasteur (Paris), 82(2), pp. 241-242.

Abhilekha P. Mantri * (1), Shiva Kumar (1), Sharadamma K.C. (1), Nischal Kandepu (1), Radhakrishna P.M. (1) and Kumara Swamy B.E. (2)

(1) Provimi Animal Nutrition India Pvt.Ltd., C7/22, KSSIDC Industrial Area, Yelahanka Newtown, Bangalore - 560 106, Karnataka, India

(2) Department of P.G. Studies and Research in Industrial Chemistry, Kuvempu University, Jnana Sahyadri, Shankaraghatta, Karnataka, India
Table 1: Cumulative Per Cent of Dose excreted until 24 hr
after Oral Administration of ICHQ (I) in Rat, Guinea Pig
and Man

 Rat (a)

Intact Urinary excretion G 2.5 [+ or -] 0.1
 ratio (%) S 10.2 [+ or -] 1.5
 T 12.7 [+ or -] 1.5
With bile fistula Urinary excretion G 4.3 [+ or -] 2.1
 ratio (%) S 9.4 [+ or -] 2.1
 T 13.7 [+ or -] 4.2
 Biliary excretion G 29.7 [+ or -] 6.2
 ratio (%) S -
 T 29.7 [+ or -] 6.2

 Guine-Pig (b) Man (c)

Intact 33.8 [+ or -] 6.1 16.4 [+ or -] 8.8
 8.1 [+ or -] 0.3 -
 41.9 [+ or -] 6.2 16.4 [+ or -] 8.8
With bile fistula 4.7 [+ or -] 2.7
 1.3 [+ or -] 0.5
 6.1 [+ or -] 3.2

 5.9 [+ or -] 0.2
 5.9 [+ or -] 0.9

G: glucuronide, S: sulfate, T: total (G+S)

(a) dose: 15 mg p.o. as CMC (Carboxymethyl cellulose) aqueous
suspension

(b) dose: 24 mg p.o. as CMC (Carboxymethyl cellulose) aqueous
suspension

(c) dose: 600 mg + CMC-Na 120 mg

- : The excretion was found very little and almost negligibly
small

Table 2: Cumulative Excretion Ration of Conjugates (Glucuronides and
Sulfates) of OX, 5-CHQ, DCHQ, and ICHQ to Doses after intravenous
administration in rat

I.V Dosage Excreted OX (%)
Form Form

 Urine Bile

Free Glu 59.9 [+ or -] 1.9 8.7 [+ or -] 0.2
 Sul 22.9 [+ or -] 2.1
Glu Glu 91.2 [+ or -] 4.9 10.5 [+ or -] 1.0
 Sul - -
Sul Glu - -
 Sul 96.7 [+ or -] 5.4 -

I.V Dosage Excreted 5-CHQ (%)
Form Form

 Urine Bile

Free Glu 28.4 [+ or -] 2.7 28.8 [+ or -] 7.6
 Sul 18.8 [+ or -] 2.7
Glu Glu 57.9 [+ or -] 4.5 31.1 [+ or -] 2.1
 Sul -
Sul Glu 16.4 [+ or -] 2.6 6.0 [+ or -] 0.4
 Sul 64.8 [+ or -] 0.6 -

I.V Dosage Excreted DCHQ(%)
Form Form

 Urine Bile

Free Glu 13.8 [+ or -] 4.3 26.9 [+ or -] 2.7
 Sul 47.9 [+ or -] 6.9 -
Glu Glu 13.0 [+ or -] 0.3 61.2 [+ or -] 3.8
 Sul 20.6 [+ or -] 1.4 -
Sul Glu 3.5 [+ or -] 0.3 7.9 [+ or -] 1.3
 Sul 86.2 [+ or -] 2.8

I.V Dosage Excreted ICHQ (%)
Form Form

 Urine Bile

Free Glu 6.4 [+ or -] 2.1 32.3 [+ or -] 0.5
 Sul 34.6 [+ or -] 2.4 -
Glu Glu 1.7 [+ or -] 1.1 37.6 [+ or -] 8.1
 Sul 9.9 [+ or -] 1.8 -
Sul Glu 2.2 [+ or -] 0.9 24.9 [+ or -] 6.0
 Sul 44.3 [+ or -] 4.3 -

Glu : Glucuronide, Sul: Sulfate

Unmetabolised form was almost not excreted

Values represent mean [+ or -] SD of 3 rats except 5 rats in the
case of MC administration.

Excretion ratios to doses until 8 hr for OX, 5-CHQ, and DCHQ and
until 10 hr for ICHQ are expressed as per cent dose

Table 3: Hydrolysis Rate Constants catalyzed by [beta]-
Glucuronidase and Arylsulfatase in Rat Liver and Kidney

Fraction Tissue

 Liver

 Mitochondrial- Microsome
 Lysosome
[beta]-Glucuronidase OX 0.149 0.176
 5CHQG 0.271 0.270
 DCHQG 0.520 0.400
 ICHQG 0.520 0.400
Arylsulfatase OX 0.010 0.019
 5CHQG 0.056 0.068
 DCHQG 0.014 0.024
 ICHQG 0.002 0.001

Fraction Tissue

 Kidney

 Mitochondrial- Microsome
 Lysosome
[beta]-Glucuronidase OX 0.027 0.016
 5CHQG 0.040 0.026
 DCHQG 0.046 0.034
 ICHQG 0.035 0.032
Arylsulfatase OX 0.007 0.008
 5CHQG 0.021 0.032
 DCHQG 0.009 0.008
 ICHQG 0.003 0.003

Table 4: Degree of Hydrolysis of conjugates in the presence of Metal
Ions

Metal ions [Cu.sup.2+] [Fe.sup.2+] [Zn.sup.2+] [Mg.sup.2+]
added (2 min) (16 min) (16 min) (16 min)
(incubation
time)

ICHQG 40.6 32.6 17.9 8.8
ICHQS 100.0 16.4 17.5 2.7
DCHQG 86.1 32.0 10.0 2.3
DCHQS 100.0 16.4 7.6 3.0
5CHQG 15.2 6.1 11.1 2.2
5CHQS 22.3 13.1 12.3 4.5
OXG 12.3 5.2 9.6 4.9
OXS 9.6 0.1 6.3 1.4

Values represent the hydrolysis ratio (%) of conjugates

Table 5: Rat, Radioactivity Percentage recovered

Dose CHQ Fed CHQ intubated
administered

 4.93 [micro]Cl 4.35 [micro]Cl

Days Urine Feces Urine Feces

1 19.3 33.5 31.8 0.3
2 4.6 32.9 5.5 53.0
3 1.2 2.0 1 6.4
4 0.4 0.4 0.3 0.6
5 0.2 Traces 0.3 0.1
6 0.2 0.2 Traces
7 0.1 0.1
8 Traces 0.1
Total 26.0 68.8 39.3 60.4
 94.8 99.7

Table 6: Calf, Radioactivity
Percentage Recovered

Dose CHQ intubated, administered
 28.19 [micro]Cl

Days Urine Feces

1 4.6 0.2
2 40.8 5.3
3 11.9 2.7
4 11.9 1.1
5 4.7 1
6 2.5 0.2
7 0.8 Traces
8 1 Traces
9 0.9 Traces
10 0.7 Traces
11 0.4 Traces
12 0.2 Traces
13 0.1 Traces
14 Traces Traces
Total 80.5 10.5
 91

Table 7: Residues in Tissues of Calf fed 3
weeks with 40 ppm of [Cl.sup.36]-CHQ

Tissues ppm (a)

Liver 0.12
Kidney 1.00
Spleen 0.23
Heart 0.07
Thymus 0.09
Muscle 0.025
Fat 0.12
Serum 0.4 [micro]g/ml
Bile 0.8 [micro]g/ml

(a) Part per million calculated as CHQ
equivalents

Table 8: Incorporation of [Cl.sup.36] in Tissues
of Rats fed 2 weeks with 80 ppm of [Cl.sup.36]-CHQ, ppm (a)

Tissues Days on medication

 1 3 6 9 12 15

Kidney 0.82 1.34 1.59 1.35 1.91 1.69
Liver 0.50 1.11 1.17 1.31 1.34
Muscle 0.25 0.39 0.49 0.37 0.64 0.51
Fat 0.20 0.24 0.35 0.35 0.50 0.45

(a) Part per million calculated as CHQ equivalents

Table 9: Incorporation of [Cl.sup.36] in Tissues
of Rats fed 2 weeks with 100 ppm of
[Cl.sup.36]-CHQ, ppm (a)

Tissues Hours after withdrawal of medication

 12 24 36 48 60 72

Kidney 0.82 0.36 0.02 0.02 0.06 0.09
Liver 0.31 0.16 0.03 0.02 0.03 0.03
Muscle 0.22 0.20 0.16 0.14 0.12 0.13
Fat 0.10 0.05 0.06 0.04 0.03 0.02

(a) Part per million calculated as CHQ equivalents
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Author:Mantri, Abhilekha P.; Kumar, Shiva; Sharadamma, K.C.; Kandepu, Nischal; Radhakrishna, P.M.; Kumara,
Publication:International Journal of Applied Chemistry
Date:May 1, 2012
Words:4882
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