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* Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA and
Department of Gynecology and Obstetrics, Emory University, Atlanta, GA
3To whom correspondence should be addressed. E-mail: abarua{at}iastate.edu.
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ABSTRACT |
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 TOP ABSTRACT CONCLUSIONSLITERATURE CITED |
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KEY WORDS: • retinoyl glucuronide • retinoic acid • neuroblastoma • pharmacology • toxicity
Discovery and background of retinoyl ß-glucuronide (RAG)
During 1956–1966, James Allen Olson and his research team, while examining the metabolites of radioactive retinal and all-trans retinoic acid (RA)3 in the bile of bile duct cannulated rats, found that a major fraction of the radioactivity in the bile was not extractable with hexane-ether, but could be extracted with butanol. Subsequently, it was shown that these water-soluble metabolites gave retinol or retinoic acid upon incubation with ß-glucuronidase, thereby indicating that the water-soluble metabolites were glucuronide derivatives of retinol and retinoic acid. Thus, Olson’s group discovered retinyl ß-glucuronide, the glucuronic acid derivative of retinol, and retinoyl ß-glucuronide (RAG), the glucuronic acid derivative of retinoic acid (1–3). The chemical structures of retinoid glucuronides and the pathway of their formation are shown in Figure 1.
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Pharmacology of RAG
The absorption, pharmacokinetics and tissue distribution of acute treatment with RAG has been studied by us and others (9). It has been shown that 3H-RAG, dosed by ip injection to rats, persisted as a major component in blood and other tissues for at least 24 h with very little hydrolysis to RA (14). In order to assess the absorption of orally administered RAG in relationship to vitamin A status of the host, RAG was given by gavage to two groups of rats raised either on a vitamin A-sufficient or a vitamin A-deficient diet. It was found that RA formed from RAG could be detected in the blood of vitamin A-deficient rats, whereas blood of vitamin A-sufficient rats showed neither RAG nor RA (15). Furthermore, it was found that the rate of formation of RA from RAG was also dependant on the vitamin A status of the host. To this end, it was subsequently shown in human studies that when RAG was given orally to healthy adult American males, with apparently normal vitamin A status, neither RAG nor RA could be detected in the plasma of these volunteers (Reida, A., Barua, A. B. & Olson, J. A., unpublished observations). Taken together, these animal and human studies suggested that there was no appreciable intestinal absorption of orally dosed RAG to go into circulation in the blood when the vitamin A status is normal.
Toxicological studies of RAG
It has been determined that treatment of pregnant Sprague Dawley (SD) rats with RAG is not at all teratogenic (16). Although some hydrolysis of RAG to RA was observed in these and other studies, the amount of RA produced is apparently not sufficient to cause any toxic effects to either the dams or fetuses. In contrast, sc injected RAG was shown to be highly teratogenic in a special strain of mice, NMRI mice (17), due apparently to extensive hydrolysis of RAG to RA. In order to determine if sc administration would also show teratogenicity in SD rats, graded doses of RAG (20 to 420 µmol/kg BW) were injected sc into pregnant rats on day 8.5 of gestation, and the effects observed on the fetuses compared with those of animals similarly dosed with RA (18,19). At dose levels of 20–160 µmol/kg BW, neither RAG nor RA showed an unwanted effect. However, a difference between RAG and RA was clearly observed at higher doses of 360 and 480 µmol/kg BW. RA at these high levels not only produced toxicity in the fetuses resulting in resorption, but the doses were toxic to the dams resulting in hypervitaminosis A and sometimes even death. On the other hand, neither the dams nor pups in the group given 360 µmol/kg BW of RAG showed any abnormality although minimal toxic effects were seen on pups from dams given 480 µmol/kg BW of the retinoid.
To assess the relative toxic and histologic effects of long-term topical applications of RA and RAG (daily for 24 wk), dorsal skin of six 21-d-old castrated male pigs was shaved and marked (2 x 2 cm) with India ink (20,21). The patches were 1.5 cm apart from each other. Each skin patch area was treated daily with a cream formulation containing various concentrations of RAG [0.16 (3.3 mmol/L), 0.8 (16.5 mmol/L) and 1.6% (33 mmol/L)] and RA [0.1 (3.3 mmol/L) and 0.5% (16.5 mmol/L)]. Towards the last 5.3 wk of the experiment, an additional patch area was gently cleansed and allowed to dry before receiving a daily application of 1.6% RAG ("washed patch") in order to simulate the daily cleaning that occurs in humans when using retinoids in topical applications. To serve as controls, one patch received no treatment (control), and another patch received blank cream containing no retinoids only (placebo).
Tissue sections prepared from biopsy samples of each site of application of the creams were evaluated and scored for epidermal proliferative changes including grading of the stratum corneum and nucleated layers. Inflammatory and vascular changes were evaluated and scored in the dermal region. Topically applied RAG cream (0.16%) resulted in significantly lower histologic scores when compared to scores from tissue treated with an equimolar concentration of RA. The highest concentration of RAG tested (1.6%) resulted in a comparable effect on the patch area to that observed with the lowest concentration of RA (0.1%). Daily cleansing of the test area before receiving 1.6% RAG (i.e., washed patch) completely eliminated negative histologic changes or clinical signs of toxicity indicating that the elevated histologic score at this concentration was due to the build-up of residual RAG on the surface of the skin. In conclusion, long-term topical RAG treatment (0.16–0.8%) in young pigs, as in humans, showed no adverse effect as was seen in the case of RA (0.1%).
Treatment of neuroblastoma with retinoids
Neuroblastoma (nb) is a tumor of the sympathetic peripheral nervous system, originating in cells derived from the neural crest. It is the most common extracranial solid tumor in children, and comprises up to 50% of malignancies among infants (22). One hallmark of this disease is the long-recognized tendency of the tumors in a certain group of patients, designated clinically as stage IVS, to undergo spontaneous regression, at times accompanied by cellular differentiation (23). This property prompted widespread interest in the use of retinoids as differentiation-inducing agents for the treatment of this disease. In 1999 Matthay et al. (24) reported the results of a prospective double-blind trial in which 13-cis retinoic acid (13-cis RA) was given for 6 mo to children with nb following consolidation therapy of their disease which included surgery, radiotherapy, chemotherapy, and/or bone-marrow transplantation. The results indicated that those patients taking 13-cis RA had 3-y event-free survival that was approximately double that of the control group. These findings have now prompted the use of long-term administration of 13-cis RA in the setting of minimal residual disease as part of the standard therapy of nb. Thus, neuroblastoma has become only the second cancer, after APL, to utilize retinoids as part of their standard therapy paradigm.
These recent encouraging clinical results have prompted a renewed interest in using retinoids in a safer and more effective manner for the treatment of nb. The present therapeutic protocol in nb calls for six 1-mo cycles of 13-cis RA administration. Each cycle consists of 14 consecutive days of oral administration followed by 14 d of a drug-free "holiday" period. The reason for this drug-free period every two wk is that severe hypervitaminosis A side effects can occur in children undergoing continuous long-term treatment with 13-cis RA (25). The lack of toxicity and systemic adverse side effects that has been demonstrated with RAG as described above has now prompted us to address the possibility that this retinoid might function as a nontoxic substitute for 13-cis RA in the treatment of nb.
Since therapy of nb will undoubtedly require retinoid administration for an extended perioid of time (>4 mo), we were interested in two aspects of long-term systemic RAG treatment: to determine the adverse side effects, if any, and to assess the consequence of long-term continuous treatment on the pharmacology and peak retinoid plasma levels that can be achieved on a daily basis. With regard to the latter, numerous studies involving chronic administration of RA have shown that this retinoid rapidly induces its own metabolism during chronic treatment by increased oxidation via the cytochrome P450 enzyme system (26). Because of this induced metabolism, plasma RA concentrations are reduced to almost undetectable levels within days of the initiation of drug therapy. This pharmacologic property of RA, its markedly increased metabolism with chronic dosing, has contributed to the limited use of this retinoid in oncologic applications. Indeed, there is now evidence that relapse from RA therapy of APL patients may, under certain circumstances, be due to a pharmacological inability to present a sustained effective drug concentration to the leukemic cells (27).
Chronic subcutaneous administration of RAG
In order to assess the possible use of RAG in long-term clinical oncology applications, we investigated both its conversion to RA and possible changes in its pharmacokinetics during chronic sc administration. We chose this route of administration for a number of reasons: i) As described above, RAG has been shown to be poorly absorbed from the gastrointestinal track following oral dosing; ii) elimination of RAG following sc injection has been shown to be relatively slow, showing a half-life of > 10 h (17,28); iii) sc injection would be a simple, nontraumatic means of chronic daily treatment of infants with nb as well as a convenient means of long-term dosing in animal models (versus oral or intravenous routes). When single sc injections of RAG were administered to mice in our study (28), the results were qualitatively similar to those of Nau et al. (17); both RAG and its hydrolysis product RA peaked at 1–2 h followed by a relatively slow decrease in the RAG concentration and a rapid fall in the RA concentration. The peak RAG concentration after a single high dose (30 µmol/kg BW) in our experiments was around 5 µmol/L with peak RA levels in the submicromolar range (
0.2 µmol/L). As opposed to the slow clearance of RAG from the blood plasma, RA was found to be rapidly cleared and was no longer detectable in the plasma after a few hours (28). The results of our chronic long-term treatment studies with RAG are summarized in Figure 2. These data showed that the peak (2 h) plasma levels of RAG were similar at all times throughout the 2 mo period. Other results indicated that both the rate of hydrolysis of RAG to RA and the rate of clearance of the parent compound remained constant during the entire dosing (28). Furthermore, retinol levels did not change significantly during the course of this chronic RAG treatment in contrast to that reported with the chronic administration of some other retinoids such as RA and 13-cis RA (29,30). Surprising, the data indicated that the small plasma concentration of RA, as a hydrolysis product of RAG, also did not decrease during the course of treatment, as opposed to decreased levels seen after chronic dosing with RA itself. Thus, it appears that the production of RA from the continuous hydrolysis of persistent RAG levels did not result in the induction of cytochrome P450 oxidative enzymes during this time.
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In our studies, there were no significant differences in weight gain, water consumption, social behavior or apparent vitality between RAG-treated and control groups of mice. Furthermore, there were no other observable clinical manifestations of retinoid toxicity from the RAG treatment, as has been reported in mice when chronically treated with other retinoids (e.g., alopecia and scaly skin) (31). Since these in vivo studies indicated that micromolar blood levels of RAG can be maintained during chronic long-term administration, we assessed whether such levels could have biological significance in terms of reduced in vivo tumor growth.
In these studies, we utilized the LA-N-5 human nb cell line, which has been shown to be sensitive to differentiation by µM concentrations of RAG in vitro (28). Two in vivo treatment protocols were utilized. In one, mice were given daily sc RAG starting 3 d before tumor injection and continued for 30 d after injection (protocol A). In the other, RAG was administered immediately after the appearance of 5-mm diameter nodules and continued for 30 d (protocol B) (30 µmol/kg BW per day in both protocols). Figure 3 shows that the proliferative capacity of the LA-N-5 cells was inhibited by the in vivo administration of RAG. Of the two protocols used, protocol A in which treatment was started before tumor cell injection appeared to be the most effective in increasing the proportion of tumor-free animals. In the presence of established tumors (protocol B), RAG also inhibited tumor growth although we could not demonstrate a concomitant increase in morphologic or biochemical differentiation of the nb cells. Our findings indicated that RAG was at least as effective as RA in inhibiting nb cell growth as previously tested in identical in vivo nude mouse models (32). In light of the recent results demonstrating the effectiveness of retinoids in preventing nb recurrences in children (24), we are encouraged by the possibility that RAG may serve as a less toxic alternative to intermittent applications of 13-cis RA which is now being utilized in the clinic.
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CONCLUSIONS |
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TOPABSTRACTCONCLUSIONSLITERATURE CITED |
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FOOTNOTES |
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2 Supported in part by NIH-DK 39733 (A.B.B.) and CA43503 (N.S.).
4 Abbreviations used: APL, acute promyelocytic leukemia; BW, body weight; 13-cis RA, 13-cis retinoic acid; nb, neuroblastoma; RA, retinoic acid; RAG, retinoyl ß-glucuronide.
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LITERATURE CITED |
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TOPABSTRACTCONCLUSIONSLITERATURE CITED |
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1. Dunagin, P. E., Meadows, E. H. & Olson, J. A. (1965) Retinoyl ß-glucuronic acid; a major metabolite of vitamin A in rat bile. Science 148:86-87.
2. Zachman, R. D. & Olson, J. A. (1964) Formation and enterohepatic circulation of water-soluble metabolite of vitamin A and vitamin A acid. Nature 203:410-412.[Medline]
3. Lippell, K. & Olson, J. A. (1968) Biosynthesis of ß-glucuronides of retinol and retinoic acid in vivo and in vitro. J. Lipid. Res. 9:168-175.[Abstract]
4. Zile, M. H., Inhorn, R. C. & DeLuca, H. F. (1982) Metabolism of all-trans retinoic acid in bile: identification of all-trans retinoyl- and 13-cis-retinoyl-glucuronides. J. Biol. Chem. 257:3544-3550.
5. Sietsema, W. K. & DeLuca, H. F. (1982) A new vaginal smear assay for vitamin A in rats. J. Nutr. 112:1481-1489.
6. Meloche, S. & Besner, J. (1986) Metabolism of isotretinoin: biliary excretion of isotretinoin glucuronide in the rat. Drug Metab. Dispos 14:246-249.[Abstract]
7. Barua, A. B. & Olson, J. A. (1985) Chemical synthesis of all-trans retinoyl ß-glucuronide. J. Lipid. Res. 26:1277-1282.[Abstract]
8. Barua, A. B. & Olson, J. A. (1986) Retinoyl ß-glucuronide: an endogenous compound of human blood. Am. J. Clin. Nutr. 43:481-485.
9. Barua, A. B. (1997.) Retinoyl ß-glucuronide: A biologically active form of vitamin A. Nutr. Rev. 55:259-267.[Medline]
10. Becker, B., Barua, A. B. & Olson, J. A. (1998) Effects of novel carbohydrate conjugates of retinoic acid on the differentiation of promyelocytic leukemia cells. Med. Biol. Environ. 26:95-101.
11. Mehta, R. C., Barua, A. B., Olson, J. A. & Moon, R. C. (1992) Retinoid glucuronides do not interact with retinoid binding proteins. Internat. J. Vitam. Nutr. Res. 62:143-147.
12. Sani, B. P., Barua, A. B., Hill, D. L., Shih, T. & Olson, J. A. (1992) Retinoyl ß-glucuronide: lack of binding to receptor proteins of retinoic acid as related to biological activity. Biochem. Pharmacol. 43:919-922.[Medline]
13. Becker, B., Barua, A. B. & Olson, J. A. (1996) All-trans retinoyl ß-glucuronide: new procedure for chemical synthesis and its metabolism in vitamin A-deficient rats. Biochem. J. 314:249-252.
14. Barua, A. B. & Olson, J. A. (1989) Chemical synthesis of all-trans-[11-3H]-retinoyl ß-glucuronide and its metabolism in rats in vivo. Biochem. J. 263:403-409.[Medline]
15. Barua, A. B., Duitsman, P. K. & Olson, J. A. (1998) The role of vitamin A status in the conversion of all-trans retinoyl ß-glucuronide to retinoic acid in male Sprague-Dawley rats. J. Nutr. Biochem. 9:8-16.
16. Gunning, D. B., Barua, A. B. & Olson, J. A. (1993) Comparative teratogenicity and metabolism of all-trans retinoic acid, all-trans retinoyl ß-glucuronide and all-trans retinoyl ß-glucose in pregnant Sprague-Dawley rats. Teratology 47:29-36.[Medline]
17. Nau, H., Elmazar, R. R., Thiel, R. & Sass, J. O. (1996) All-trans retinoyl ß-glucuronide is a potent teratogen in the mouse because of extensive metabolism to all-trans retinoic acid. Teratology 54:150-156.[Medline]
18. Ueltschy, A., Gunning, D. B., Barua, A. B. & Olson, J. A. (1999) Subcutaneously administered retinoyl ß-glucuronide (20 mol/kg bw) is not teratogenic in Sprague-Dawley rats. FASEB J 13:A898.
19. Ueltschy, A., Gunning, D. B., Barua, A. B. & Olson, J. A. (2002) Effects of subcutaneously injected graded doses of all-trans retinoic acid and all-trans retinoyl ß-glucuronide on the outcome of pregnancy in Sprague-Dawley rats. Internat. J. Vit. Nutr. Res. 72:229-235.
20. Gunning, D. B., Barua, A. B., Ueltschy, A., Romans, D. & Olson, J. A. (1999) Effects of daily topical treatments with creams containing all-trans retinoyl ß-glucuronide on pig skin. FASEB. J. 13:A896.
21. Gunning, D. B., Barua, A. B., Myers, R. K., Ueltschy, A., Romans, D. & Olson, J. A. (2002) Comparative histological effects of daily topical application of creams containing all-trans retinoic acid or all-trans retinoyl ß-glucuronide on pig skin. Skin Pharmacol. Appl. Skin Physiol. 15:205-212.[Medline]
22. Gale, G, D’Angio, G., Uri, A., Chatten, J. & Coop, C. E. (1982) Cancer in neonates: The experience at the Children’s Hospital of Philadelphia. Pediatrics 70:409-413.
23. McWilliams, N. B. (1986) IVS neuroblastoma: Treatment controversy revisited. Med. Pediatr. Oncol. 14:41-44.[Medline]
24. Matthay, K. K., Villablanca, J. G., Seeger, R. C., Stram, D. O., Harris, R. E., Ramsay, N. K., Swift, P., Shimada, H., Black, C. T., Brodeur, G. M., Gerbing, R. B. & Reynolds, C. P. (1999) Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children’s Cancer Group. N. Engl. J. Med. 341:1165-1173.
25. Ruiz-Maldonado, R., Tamayo-Sanchez, L. & Orozco-Covarrubias, M. L. (1998) The use of retinoids in the pediatric patient. Dermatol. Clin. 16:553-569.[Medline]
26. Regazzi, M. B., Iacona, I., Gervasutti, C., Lazzarino, M. & Toma, S. (1997) Clinical pharmacokinetics of tretinoin. Drug Dispos. 32:382-402.
27. Cornic, M., Delva, L., Castaigne, S., Lefebvre, P., Balitrand, N., Degos, L. & Chomienne, C. (1994) In vitro all-trans retinoic acid (ATRA) sensitivity and Cellular Retinoic Acid Binding Protein (CRABP) levels in relapse leukemic cells after remission induction by ATRA in acute promyelocytic leukemia. Leukemia 8:914-917.[Medline]
28. Sidell, N., Sawatsri, S., Connor, M. J., Barua, A. B., Olson, J. A. & Wada, R. K. (2000) Pharmacokinetics of chronically administered all-trans retinoyl ß-glucuronide in mice. Biochim. Biophys. Acta. 1502:264-272.[Medline]
29. Gerber, L. E. & Erdman, J. W., Jr (1980) Comparative effects of all-trans and 13-cis retinoic acid administration on serum and liver lipids in rats. J. Nutr. 110:343-351.
30. Barua, A. B., Duitsman, P. K., Kostic, D., Barua, M. & Olson, J. A. (1997) Reduction of serum retinol levels following a single oral dose of retinoic acid in humans. Int. J. Vitam. Nutr. Res. 67:423-425.[Medline]
31. Lindamood, C., Giles, H. D. & Hill, D. L. (1987) Preliminary toxicity profile of arotinoids SMR-2 and SMR-6 in male B6D2F1 mice. Fund. Appl. Toxicol. 8:517-530.[Medline]
32. Abemayor, E., Chang, B. & Sidell, N. (1990) Effects of retinoic acid on the in vivo growth of human neuroblastoma cells. Cancer Lett 55:1-5.[Medline]
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