* Laboratoires Merck Sharp and Dohme-Chibret, Route de Marsat, Riom, 63963 Clermont-Ferrand Cedex 9, France;
Banyu Development Research Laboratories, Menuma, Japan; and
Merck Research Laboratories, West Point, Pennsylvania
Received May 1, 2001; accepted October 24, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: selective and nonselective muscarinic receptor antagonists; lens; epithelial proliferation; dose-related response; rat.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In France, the animal facilities have animal care and use programs accredited by AAALAC International and operated in accordance with current standards. The animal facilities in Japan conform with similar guidelines in that country. All studies were approved by the Institutional Animal Care and Use Committee of Merck Research Laboratories, West Point, PA.
Chemicals.
Compound A, 2(R)-N-[1-(6-aminopyridin-2-ylmethyl) piperdin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl)]-2-hydroxy-2-phenylacetamide, was brought forward as a research candidate by Banyu Pharmaceutical Co. Ltd (Tsukuba, Ibaraki Pref., Japan) and synthesized at Merck Research Laboratories, Rahway, New Jersey (Hirose et al., 2001; Mitsuya et al., 2000
). Atropine was used as the sulfate salt and was obtained from MSD-Chibret, France. Tolterodine was used as the tartrate salt and manufactured by J. Star Research, Inc., South Plainfield, New Jersey. The purity of compound A, atropine, and tolterodine was greater than 99% by high-pressure liquid chromatography.
Experimental Designs and Treatments
Animals were randomly distributed into groups of 15 to 40 animals. Compounds were given by gavage in suspension in 0.5% methylcellulose in deionized water (compound A and tolterodine) or in solution in deionized water (atropine) at a volume of 5 ml/kg. In each study, a similar size group was dosed with the vehicle and served as a control group. All studies were performed in France except the 53-week oral toxicity studies in rats with compound A, which was done in Japan. The following studies were conducted.
Compound A.
Two toxicity studies were conducted in which female and male rats were treated for 14 (15/sex/group) or 53 (30/sex/group) weeks at 10, 30, or 100 mg/kg/day. To evaluate the reversibility of the lenticular change, another study was performed in female rats distributed into two groups of 60 control and 60 treated rats which received 100 mg/kg/day of compound A. This dose level was selected because it had been shown to produce lenticular changes in a large proportion of animals of both sexes. Ten control and ten treated animals were euthanized at the end of a 13-week treatment period, and all remaining rats were euthanized 26 weeks after cessation of treatment.
Atropine.
The first study was performed in which female and male rats were treated for 27 weeks (30/sex/group) at 125 mg/kg/day. To determine if increased illumination of the lens due to prolonged mydriasis may have contributed to the lenticular change seen with atropine, another study was carried out in female and male rats treated with atropine for 14 weeks at 125 mg/kg/day. In this study, rats were distributed into two groups of 60 control (30/sex each) and 60 treated (30/sex each) rats maintained in a standard lighting environment (approximately 350 lux) or in a low-light environment (approximately 15 lux). The light intensity was measured 1 m above the floor in the center of the animal room. The light intensity was also measured in the center of the cages, where it was approximately 20 lux in a standard lighting environment or 1 lux in a low-light environment.
Tolterodine.
One study was conducted in which female or male rats were treated for 14 weeks at 30 or 60 mg/kg/day (30/group) and at 80 or 100 mg/kg/day (40/group), respectively. Treatment was discontinued at 80 and 100 mg/kg/day in drug week 10 due to high mortality.
Ophthalmology.
Eye examinations were performed using indirect ophthalmoscopes of various brands with interposition of a 28 diopter Nikon lens, and/or a hand-held slit lamp biomicroscope (Kowa Co., Ltd, Tokyo, Japan). Before examination, pupils were dilated by the instillation of 0.5% tropicamide (Mydriaticum; MSD-Chibret, Paris, France). Ocular examinations were performed every 2 to 15 weeks, depending on the type of study. Pupillary response to light was generally evaluated 0.5, 2, 6, and 24 h postdosing once a week using a pen light.
Necropsy and pathology.
At study termination or interim necropsy, animals were euthanized by exsanguination after carbon dioxide inhalation. Immediately after euthanasia and exsanguination, both eyes were sampled and fixed in 10% neutral-buffered formalin, routinely processed, embedded in paraffin, cut in 5-µm-thick sections, and stained with hematoxylin and eosin (H&E). Due to the very small size of the lesion, serial sections were often necessary to visualize the change seen during the ophthalmologic evaluations.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Similarly located drug-induced (cationic amphiphilic drugs) lens epithelial changes have been observed in Wistar and Lewis rats, but these were accompanied by lesions of the cortical fibers (Drenckhahn, 1978).
It is known that in the normal rat lens, the mitotic index is higher in the peripheral zone than in the central lens epithelial zone. However, in several cases such as UV irradiation of pigmented or nonpigmented rats (Hatano and Kojima, 1996; Michael et al, 2000
; Wegener, 1995
; Wu et al, 1997
) or galactose (Terubayashi et al., 1993
) or cationic amphiphilic drug treatment (Drenckhahn, 1978
), the mitotic index was higher in the central zone. This shows that some treatment-induced lenticular changes typically involve the anterior, polar area in pigmented or nonpigmented rats, whereas drug-induced lens changes in man involve the peripheral lens epithelium. Focal epithelial proliferation has been reported in man, either as a congenital change or as a consequence of trauma, but it was always associated with degeneration of the underlying lens fibers (Duke-Elder, 1969
).
Because mydriasis and/or incomplete pupillary responses were observed with the three drugs, it was hypothesized that increased illumination of the lens could result in epithelial proliferation. Indeed, rats with prolonged mydriasis or incomplete pupillary response could be more prone to UV irradiation, known to produce changes in the central lens epithelium (Michael et al., 2000; Wegener, 1995
; Wu et al., 1997
). It has been shown that absorbed UV photons excite lens molecules creating free radicals, and the resulting oxidative stress contributes to the pathogenesis of cataracts (Spector et al., 1995
). The results of the study conducted in our laboratory, comparing the effects of 125 mg/kg/day of atropine given orally to two groups of rats maintained in either a low-light intensity or a standard light intensity environment did not support the hypothesis of a light-induced change, as both groups developed similar lens changes, both in incidence and severity. These results were in agreement with those obtained with tolterodine, which had shown no evidence of any correlation between the incidence and severity of mydriasis and that of the lens changes. Another hypothesis was that the lens epithelial proliferation could be due to mechanical forces exerted on the lens, as muscarinic receptor antagonists are known to paralyze accommodation (Bito and Harding, 1965
). As the rat is a species in which the ciliary muscle is very poorly developed (Cole, 1974
) and which hardly accommodates under normal circumstances, this mechanism is not considered to be a likely cause of the change in this species. The possibility that the lesion could be due to chemical modifications of the aqueous humor can be considered. Indeed, in the rabbit, atropine has been shown to decrease the flow rate of aqueous humor and to produce very slight decreases in the concentration of glucose and potassium; however, no definite correlation was found with increases in mitosis of the lenticular epithelium in that species (Bito et al., 1965
). Furthermore, other compounds, such as carbonic anhydrase inhibitors, which also decrease aqueous humor flow and alter aqueous humor composition (Bar-Ilan et al., 1984
) have been studied in rats (Ponticello et al., 1998
), and no changes in the lens epithelium were reported. Therefore, there is no evidence in the literature to support a possible chemical modification of the aqueous humor as a cause for the change.
Because these opacities are induced by structurally unrelated muscarinic receptor antagonists, the lenticular changes may be the result of muscarinic receptor inhibition. For such an effect, the compound and/or metabolite(s) must be present in the aqueous humor and/or in the lens epithelium. However, the presence of muscarinic receptors has not been reported in the lens of rats nor evaluated in our laboratory. M3 muscarinic receptors have been reported in human lens epithelium (Collison et al., 2000; Gupta et al., 1994
). However, all nonspecific muscarinic receptor antagonists have sufficient M3 muscarinic activity to block these receptors, and no lens changes have been reported in man with any of these drugs.
Compound A, as well as other muscarinic receptor antagonists such as atropine and tolterodine, induced central epithelial lens proliferation in Sprague-Dawley rats. Several hypotheses based on indirect or direct mechanisms were considered to explain this class effect, although none has thus far been supported. The change appears specific to the rat, a species prone to develop polar anterior subcapsular changes spontaneously, and has not been seen in mice or dogs, species that also had a long duration mydriasis. Due to its very small size, this lens opacity is not as readily identified with the indirect ophthalmoscope as with the slit lamp biomicroscope, and serial sections of the eye aid in locating the lesion for histologic evaluation. As this change has been observed in rats treated with marketed muscarinic receptor antagonists for which there has been no report of human adverse lens reactions after prolonged treatment (Brodstein et al., 1984; North, 1987
) and as the plasma profile of compound A is similar in man and dog, a species which did not develop the lens changes, it is not considered relevant to the risk evaluation of this class of compounds.
![]() |
NOTES |
---|
Part of this work (atropine only) was presented as a poster at the 2001 Society of Toxicology Annual Meeting in San Francisco, California.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bito, L. Z., Davson, H., and Snider, N. (1965). The effects of autonomic drugs on mitosis and DNA synthesis in the lens epithelium and on the composition of the aqueous humour. Exp. Eye Res. 4, 5461.[ISI]
Bito, L. Z., and Harding, C. V. (1965). Patterns of cellular organization and cell division in the epithelium of the cultured lens. Exp. Eye Res. 4, 146161.[Medline]
Brodstein, R. S., Brodstein, D. E., Olson, R. J., Hunt, S. C., and Williams, R. R. (1984). The treatment of myopia with atropine and bifocals. A long-term prospective study. Ophthalmology 91, 13731379.[ISI][Medline]
Cole, D. F. (1974). Comparative aspects of the intraocular fluid. In The Eye: Comparative Physiology. (H. Davson, Ed.), Vol. 5, pp.71161. Academic Press, New York.
Collison, D. J., Coleman, R. A., James, R. S., Carey, J. R.-S., and Duncan, G. (2000). Characterization of muscarinic receptors in human lens cells by pharmacologic and molecular techniques. Invest. Ophthalmol. Vis. Sci. 41, 26332641.
Drenckhahn, D. (1978). Anterior polar cataract and lysosomal alterations in the lens of rats treated with the amphiphilic lipidosis-inducing drugs chloroquine and chlorphentermine. Virchows Arch. B Cell Path. 27, 255266.[ISI]
Duke-Elder, S. (1969). Diseases of the lens: The capsule, subcapsular epithelium and subcapsular cataract. In System of Ophthalmology. (S. Duke-Elder, Ed.), Vol. 11, pp. 1942. Henry Klimpton, London.
Durand, G., Hubert, M. F., Kuno, H., Cook, W., Stabinski, L., Darbes, J., and Virat, M. (2001). Spontaneous polar anterior subcapsular lenticular opacity in Sprague-Dawley rats. Comp. Med. 51, 176179.[ISI][Medline]
Eglen, R. M., and Watson, N. (1996). Selective muscarinic receptor agonists and antagonists. Pharmacol. Toxicol. 78, 5968.[ISI][Medline]
Gupta, N., Drance, S. M., McAllister, R., Prasad, S., Rootman, J., and Cynader, M. S. (1994). Localization of M3 muscarinic receptor subtype and mRNA in the human eye. Ophthalmic Res. 26, 207213.[ISI][Medline]
Hatano, T., and Kojima, M. (1996). UV-B induced cataract model in brown-Norway rat eyes combined with preadministration of buthionine sulfoximine. Ophthalmic Res. 28(Suppl. 2), 5463.[ISI][Medline]
Hirose, H., Aoki, I., Kimura, T., Fujikawa, T., Numazawa, T., Sasaki, K., Sato, A., Hasegawa, T., Nishikibe, M., Mitsuya, M., Ohtake, N., Mase, T., and Noguchi, K. (2001). Pharmacological properties of (2R)-N-[1-(6-aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl)]-2-hydroxy-2-phenylacetamide: A novel muscarinic antagonist with M(2)-sparing antagonist activity. J. Pharmacol. Exp. Ther. 297, 790797.
Michael, R., Vrensen, G. F., van Marle, J., Lofgren, S., and Soderberg, P. G. (2000). Repair in the rat lens after threshold ultraviolet radiation injury. Invest. Ophthalmol. Vis. Sci. 41, 204212.
Mitsuya, M., Kobayashi, K., Kawakami, K., Satoh, A., Ogino, Y., Kakikawa, T., Ohtake, N., Kimura, T., Hirose, H., Sato, A., Numazawa, T., Hasegawa, T., Noguchi, K., and Mase, T. (2000). A potent, long-acting, orally active 2(R)-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide: Novel muscarinic M(3) receptor antagonist with high selectivity for M(3) over M(2) receptors. J. Med. Chem. 43, 50175029.[ISI][Medline]
Nilvebrant, L., Hallen, B., and Larsson, G. (1997). Tolterodinea new bladder selective muscarinic receptor antagonist: Preclinical pharmacological and clinical data. Life Sci. 60, 11291136.[ISI][Medline]
North, R. V., and Kelly, M. E. (1987). A review of the uses and adverse effects of topical administration of atropine. Ophthalmic Physiol. Opt. 7, 109114.[ISI][Medline]
Ogawa, M., Takayama, F., Kubota, M., Tanaka, Y., Takano, T., Yasumori, T., Ohzone, Y., and Uohama, K. Pharmacokinetics of J-104135, muscarinic M3 receptor antagonist, in rats and dogs (2001): Comparison with the data of animal study and Phase I single dose study. Presented at Pharmaceutical Society of Japan 121st Annual meeting, Sapporo, Japan, March 2830, 2001.
Ponticello, G. S., Sugrue, M. F., Durand-Cavagna, G., and Plazonnet, B. (1998). Dorzolamide, a 40-year wait: From an oral to a topical carbonic anhydrase inhibitor for the treatment of glaucoma. In Integration of Pharmaceutical Discovery and Development: Case Studies (Borchardt et al., Eds.), pp. 555574. Plenum Press, New York.
Spector, A., Wang, G. M., Wang, R. R., Li, W. C., and Kleiman, N. J. (1995). A brief photochemically induced oxidative insult causes irreversible lens damage and cataract. II. Mechanism of action. Exp. Eye. Res. 60, 483493.[ISI][Medline]
Terubayashi, H., Tsuji, T., Okamoto, S., Ikebe, H., and Akagi, Y. (1993). Proliferative changes of lens epithelial cells in rat and mouse galactose cataractsexamination using whole-mount preparations. Jpn. J. Ophthalmol. 37, 100107.[ISI][Medline]
Wegener, A. R. (1995). In vivo studies on the effect of UV-radiation on the eye lens in animals. Doc. Ophthalmol. 88, 221232.[ISI]
Wu, K., Shui, Y. B., Kojima, M., Murano, H., Sasaki, K., and Hockwin, O. (1997). Location and severity of UVB irradiation damage in the rat lens. Jpn. J. Ophthalmol. 41, 381387.[ISI][Medline]