Toxicological Sciences 66, 166-172 (2002)
Copyright © 2002 by the Society of Toxicology
SAFETY EVALUATION |
Muscarinic Receptor Antagonist-Induced Lenticular Opacity in Rats
* 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 |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Investigations on compound A, an M2-sparing M3 muscarinic receptor antagonist, showed that focal polar anterior subcapsular lenticular opacities, characterized by focal epithelial proliferation, developed in Sprague-Dawley rats. The incidence and bilateral localization of this change increased generally with dose and time, though plateauing after 8 months of treatment; however the severity progressed very slightly. Over a 1-year period, no anterior cortical lens fiber changes or other histological ocular changes developed. A decreased severity of the change and apoptosis suggested some regression after a 26-week recovery period. Two nonselective muscarinic receptor antagonists, atropine and tolterodine, induced similar lenticular changes in rats. A hypothesis in relation to an indirect effect of the drug, such as increased illumination of the lens due to mydriasis observed with all these compounds, was investigated and disproven. Because these opacities are induced by structurally unrelated muscarinic receptor antagonists (atropine and tolterodine), it is likely that these lenticular changes are the result of muscarinic receptor inhibition. However, hypotheses regarding a direct effect of the drug on muscarinic receptors in the lens epithelium, possibly mediated by drug and/or metabolite(s) in the aqueous humor and/or lens epithelium, remain to be investigated. This lenticular opacity is similar to that observed spontaneously in Sprague-Dawley rats, although the latter occur at a lower incidence. No such lenticular opacities have been reported in other animal species, including man, after treatment with muscarinic receptor antagonists.
Key Words: selective and nonselective muscarinic receptor antagonists; lens; epithelial proliferation; dose-related response; rat.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Muscarinic receptor antagonists, the prototype of which is atropine, have been marketed for many years. They have been used by local administration to achieve mydriasis or cycloplegia, or by systemic administration for the treatment of a wide variety of clinical conditions (including peptic ulcer, motion sickness, urinary incontinence, and chronic obstructive pulmonary disease). There was a renewed interest in the development of these drugs after five muscarinic receptor subtypes were characterized (Eglen and Watson, 1996
). Compound A, an M2-sparing M3 muscarinic receptor antagonist (Hirose et al., 2001) evaluated for the treatment of urinary incontinence and chronic obstructive pulmonary disease, was studied in our laboratories in a number of acute, subacute, and chronic toxicity studies in rats, mice, and dogs to determine its toxicity profile. During these studies, lenticular changes were found in rats, but not in mice or dogs. The purpose of this paper is to describe the effects of compound A and of two marketed nonselective muscarinic receptor antagonists (Nilvebrant et al., 1997), atropine and tolterodine, on the lens of rats, to assess the progression and the reversibility of the lenticular lesion, and to propose mechanisms for the genesis of this change. This paper also addresses the potential significance of these changes in the context of risk evaluation. |
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Animals.
Male and female Crl:CD®(SD)IGS BR (Sprague-Dawley) rats, weighing approximately 150 g, were 5 to 6 weeks old at study initiation. They were obtained from Charles River France (Saint-Aubin-lès-Elbeuf, France) or Charles River Japan Inc. (Tsukuba Breeding Center, Ibaraki Pref., Japan). They were received and maintained throughout the studies under VAF (virus antibody free) conditions. All rats were housed individually in stainless-steel cages, fed a commercial diet (16 g/day for females and 22 g/day for males), and were provided tap water ad libitum. In France, animals received UAR A04C Certified Rodent Diet (UAR, Villemoison-sur-Orge, France), and in Japan, animals received PMI Certified Rodent Diet #5002 (PMI Nutrition International, Brentwood, MO). The light intensity at 1 m above the floor was approximately 350 lux in France and 540 lux in Japan. Animal rooms were air conditioned and maintained at a temperature of 22 ± 3°C and a relative humidity of approximately 50 ± 20% with approximately 15 air changes per hour and a 12-h light cycle.
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 |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In the 14-week oral toxicity study with compound A at 10, 30, or 100 mg/kg/day, eye examinations performed with only the indirect ophthalmoscope did not show any lenticular changes in drug weeks 6 or 11. However, lenticular epithelial proliferation was observed histologically at 30 and 100 mg/kg/day in a few animals. When eye examinations were performed with the slit lamp and the indirect ophthalmoscope in all subsequent compound A, atropine, or tolterodine studies, lenticular changes were noted at all examinations, which correlated with the lenticular epithelial proliferation observed histologically (Tables 1, 2, and 3
).
|
|
|
The lesion was more easily detected with the slit lamp biomicroscope than with the indirect ophthalmoscope and was similar with all compounds; its appearance consisted of a unilateral or bilateral focal (generally pinpoint size) anterior subcapsular lenticular opacity (Fig. 1
) located in the central portion of the lens. With time in some animals, the change extended slowly around the anterior suture lines (Fig. 2). Light microscopically, the lesion consisted of focal lenticular epithelial proliferation of varying degrees of severity, in some cases with extension around the suture center (Fig. 3). The change was always of a very small size and limited to the epithelium without any changes in the anterior cortical fibers. The incidence and bilateral localization increased generally with dose and time and plateaued after 8 months of treatment with compound A. The severity of the change progressed very slightly with time and there was no sex difference. However, the no-effect level in the 53-week study with compound A was 10 mg/kg/day in males and was less than 10 mg/kg/day in females. The no-effect levels were not determined for atropine and tolterodine. The incidence of these lenticular changes was not correlated with any other ocular change. However, rats treated with compound A, atropine, or tolterodine had mydriasis (no reaction to the light) and/or incomplete pupillary responses to light (partial reaction to the light). The pupillary responses to light throughout a 14-week period are reported in Table 4.
|
|
|
|
No drug-related mortality was observed with compound A. A few drug-related deaths occurred with atropine at 125 mg/kg/day, and high mortality was observed with tolterodine at 80 and 100 mg/kg/day. Decreases in body weight gain were observed with all compounds at all tested dose levels. In rats treated with compound A at 100 mg/kg/day for 13 weeks and thereafter kept without treatment for 26 weeks, the lenticular epithelial proliferation showed partial regression 26 weeks after cessation of treatment. Although there was no substantial change in the incidence of lens opacities at ophthalmological examination during the recovery period, histologically there was a decrease in the severity of the lenticular epithelial proliferation associated in some animals with apoptosis (Table 5
).
|
There was no difference in the incidence of anterior subcapsular lens opacities between rats treated with atropine at 125 mg/kg/day and maintained either in a standard (350 lux) or in a low-light environment (15 lux), showing that the lens opacities were not related to light-induced damage.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Muscarinic receptor antagonists, M3 selective or non-selective, induced an increased incidence of lenticular epithelial proliferation in rats, which suggests a class-related effect. The lens epithelial proliferation is minute (generally pinpoint size) and located in the anterior polar region of the lens. A similar change occurs spontaneously in the Sprague-Dawley Crl:CD®(SD)IGS BR rat and has been reported in our laboratories with an incidence up to 10%, depending on the age of the animals (Durand et al, 2001
). No such epithelial lenticular changes have been observed in mice receiving up to 300 mg/kg/day of compound A for 14 weeks or in dogs receiving up to 5 mg/kg/day for 14 weeks or up to 2.5 mg/kg/day of the same compound for 53 weeks. Dogs were exposed to the maximum tolerated dose based on the presence of keratoconjunctivitis sicca resulting from drug-related reduced lacrimal secretion. However, bioavailability was much higher in dogs than in rats (Ogawa et al., 2001). In mice and dogs, prolonged mydriasis, similar to that in rats, was also observed.
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 |
|---|
1To whom correspondence should be addressed. Fax: (33) 4 73 38 56 91. E-mail: genevieve_durand{at}merck.com.
Part of this work (atropine only) was presented as a poster at the 2001 Society of Toxicology Annual Meeting in San Francisco, California.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bar-Ilan, A., Pessah, N. I., and Maren, T. H. (1984). The effects of carbonic anhydrase inhibitors on aqueous humor chemistry and dynamics. Invest. Ophthalmol. Vis. Sci. 25, 1198–1205.
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, 54–61.
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, 146–161.[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, 1373–1379.[Web of Science][Medline]
Cole, D. F. (1974). Comparative aspects of the intraocular fluid. In The Eye: Comparative Physiology. (H. Davson, Ed.), Vol. 5, pp.71–161. 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, 2633–2641.
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, 255–266.
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. 19–42. 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, 176–179.[Web of Science][Medline]
Eglen, R. M., and Watson, N. (1996). Selective muscarinic receptor agonists and antagonists. Pharmacol. Toxicol. 78, 59–68.[Web of Science][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, 207–213.[Web of Science][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), 54–63.
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, 790–797.
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, 204–212.
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, 5017–5029.[Web of Science][Medline]
Nilvebrant, L., Hallen, B., and Larsson, G. (1997). Tolterodine—a new bladder selective muscarinic receptor antagonist: Preclinical pharmacological and clinical data. Life Sci. 60, 1129–1136.[Web of Science][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, 109–114.[Web of Science][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 28–30, 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. 555–574. 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, 483–493.[Web of Science][Medline]
Terubayashi, H., Tsuji, T., Okamoto, S., Ikebe, H., and Akagi, Y. (1993). Proliferative changes of lens epithelial cells in rat and mouse galactose cataracts—examination using whole-mount preparations. Jpn. J. Ophthalmol. 37, 100–107.[Medline]
Wegener, A. R. (1995). In vivo studies on the effect of UV-radiation on the eye lens in animals. Doc. Ophthalmol. 88, 221–232.
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, 381–387.[Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  W. M. Lee Drug-Induced Hepatotoxicity N. Engl. J. Med., July 31, 2003; 349(5): 474 - 485. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||