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Occurrence of corneal opacities in rats after acute administration of l-α-acetylmethadol
TOXICOLOGY
AND
Occurrence
DAVID
APPLIED
56, 155-163(1980)
PHARMACOLOGY
of Cornea1 Opacities in Rats after Acute Administration of I-a-Acetylmethadoi
L. ROERIG,’
ANDREW
T. HASEGAWA, AND RICHARD
GERALD J. HARRIS, I. H. WANG
KENNETH
L. LYNCH.
Resrrrrch Service and the Drug Treutment Center. Wood Vetertrns Administration Medico/ Ccntct MiluuuAee. Wisconsin 53193. rend the Deportments of Pharmrrcology trnd Ophth~dmolo,~~. The, Medical Co//c~,qe of Wisconsin, MilwrruLcTc,. Wiscorl tin 53226
Received
March
24, 1980: accepted
.Irrly
IS, 1980
Occurrence of Comeal Opacities in Rats after Acute Administration of I-a-Acetylmethadol. ROERIG. D. L.. HASEGAWA, A. T.. HARRIS, G. J.. LYNCH. K. L..AND WANG, R. I. H. (1980). Toxicol. A&. Pharmacol. 56, 155- 163. The ability of I-a-acetylmethadol (LAAM) to induce ocular opacities was studied in rats. Between the third and fifth days after subcutaneous or oral administration of a single dose of LAAM, a dose-dependent incidence of opacification of the anterior segments of rat eyes was observed. Observation of the eyes of LAAM-treated rats, using a dissection microscope and a slit beam biomicroscope. indicated that the opacities were localized to the cornea and the depth of cornea1 involvement varied from patches of grayish epithelium to full-thickness involvement. Light microscopy ofeye sections from rats given LAAM. 20 mgikg orally. revealed cornea1 changes ranging from thickening and loss of regularity of epithelial cell layers to stromal vascularization and spindle cell infiltration. The SCand oral ED-50 for LAAM-induced cornea1 opacities was found to be 4.7 (4.2-5.3) and 12.6 (9.8- 16. I) mgikg. respectively. This is similar to 01 lower than the ED-50 for pharmacological effects of LAAM such as analgesia and mydriasis. The mechanism(s) by which LAAM initiates the cornea1 opacities is unknown. However. in rats made tolerant to morphine by implantation of a morphine pellet. we observed a threefold tolerance to LAAM-induced cornea1 opacities, suggesting a central nervous system mechanism in the development of the observed cornea1 changes.
Drug-induced opacities of animal eyes have been reported after administration of several narcotic analgesics (Weinstock et rll., 1958, 1961, 1967, 1969a,b: Smith et al., 1966, 1967: Fabian et al., 1967). Weinstock and Stewart (1967) reported that the administration of morphine, heroin, levorphan, pethidine, and methadone resulted in lenticular opacities in mice at doses above the analgesic ED50 but below the LD50. These lenticular opacities were transient ’ Address correspondence and reprint requests to: David L. Roerig. Ph.D.. Research ServiceilSl, Wood Veterans Administration Medical Center. 5000 West National Avenue, Milwaukee, Wise. 53193.
and disappeared as other effects of these drugs subsided (30-75 min). Lenticular opacities have also been observed in rats but at doses which were more toxic. and the opacities appeared more slowly (Weinstock et ~1.. 1958). Smith et ~1. (1967) reported that tolerance develops to the lenticular effects of opiates. Fabian et ~1. ( 19671.on the other hand. reported cornea1 opacities in rats after high doses of morphine. These cornea1 opacities were the result of irreversible cornea1 damage and developed within 4 to 5 days of morphine administration. Recently, we observed ocular opacities in rats after administration of the long-acting
1.56
ROERIG ET AL.
narcotic analgesic I-a-acetylmethadol (LAAM). Opacification of the anterior segments of rat eyes was observed after both subcutaneous and oral LAAM administration and was not observable until approximately 3 days after drug administration. Examination of these rats at 4 weeks post-LAAM administration indicated that the opacities were not reversible. Since this represents a toxic effect of LAAM in the rat, the present study was designed to investigate the incidence and anatomical localization of these ocular changes. METHODS I-cY-Acetylmethadol (LAAM) as the hydrochloride salt was obtained from the Research Technology Branch of the National Institutes on Drug Abuse and all doses of LAAM are expressed as the salt form. Morphine pellets containing 75 mg of morphine as the free base were prepared as described by Cicero ef al. (1973). Treatment of animals. Male Sprague-Dawley rats (King Animals, Oregon, Wise.) weighing 150-175 g were used. The rats were maintained in light-controlled, air-conditioned quarters with free access to food and water. A single dose of LAAM was administered either SC in saline (2.0 ml/kg) or orally in water (10.0 ml/kg). The doses of LAAM studied in these experiments ranged from 2 to IO mgikg and 8 to 40 mgikg for subcutaneous and oral LAAM administration, respectively, and were selected based on our previous determination of the analgesic ED50 and LDSO of LAAM in the rat (Roerig et al., 1977). For morphine pellet implantation the rats were anesthetized with ether and a single pellet implanted SCon the right rear side 72 hr before LAAM administration. Ocular examination. To better define the ocular opacities visible to the naked eye and mild changes not grossly visible, the rats eyes were examined using a dissection microscope (20x, American Optical Co., Buffalo, N.Y.) and a portable slit beam biomicroscope (10x, Kowa Co., Lt., Nagoya, Japan). To determine the dose-related incidence of the ocular opacities, the rats were examined at 5 days after LAAM administration using the dissection microscope. To determine the effect of LAAM on pupil size the rats were examined at 30-min intervals for at least 6 hr after LAAM administration. The pupil diameter was measured using the micrometer disk in the eyepiece of the dissection microscope and expressed as a percentage change from the predrug diameter. The ED50 and 95% confidence limits for the cornea1
opacity and the mydriatic effect of LAAM were calculated using the method of Litchfield and Wilcoxon (1949). The eyes of rats given 20 mgikg LAAM orally were also examined by light microscopy 2 weeks after LAAM administration and compared to a control group that received only water. These rats were killed by asphyxiation in CO, and the eyes removed and fixed in 10% formalin. The eyes were embedded in paraffin and S-pm sections taken from the center of the eye were stained with standard hemotoxylin and eosin procedure for histologic evaluation.
RESULTS The ocular opacities in LAAM-treated rats appeared to involve the anterior segments of the eyes with one or both eyes being involved. They became observable between the third and fifth day after LAAM administration. Using a dissection microscope, the opacities were found to be localized in the cornea with greatest involvement in the central, nasal or inferonasal regions. With the obliquely directed slit beam of the biomicroscope, the depth of the cornea1 involvement could be observed. The mildest degree of involvement was a geographic patch of grayish epithelium. The corneas of some animals revealed subepithelial involvement with whitish vascularized plaques: however, in these rats there was no significant conjunctival vascular engorgement or purulent discharge. In a few rats, frank cornea1 ulceration or full-thickness cornea1 perforation and endophthalmitis was observed. In these latter animals, some element of infection was present. Rats given 20 mg/kg LAAM (orally) were reexamined 2 weeks and some at 4 weeks after LAAM administration. None of the cornea1 opacities observed at 5 days had disappeared, and there was some tendency toward deeper involvement of the cornea. However, eyes which were not involved by 5 days showed no cornea1 opacities at 2 weeks. Effect of LAAM dose on cornea1 opacities. In order to determine if the incidence
LAAM-INDUCED
CORNEAL
of cornea1 opacities was related to the dose of LAAM, rats were given LAAM by the SC or oral route. Figure 1 shows the effect of dose on the incidence of cornea1 opacities in rats. The cornea1 opacities are expressed as the percentage of animals that developed opacities in either one or both eyes. The ED.50 and 95% confidence limits calculated from these curves were 4.7 (4.25.3) and 12.6 (9.8-16.1) mg/kg for SC and oral LAAM, respectively. No attempt was made at this time to relate the depth of cornea1 involvement to the LAAM dose. Tolerance to the cornea1 opacities was studied by determining the incidence of cornea1 opacities after SC LAAM in rats made tolerant to morphine by implantation of a morphine pellet. Morphine-tolerant rats were used since the chronic administration of LAAM necessary to achieve tolerance would probably result in some irreversible cornea1 opacities, making it difficult to determine if tolerance developed to cornea1 opacities. When LAAM was administered SC to morphine-tolerant rats, we again observed a dose-related incidence of 99so6070505040JOzoTO-
l-. -,-
,
1
I
I
5
10 DOSE
;o
;o
VA0
mg/kg
FIG. 1. Percentage of rats (probit scale) which developed cornea1 opacities in one or both eyes as a function of LAAM dose (log scale). All animals were examined 5 days after LAAM administration using the dissection microscope. LAAM administered SC (0). LAAM administered orally (O), LAAM administered SC to morphine-tolerant rats (0). At least six animals were used per dose. Asterisks indicate points calculated from 0 to 100% response.
157
OPACITIES
I 5
Al
DOSE
;0
$0
100
mg’kg
FIG. 2. Percentage change (probit scale) in pupil diameter of rats as a function of LAAM dose (log scale). Pupil diameter measured 3 hr post-LAAM administration (time of maximum dilatation). LAAM administered SC (0). LAAM administered orally I. )). At least six rats were used per dose.
cornea1 opacity (Fig. 1). However, the dose-response curve was shifted significantly to the right and the ED50 for cornea1 opacity was increased approximately threefold to 12.0 (8.6-16.7) mgikg. No cornea1 opacities were observed in the morphine-tolerant rat prior to LAAM administration. Ej$Jct o$ LAAM ot1 pupil ditrtnrtcJr-. In rats, the administration of LAAM also resulted in dilatation of the pupil which reached a maximum between 2 and 3 hr after LAAM administration. Figure 2 shows the percentage increase in pupil diameter with LAAM dose. The ED50 of LAAM for this mydriatic effect after SC and oral LAAM administration was found to be 6.2 (4.8-8.0) and 31.5 (21.0-47.3) mgikg, respectively. Depending upon the dose, the pupil diameter returned to predrug diameter by h-10 hr after administration. However. pupil diameter did not stabilize at the predrug diameter but continued to decrease. Twenty-four hours after LAAM administration miosis was observed which was marked at the higher doses. This miotic effect slowly disappeared 36-48 hr after drug administration. It should be noted that at the mydriatic
1.58
ROERIG
ET AL.
LAAM-INDUCED
CORNEAL
OPACITIES
FIG. 3. (A) Section through central cornea of a control rat. The epithelium is five or six layers thick with regular progression from basal cuboidal to surface flattened cells. The underlying stroma i\ avascular and shows artefactitious clefting (magnification, 400x 1. (B) Section through central cornea of a LAAM-treated rat. The epithelium is markedly thickened with an irregular surface and loss of regular stratification (magnification, 400x1. (Cl Low power view of full-thickness central cornea of LAAM-treated rat. A large central clear bleb separates the partially disrupted epithelium from a thickened basement membrane (magnification, 200x 1. (D) Section through central cornea of L.4AMtreated rat. The epithelium has regained an almost normal appearance. The basement membrane is crinkled and a subepithelial and superficial stromal vascular pannus is present (magnification. 400Y 1
159
160
ROERIG
ET AL.
FIG. 3-Continued.
ED50 doses approximately 15% of the rats died in the first 24 hr after SC LAAM administration and approximately 50% died after oral LAAM administration. No deaths, however, were observed after doses of LAAM at the ED50 for the cornea1 opacification effect. Pathological changes in the cornea after LAAM administration. Figure 3 shows a series of light micrographs of rat eyes 2 weeks after LAAM administration. An oral dose of 20 mg/kg LAAM was selected since this is well above the ED50 for the LAAMinduced cornea1 opacities and resulted in more accentuated cornea1 change. Furthermore, this dose is sufficiently below our previously reported LD50 for LAAM of 29 mg/kg that no deaths occurred in these rats. Examination by light microscopy demonstrated pathological changes limited to the cornea. When compared with control eyes (Fig. 3A), mild involvement (Fig. 3B) consisted of thickening and loss of regularity
of epithelial cell layers. In one eye, the epithelium was elevated off a thickened, hyalinized basement membrane by macrobleb formation (Fig. 3C). Deeper cornea1 involvement included subepithelial and superficial stromal vascularization and spindlecell infiltration (Fig. 3D). Inflammatory cell numbers were small. DISCUSSION The development of cornea1 abnormalities in LAAM-treated rats represents a toxic effect of LAAM occurring at relatively low doses. We previously reported EDSOs for LAAM analgesia (hot plate method) in the rat after SCand oral administration of 5.6 and 24.5 mg/kg, respectively (Roerig et al., 1977). The EDSOs for the LAAM-induced cornea1 opacities (SC, 4.7 mg/kg; and oral, 12.0 mg/kg) are similar to or lower than those for analgesia and are considerably less
LAAM-INDUCED
CORNEAL
OPACITIES
I0 I
than the LD50 for LAAM in the rat (SC, Fabian ei (I/. (1967). These investigators 8.5 mg/kg: and oral, 29 mg/kg, Roerig et administered morphine to rats by SC injecLll. ( 1977). tions on a daily basis (dose range 20 to X0 Another ocular effect of LAAM ob- mg/kg/day). Four to five days after the start served in the present study in the rat was of morphine, cornea1 opacities were grossly its effect on pupil diameter. The effect of visible and histological examination of eyes narcotic analgesics on pupil diameter in ani- from these animals revealed changes in the mals is varied and species dependent cornea! epithelium very similar to those ob(Krueger rt al., 1971). With LAAM we ob- served with LAAM in the present study. served a dose-related increase in pupil Fabian ef ~1. also reported that morphinediameter (Fig. 2) and the time of maximum induced cornea1 opacities were permanent pupil dilation after LAAM and the duration since they had not disappeared by IO mont hx of mydriasis were similar to that for anal- after morphine administration. gesia. While the ED50 for the mydriatic efThe cornea! changes induced by LAAM fect of LAAM after SC administration (6.2 administration might result from several mg/kg) is only slightly larger than the ED50 pathophysiologica! mechanisms. Qualitafor analgesia and cornea1 opacification it is tive or quantitative alterations in the precloser to the LD-50 of 8.5 mg/kg. After oral cornea! tear film or decreased blinking can administration the ED50 for mydriasis lead to epithelia! dessication and a break(31.5 mgikg) is approximately the same as down of this barrier layer. Fabian (‘I trf. the LD50 (29 mgikg) and. as previously (1967) suggested that excessive evaporation mentioned, a significant number of rats died from the cornea! surface during prolonged in the 24-hr period after a dose of LAAM at sedation and analgesia after high doses of’ the mydriatic ED50. It was also observed morphine may be an important factor in that miosis followed the initial LAAMmorphine-induced cornea1 opacities, In induced mydriasis; however. the rela- support of this, they reported that moistentionship of this miosis to the cornea1 opaci- ing the eyes of the rats with warm saline ties is not known. Miosis was observed in during the period of deep sedation markedly a!! rats 24 hr after LAAM administration reduced the incidence of cornea1 opacities even in those that did not develop cornea1 In genera!. transient minor insults to the opacities. cornea can result in partial thickness defect\ We found no evidence of lenticular in the epithelium. and repair is accomchanges after LAAM administration, as has plished by migration of healthy epithelial been reported with other narcotic anal- cells from the margins of the defect and 1-t’. gesics. The LAAM-induced opacities generation from the basal epithelia! layer Ir’ resulted from permanent damage to the the basal layer is partially damaged, the recornea! epithelium which was not ob- generated epithelium may be thickened and servable until about 3 days after LAAM ad- disorganized (Fig. 3B). If the basal layer i\ ministration. In contrast, opioid-induced more severely involved, inflammatory inlenticular opacities are transient in nature, jury may extend into the underlying basepersist only as long as other pharmacoment membrane and cornea1 stroma before logical effects and are observed at doses reepitheliazation can occur. Such deeper we!! above the analgesic ED-50 (Weinstock involvement is evidenced by the presence 01 et (~1.q 1958: Weinstock and Stewart, 1961). blood vessels in the stroma. which form There are. however, considerable simiduring the repair process (Fig. 3D). Permalarities between the LAAM-induced cornea! nent opacification of the cornea can thereopacities reported here and the morphinefore result despite the ultimate regeneration induced cornea! opacities reported by of a near normal epithelium. The paucitv of
162
ROERIG ET AL.
inflammatory cells in the specimens examined suggests that any acute inflammation process had subsided by 2 weeks after LAAM administration. While an epithelial defect exists, the stroma is vulnerable to infection. Full-thickness perforation by pathogens and frank intraocular infection (endopthalamitis) may occur and, indeed, was observed in the eyes of a few rats at 1 week after LAAM administration. Another possible mechanism for the observed cornea1 changes might involve denervation of the cornea. The sensory innervation of the cornea is mediated by the trigeminal nerve, which has a trophic effect on the cornea1 epithelium. Denervation can produce cornea1 changes similar to those observed in LAAM-treated rats (Duke-Elder and Leigh, 1965). The manner in which LAAM could initiate one of the above mechanisms is unknown. A direct effect of LAAM in tear formation, blinking, or cornea1 innervation is possible. Although LAAM might have a direct chemical effect on the cornea1 epithelium, the approximately threefold cross-tolerance to LAAM-induced cornea1 opacities in morphine-tolerant rats would tend to rule out this mechanism in favor of a central nervous system (CNS) mechanism in the development of the cornea1 opacities. The data of Fabian et al. (1967) also suggests that morphine-induced cornea1 opacities were related to some CNS effect of morphine, since administration of the narcotic antagonist, nalorphine, immediately after morphine completely inhibited the occurrence of cornea1 opacities. In their experiments, the morphine dose was large enough to produce a period of marked respiratory depression, sedation and exophthalmos lasting approximately 3 hr during which time drying of the cornea1 surface was observed. Nalorphine greatly reduced both the depth and duration of this sedation. With LAAM at an analgesic ED50 dose, analgesia and sedation are protracted for about 6 hr. It is possible that during
this protracted period sufficient drying of the cornea can result in the initial cornea1 epithelial damage. If this is the case, tolerance to LAAM-induced cornea1 opacities in morphine-tolerant rats would be expected since recent work in our laboratory has demonstrated cross-tolerance to CNS effects of LAAM such as analgesia (Cfold cross-tolerance) and lethality (lo-fold crosstolerance) in morphine-tolerant rats (Roerig et al., 1980). ACKNOWLEDGMENT We gratefully acknowledge the excellent technical assistance of Marilyn Fintak, H.T. (A.S.C.P.), for the histologic preparations.
REFERENCES CICERO, T. J., AND MEYER, E. R. (1973). Morphine pellet implantation in rats. Quantitative assessment of tolerance and dependence. J. Pharmacol. Exp. Thu. 184, 404-408. DUKE-ELDER, S., AND LEIGH, A. G. (1965). Diseases of the outer eye. In System of Ophthalmology (S. Duke-Elder, ed.), Vol. 8, p. 807. Mosby, St. Louis, MO. FABIAN, R. J., BOND, J. M., AND DROBECK, H. P. (1967). Induced comeal opacities in the rat. Brit. J. Ophthalmol. 51, 124- 129. KRUEGER, H., EDDY, N. B., AND SUMWALT, M. (1941). The Pharmacology of the Opium Alkaloids. Public Health Report, Suppl. No. 165, Part I, pp. 70-71. LITCHFIELD, J. T., AND WILCOXON, F. (1949). A simplified method of evaluating dose effect experiments. J. Pharmacol. Exp. Ther. %, 99-113. ROERIG,
D.
L.,
HASEGAWA,
A.
T.,
AND
WANG,
R. I. H. (1977). Effect of alteration of metabolism on the analgesic activity, toxicity, distribution and excretion of I-a-acetylmethadol in the rat. J. Pharmacol. ROERIG, D.
Exp. Ther. 203, 377-387. L., HASEGAWA, A. T.,
AND
WANG,
R. I. H. (1980). Cross tolerance to I-a-acetylmethadol (LAAM) analgesia and lethality in morphine and methadone-tolerant rats. Fed. Proc. 39, 301. SMITH,
A. A.,
KARMIN,
M.,
AND GAVITI‘,
J. (1966).
Interaction of catecholamines with levorphenol and morphine in the mouse eye. J. Pharmacol. Exp. Ther. 151, 103-109.
LAAM-INDUCED SMITH,
A.
Tolerance Pharmacol. WEINSTOCK,
A.,
CORNEAL
OPACITIES
163
KARMIN, M., AND GAVITT, J. (1967). to the lenticular effects of opiates. J. Exp. Ther. 156, 85-91.
WEINSTOCK,
M.,
M.. AND MARSHALL, A. S. ( 1969a). ‘I he influence of the sympathetic nervous system on the action of drugs on the lens. J. Pharmtrc~~d. .Crp. Ther. 166,8- 13. WEINSTOCK, M.. AND MARSHALL, A. S. (1969bi. Factors influencing the incidence of reversible lens opacities in solitary and aggregated mice J Pharmacol. Exp. Thrr. 170, 168-172.
STEWART,
H.
C.,
AND
BUTTER-
WORTH. K. R. (19%). Lenticular effect in mice of some morphine-like drugs. Nature (London) 182, 1519-1520. WEINSTOCK, M.. AND STEWART, H. C. (1961). Occurrence in rodents of reversible drug-induced opacities of the lens. &it. J. Ophthalmol. 45, 408-414.
M.. AND SCOTT, J. D. (1967). Effect of various agents in drug-induced opacities of the Ien\.
Exp.
Eye Res.
WEINSTOCK,
6, 368-375.
AND
Occurrence
DAVID
APPLIED
56, 155-163(1980)
PHARMACOLOGY
of Cornea1 Opacities in Rats after Acute Administration of I-a-Acetylmethadoi
L. ROERIG,’
ANDREW
T. HASEGAWA, AND RICHARD
GERALD J. HARRIS, I. H. WANG
KENNETH
L. LYNCH.
Resrrrrch Service and the Drug Treutment Center. Wood Vetertrns Administration Medico/ Ccntct MiluuuAee. Wisconsin 53193. rend the Deportments of Pharmrrcology trnd Ophth~dmolo,~~. The, Medical Co//c~,qe of Wisconsin, MilwrruLcTc,. Wiscorl tin 53226
Received
March
24, 1980: accepted
.Irrly
IS, 1980
Occurrence of Comeal Opacities in Rats after Acute Administration of I-a-Acetylmethadol. ROERIG. D. L.. HASEGAWA, A. T.. HARRIS, G. J.. LYNCH. K. L..AND WANG, R. I. H. (1980). Toxicol. A&. Pharmacol. 56, 155- 163. The ability of I-a-acetylmethadol (LAAM) to induce ocular opacities was studied in rats. Between the third and fifth days after subcutaneous or oral administration of a single dose of LAAM, a dose-dependent incidence of opacification of the anterior segments of rat eyes was observed. Observation of the eyes of LAAM-treated rats, using a dissection microscope and a slit beam biomicroscope. indicated that the opacities were localized to the cornea and the depth of cornea1 involvement varied from patches of grayish epithelium to full-thickness involvement. Light microscopy ofeye sections from rats given LAAM. 20 mgikg orally. revealed cornea1 changes ranging from thickening and loss of regularity of epithelial cell layers to stromal vascularization and spindle cell infiltration. The SCand oral ED-50 for LAAM-induced cornea1 opacities was found to be 4.7 (4.2-5.3) and 12.6 (9.8- 16. I) mgikg. respectively. This is similar to 01 lower than the ED-50 for pharmacological effects of LAAM such as analgesia and mydriasis. The mechanism(s) by which LAAM initiates the cornea1 opacities is unknown. However. in rats made tolerant to morphine by implantation of a morphine pellet. we observed a threefold tolerance to LAAM-induced cornea1 opacities, suggesting a central nervous system mechanism in the development of the observed cornea1 changes.
Drug-induced opacities of animal eyes have been reported after administration of several narcotic analgesics (Weinstock et rll., 1958, 1961, 1967, 1969a,b: Smith et al., 1966, 1967: Fabian et al., 1967). Weinstock and Stewart (1967) reported that the administration of morphine, heroin, levorphan, pethidine, and methadone resulted in lenticular opacities in mice at doses above the analgesic ED50 but below the LD50. These lenticular opacities were transient ’ Address correspondence and reprint requests to: David L. Roerig. Ph.D.. Research ServiceilSl, Wood Veterans Administration Medical Center. 5000 West National Avenue, Milwaukee, Wise. 53193.
and disappeared as other effects of these drugs subsided (30-75 min). Lenticular opacities have also been observed in rats but at doses which were more toxic. and the opacities appeared more slowly (Weinstock et ~1.. 1958). Smith et ~1. (1967) reported that tolerance develops to the lenticular effects of opiates. Fabian et ~1. ( 19671.on the other hand. reported cornea1 opacities in rats after high doses of morphine. These cornea1 opacities were the result of irreversible cornea1 damage and developed within 4 to 5 days of morphine administration. Recently, we observed ocular opacities in rats after administration of the long-acting
1.56
ROERIG ET AL.
narcotic analgesic I-a-acetylmethadol (LAAM). Opacification of the anterior segments of rat eyes was observed after both subcutaneous and oral LAAM administration and was not observable until approximately 3 days after drug administration. Examination of these rats at 4 weeks post-LAAM administration indicated that the opacities were not reversible. Since this represents a toxic effect of LAAM in the rat, the present study was designed to investigate the incidence and anatomical localization of these ocular changes. METHODS I-cY-Acetylmethadol (LAAM) as the hydrochloride salt was obtained from the Research Technology Branch of the National Institutes on Drug Abuse and all doses of LAAM are expressed as the salt form. Morphine pellets containing 75 mg of morphine as the free base were prepared as described by Cicero ef al. (1973). Treatment of animals. Male Sprague-Dawley rats (King Animals, Oregon, Wise.) weighing 150-175 g were used. The rats were maintained in light-controlled, air-conditioned quarters with free access to food and water. A single dose of LAAM was administered either SC in saline (2.0 ml/kg) or orally in water (10.0 ml/kg). The doses of LAAM studied in these experiments ranged from 2 to IO mgikg and 8 to 40 mgikg for subcutaneous and oral LAAM administration, respectively, and were selected based on our previous determination of the analgesic ED50 and LDSO of LAAM in the rat (Roerig et al., 1977). For morphine pellet implantation the rats were anesthetized with ether and a single pellet implanted SCon the right rear side 72 hr before LAAM administration. Ocular examination. To better define the ocular opacities visible to the naked eye and mild changes not grossly visible, the rats eyes were examined using a dissection microscope (20x, American Optical Co., Buffalo, N.Y.) and a portable slit beam biomicroscope (10x, Kowa Co., Lt., Nagoya, Japan). To determine the dose-related incidence of the ocular opacities, the rats were examined at 5 days after LAAM administration using the dissection microscope. To determine the effect of LAAM on pupil size the rats were examined at 30-min intervals for at least 6 hr after LAAM administration. The pupil diameter was measured using the micrometer disk in the eyepiece of the dissection microscope and expressed as a percentage change from the predrug diameter. The ED50 and 95% confidence limits for the cornea1
opacity and the mydriatic effect of LAAM were calculated using the method of Litchfield and Wilcoxon (1949). The eyes of rats given 20 mgikg LAAM orally were also examined by light microscopy 2 weeks after LAAM administration and compared to a control group that received only water. These rats were killed by asphyxiation in CO, and the eyes removed and fixed in 10% formalin. The eyes were embedded in paraffin and S-pm sections taken from the center of the eye were stained with standard hemotoxylin and eosin procedure for histologic evaluation.
RESULTS The ocular opacities in LAAM-treated rats appeared to involve the anterior segments of the eyes with one or both eyes being involved. They became observable between the third and fifth day after LAAM administration. Using a dissection microscope, the opacities were found to be localized in the cornea with greatest involvement in the central, nasal or inferonasal regions. With the obliquely directed slit beam of the biomicroscope, the depth of the cornea1 involvement could be observed. The mildest degree of involvement was a geographic patch of grayish epithelium. The corneas of some animals revealed subepithelial involvement with whitish vascularized plaques: however, in these rats there was no significant conjunctival vascular engorgement or purulent discharge. In a few rats, frank cornea1 ulceration or full-thickness cornea1 perforation and endophthalmitis was observed. In these latter animals, some element of infection was present. Rats given 20 mg/kg LAAM (orally) were reexamined 2 weeks and some at 4 weeks after LAAM administration. None of the cornea1 opacities observed at 5 days had disappeared, and there was some tendency toward deeper involvement of the cornea. However, eyes which were not involved by 5 days showed no cornea1 opacities at 2 weeks. Effect of LAAM dose on cornea1 opacities. In order to determine if the incidence
LAAM-INDUCED
CORNEAL
of cornea1 opacities was related to the dose of LAAM, rats were given LAAM by the SC or oral route. Figure 1 shows the effect of dose on the incidence of cornea1 opacities in rats. The cornea1 opacities are expressed as the percentage of animals that developed opacities in either one or both eyes. The ED.50 and 95% confidence limits calculated from these curves were 4.7 (4.25.3) and 12.6 (9.8-16.1) mg/kg for SC and oral LAAM, respectively. No attempt was made at this time to relate the depth of cornea1 involvement to the LAAM dose. Tolerance to the cornea1 opacities was studied by determining the incidence of cornea1 opacities after SC LAAM in rats made tolerant to morphine by implantation of a morphine pellet. Morphine-tolerant rats were used since the chronic administration of LAAM necessary to achieve tolerance would probably result in some irreversible cornea1 opacities, making it difficult to determine if tolerance developed to cornea1 opacities. When LAAM was administered SC to morphine-tolerant rats, we again observed a dose-related incidence of 99so6070505040JOzoTO-
l-. -,-
,
1
I
I
5
10 DOSE
;o
;o
VA0
mg/kg
FIG. 1. Percentage of rats (probit scale) which developed cornea1 opacities in one or both eyes as a function of LAAM dose (log scale). All animals were examined 5 days after LAAM administration using the dissection microscope. LAAM administered SC (0). LAAM administered orally (O), LAAM administered SC to morphine-tolerant rats (0). At least six animals were used per dose. Asterisks indicate points calculated from 0 to 100% response.
157
OPACITIES
I 5
Al
DOSE
;0
$0
100
mg’kg
FIG. 2. Percentage change (probit scale) in pupil diameter of rats as a function of LAAM dose (log scale). Pupil diameter measured 3 hr post-LAAM administration (time of maximum dilatation). LAAM administered SC (0). LAAM administered orally I. )). At least six rats were used per dose.
cornea1 opacity (Fig. 1). However, the dose-response curve was shifted significantly to the right and the ED50 for cornea1 opacity was increased approximately threefold to 12.0 (8.6-16.7) mgikg. No cornea1 opacities were observed in the morphine-tolerant rat prior to LAAM administration. Ej$Jct o$ LAAM ot1 pupil ditrtnrtcJr-. In rats, the administration of LAAM also resulted in dilatation of the pupil which reached a maximum between 2 and 3 hr after LAAM administration. Figure 2 shows the percentage increase in pupil diameter with LAAM dose. The ED50 of LAAM for this mydriatic effect after SC and oral LAAM administration was found to be 6.2 (4.8-8.0) and 31.5 (21.0-47.3) mgikg, respectively. Depending upon the dose, the pupil diameter returned to predrug diameter by h-10 hr after administration. However. pupil diameter did not stabilize at the predrug diameter but continued to decrease. Twenty-four hours after LAAM administration miosis was observed which was marked at the higher doses. This miotic effect slowly disappeared 36-48 hr after drug administration. It should be noted that at the mydriatic
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FIG. 3. (A) Section through central cornea of a control rat. The epithelium is five or six layers thick with regular progression from basal cuboidal to surface flattened cells. The underlying stroma i\ avascular and shows artefactitious clefting (magnification, 400x 1. (B) Section through central cornea of a LAAM-treated rat. The epithelium is markedly thickened with an irregular surface and loss of regular stratification (magnification, 400x1. (Cl Low power view of full-thickness central cornea of LAAM-treated rat. A large central clear bleb separates the partially disrupted epithelium from a thickened basement membrane (magnification, 200x 1. (D) Section through central cornea of L.4AMtreated rat. The epithelium has regained an almost normal appearance. The basement membrane is crinkled and a subepithelial and superficial stromal vascular pannus is present (magnification. 400Y 1
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FIG. 3-Continued.
ED50 doses approximately 15% of the rats died in the first 24 hr after SC LAAM administration and approximately 50% died after oral LAAM administration. No deaths, however, were observed after doses of LAAM at the ED50 for the cornea1 opacification effect. Pathological changes in the cornea after LAAM administration. Figure 3 shows a series of light micrographs of rat eyes 2 weeks after LAAM administration. An oral dose of 20 mg/kg LAAM was selected since this is well above the ED50 for the LAAMinduced cornea1 opacities and resulted in more accentuated cornea1 change. Furthermore, this dose is sufficiently below our previously reported LD50 for LAAM of 29 mg/kg that no deaths occurred in these rats. Examination by light microscopy demonstrated pathological changes limited to the cornea. When compared with control eyes (Fig. 3A), mild involvement (Fig. 3B) consisted of thickening and loss of regularity
of epithelial cell layers. In one eye, the epithelium was elevated off a thickened, hyalinized basement membrane by macrobleb formation (Fig. 3C). Deeper cornea1 involvement included subepithelial and superficial stromal vascularization and spindlecell infiltration (Fig. 3D). Inflammatory cell numbers were small. DISCUSSION The development of cornea1 abnormalities in LAAM-treated rats represents a toxic effect of LAAM occurring at relatively low doses. We previously reported EDSOs for LAAM analgesia (hot plate method) in the rat after SCand oral administration of 5.6 and 24.5 mg/kg, respectively (Roerig et al., 1977). The EDSOs for the LAAM-induced cornea1 opacities (SC, 4.7 mg/kg; and oral, 12.0 mg/kg) are similar to or lower than those for analgesia and are considerably less
LAAM-INDUCED
CORNEAL
OPACITIES
I0 I
than the LD50 for LAAM in the rat (SC, Fabian ei (I/. (1967). These investigators 8.5 mg/kg: and oral, 29 mg/kg, Roerig et administered morphine to rats by SC injecLll. ( 1977). tions on a daily basis (dose range 20 to X0 Another ocular effect of LAAM ob- mg/kg/day). Four to five days after the start served in the present study in the rat was of morphine, cornea1 opacities were grossly its effect on pupil diameter. The effect of visible and histological examination of eyes narcotic analgesics on pupil diameter in ani- from these animals revealed changes in the mals is varied and species dependent cornea! epithelium very similar to those ob(Krueger rt al., 1971). With LAAM we ob- served with LAAM in the present study. served a dose-related increase in pupil Fabian ef ~1. also reported that morphinediameter (Fig. 2) and the time of maximum induced cornea1 opacities were permanent pupil dilation after LAAM and the duration since they had not disappeared by IO mont hx of mydriasis were similar to that for anal- after morphine administration. gesia. While the ED50 for the mydriatic efThe cornea! changes induced by LAAM fect of LAAM after SC administration (6.2 administration might result from several mg/kg) is only slightly larger than the ED50 pathophysiologica! mechanisms. Qualitafor analgesia and cornea1 opacification it is tive or quantitative alterations in the precloser to the LD-50 of 8.5 mg/kg. After oral cornea! tear film or decreased blinking can administration the ED50 for mydriasis lead to epithelia! dessication and a break(31.5 mgikg) is approximately the same as down of this barrier layer. Fabian (‘I trf. the LD50 (29 mgikg) and. as previously (1967) suggested that excessive evaporation mentioned, a significant number of rats died from the cornea! surface during prolonged in the 24-hr period after a dose of LAAM at sedation and analgesia after high doses of’ the mydriatic ED50. It was also observed morphine may be an important factor in that miosis followed the initial LAAMmorphine-induced cornea1 opacities, In induced mydriasis; however. the rela- support of this, they reported that moistentionship of this miosis to the cornea1 opaci- ing the eyes of the rats with warm saline ties is not known. Miosis was observed in during the period of deep sedation markedly a!! rats 24 hr after LAAM administration reduced the incidence of cornea1 opacities even in those that did not develop cornea1 In genera!. transient minor insults to the opacities. cornea can result in partial thickness defect\ We found no evidence of lenticular in the epithelium. and repair is accomchanges after LAAM administration, as has plished by migration of healthy epithelial been reported with other narcotic anal- cells from the margins of the defect and 1-t’. gesics. The LAAM-induced opacities generation from the basal epithelia! layer Ir’ resulted from permanent damage to the the basal layer is partially damaged, the recornea! epithelium which was not ob- generated epithelium may be thickened and servable until about 3 days after LAAM ad- disorganized (Fig. 3B). If the basal layer i\ ministration. In contrast, opioid-induced more severely involved, inflammatory inlenticular opacities are transient in nature, jury may extend into the underlying basepersist only as long as other pharmacoment membrane and cornea1 stroma before logical effects and are observed at doses reepitheliazation can occur. Such deeper we!! above the analgesic ED-50 (Weinstock involvement is evidenced by the presence 01 et (~1.q 1958: Weinstock and Stewart, 1961). blood vessels in the stroma. which form There are. however, considerable simiduring the repair process (Fig. 3D). Permalarities between the LAAM-induced cornea! nent opacification of the cornea can thereopacities reported here and the morphinefore result despite the ultimate regeneration induced cornea! opacities reported by of a near normal epithelium. The paucitv of
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inflammatory cells in the specimens examined suggests that any acute inflammation process had subsided by 2 weeks after LAAM administration. While an epithelial defect exists, the stroma is vulnerable to infection. Full-thickness perforation by pathogens and frank intraocular infection (endopthalamitis) may occur and, indeed, was observed in the eyes of a few rats at 1 week after LAAM administration. Another possible mechanism for the observed cornea1 changes might involve denervation of the cornea. The sensory innervation of the cornea is mediated by the trigeminal nerve, which has a trophic effect on the cornea1 epithelium. Denervation can produce cornea1 changes similar to those observed in LAAM-treated rats (Duke-Elder and Leigh, 1965). The manner in which LAAM could initiate one of the above mechanisms is unknown. A direct effect of LAAM in tear formation, blinking, or cornea1 innervation is possible. Although LAAM might have a direct chemical effect on the cornea1 epithelium, the approximately threefold cross-tolerance to LAAM-induced cornea1 opacities in morphine-tolerant rats would tend to rule out this mechanism in favor of a central nervous system (CNS) mechanism in the development of the cornea1 opacities. The data of Fabian et al. (1967) also suggests that morphine-induced cornea1 opacities were related to some CNS effect of morphine, since administration of the narcotic antagonist, nalorphine, immediately after morphine completely inhibited the occurrence of cornea1 opacities. In their experiments, the morphine dose was large enough to produce a period of marked respiratory depression, sedation and exophthalmos lasting approximately 3 hr during which time drying of the cornea1 surface was observed. Nalorphine greatly reduced both the depth and duration of this sedation. With LAAM at an analgesic ED50 dose, analgesia and sedation are protracted for about 6 hr. It is possible that during
this protracted period sufficient drying of the cornea can result in the initial cornea1 epithelial damage. If this is the case, tolerance to LAAM-induced cornea1 opacities in morphine-tolerant rats would be expected since recent work in our laboratory has demonstrated cross-tolerance to CNS effects of LAAM such as analgesia (Cfold cross-tolerance) and lethality (lo-fold crosstolerance) in morphine-tolerant rats (Roerig et al., 1980). ACKNOWLEDGMENT We gratefully acknowledge the excellent technical assistance of Marilyn Fintak, H.T. (A.S.C.P.), for the histologic preparations.
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WORTH. K. R. (19%). Lenticular effect in mice of some morphine-like drugs. Nature (London) 182, 1519-1520. WEINSTOCK, M.. AND STEWART, H. C. (1961). Occurrence in rodents of reversible drug-induced opacities of the lens. &it. J. Ophthalmol. 45, 408-414.
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