The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Original Article
Drug-induced lenticular opacity and accumulation of cholesterol-related substances in the lens cortex of dogs
Hiroshi IwasakiMasaki WakamatsuKazunari SugiharaKyohei KamioSatoshi TsujiJunya MoritaYasuhiro KuriharaTomoko IzumiTomohiro NishimotoKohnosuke KinoshitaYutaka NakanishiMinoru Sasaki
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2020 Volume 45 Issue 4 Pages 201-218

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Abstract

TP0446131, developed as an antidepressant agent, was found to cause lenticular opacity in a 13-week repeated-dose study in dogs. Histopathologically, the lenticular opacity was observed as a degeneration of the lens fibers, characterized by irregularity in the ordered arrangement of the fibers which is necessary to maintain the transparency of the lens, and was considered to manifest clinically as cataract. To evaluate the development mechanism of the lenticular opacity, the chemical constituents of the lens, which is known to be associated with the development of cataract, were examined. The results of liquid chromatography-tandem mass spectrometry analysis revealed an increase in the amplitudes of 3 unknown peaks in a dose- and time-dependent manner in the lens, with no remarkable changes in the other chemical components tested. In addition, the content of cholesterol, alterations of which have been reported to be associated with cataract, remained unchanged. The mass spectral data and chromatographic behavior of the 3 peaks indicated that these peaks corresponded to sterol-related substances, and that one of them was 7-dehydrocholesterol, a precursor of cholesterol biosynthesis. This finding suggested that TP0446131 exerts some effects on the cholesterol biosynthesis pathway, which could be involved in the development of the cataracts. Furthermore, increases in the levels of these sterol-related substances were also detected in the serum, and were, in fact, noted prior to the onset of the cataract, suggesting the possibility that these substances in the serum could be used as potential safety biomarkers for predicting the onset of cataract induced by TP0446131.

INTRODUCTION

Cataract, which is characterized by clouding or loss of transparency of the optic lens, resulting in a decrease of vision (Bassnett et al., 2011), can arise from many causes, including aging, trauma, radiation, diabetes mellitus, and other diseases (Bron et al., 1993; Moreau and King, 2012; Thompson and Lakhani, 2015). While drug-induced cataracts are rare, a wide variety of approved drugs, including steroid preparations (Jobling and Augusteyn, 2002), hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors (MacDonald et al., 1988; Gerson et al., 1990), glucose-elevating agents (Schiavo et al., 1975), cytotoxic chemotherapeutic agents (Saito et al., 2015), and an iron chelator (Wang et al., 1992), have been shown in non-clinical studies to have cataract-inducing potential. Cataract has been reported to develop even during clinical use of some of these drugs (Cenedella, 1996).

In the lens, lens fibers are constantly produced to sustain a clear lens (Bassnett et al., 2011). The lens epithelial cells proliferate in the germinative zone, which is located just anterior to the equator of lens. The proliferating cells migrate posteriorly to the bow area of the lens and differentiate into lens fiber cells. The cells change in shape from low cuboidal to high columnar, and finally elongate towards both the anterior and posterior poles of the lens, forming crescent fibers. The lens owes its transparency to the high degree of ordering of the lens fibers in the syncytium, the cells losing their nuclei and organelles during the differentiation process and beginning to express transparent proteins, the crystallins. The lens functions are retained throughout life by the addition of new lens fibers in place of old ones (Rao, 2008; Song et al., 2009). Since the lens fibers elongate to fit the thickness of the lens, a large amount of cholesterol is needed as a constituent of the cell membrane. However, the lens receives very little supply of cholesterol from the blood via aqueous humor in the anterior chamber, and the epithelial cells have to obtain most of its cholesterol by de novo synthesis (Cenedella, 1996).

Inhibitors of the cholesterol biosynthesis pathway have been reported to induce cataract in both animals and humans. The statins, which belong to the drug class of HMG-CoA reductase inhibitors, are well known to induce cataract in dogs (Gerson et al., 1990; MacDonald et al., 1988; Walsh et al., 1996). The oxidosqualene cyclase inhibitors have been reported to induce cataract in mice, dogs, and hamsters (Cenedella and Bierkamper, 1979; Pyrah et al., 2001), and a Δ7-reductase inhibitor to induce cataract in rats (Xu et al., 2011). E2012, a gamma secretase modulator had been developed for the treatment of Alzheimer’s disease, induced cataract in rats, and was also found to inhibit 3β-hydroxysterol Δ24-reductase as an off-target effect (Nakano-Ito et al., 2014). Triparanol, an inhibitor of the same reductase, is known as an inducer of cataract in animal and humans (Kirby, 1967; Harris and Gruber, 1969; von Sallmann et al., 1963). As a pathological characteristic of the cataract induced by these inhibitors of the cholesterol biosynthesis pathway, the opaque area of the lens seems to expand from the equator of the lens to the center of the cortex, as it is the newly differentiated lens fibers that are affected.

TP0446131 [3-Methoxy-N-{1-[(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)methyl]piperidin-4-yl}benzamide], a melanin-concentrating hormone receptor antagonist, is under development as an antidepressant agent. In a 13-week repeated-dose toxicity study conducted in dogs, both male and female dogs in the highest dose group (30 mg/kg/day) were detected to have developed lenticular opacity by ophthalmological examination conducted at Week 13 of dosing. The plasma drug exposure (AUC0-24hr) in the highest-dose group is 1290-fold higher than that observed at the minimal effective dose in the forced swimming test in rats. By contrast, no lenticular opacity was observed in the 13-week repeated-dose toxicity study conducted in rats, even though the plasma drug concentrations in the rats were higher than those observed in the highest-dose group in dogs. There have been no other reports of the development of lenticular opacity in laboratory animals after repeated doses of a melanin-concentrating hormone receptor antagonist, and the presence of this receptor has not been reported in the lens. Based on the above, it is considered that the lenticular opacity detected in the dogs may have been induced by an indeterminate off-target effect of TP0446131.

In the present study, to characterize the lenticular opacity observed in the dogs treated with the drug, male dogs were administered TP0446131 once or repeatedly up to 13 weeks. The occurrence and progression of cataracts, and the changes occurring after withdrawal of TP0446131 were examined by weekly ophthalmological examination as well as histopathological examination. The blood and ocular tissues were collected to investigate the biochemical changes associated with the cataracts. Interestingly, changes in the contents of sterol-related substances were observed in the lens cortex of the dogs treated with TP0446131. This finding suggests that some effects of the drug on the cholesterol biosynthesis pathway may have been involved in the development of the drug-induced cataracts. Since alterations in the levels of the same sterol-related substances were found even prior to the onset of the cataract, it is considered that the serum levels of these substances may potentially serve as predictive safety biomarkers for the development of cataract induced by TP0446131.

MATERIALS AND METHODS

Animals

Male beagle dogs that were 7 to 9 months old (Beijing Marshall Biotechnology Co., Ltd., Beijing, China) at the initiation of dosing were used for the studies. The dogs were housed in individual pen-type cages and fed approximately 250 g/day of a pellet diet (DS-A, Oriental Yeast Co., Ltd., Tokyo, Japan). Drinking water was supplied ad libitum through an automatic water supply system. To minimize the study design bias, the dogs were selected based on the results of pre-dose ophthalmologic examinations, and were divided into different dose groups in such a way that the mean body weights in the groups were approximately equal in each study. All animal experiments were conducted in accordance with the “Guideline for Animal Experimentation” specified by the Research Center, Taisho Pharmaceutical Co., Ltd.

Drug administration

TP0446131 was dissolved in 0.5% methylcellulose (MC) 4000 solution (50 mmol/L citrate buffer solution, pH 3.5) as a vehicle. The dosing formulation was administered to the animals via oral gavage. The dosing volume (5 mL/kg) for each dog was calculated based on the latest body weight measured.

Study design

To investigate the time of onset and potential pathogenesis of drug-induced cataract and to explore the biochemical changes associated with the cataract in the blood and ocular tissues of dogs, TP0446131 was administered once, consecutively for 14 days, or over the long-term for 13 weeks to dogs.

In the single-dose group, 6 dogs were administered TP0446131 once at the dose level of 100 mg/kg. At 4 or 24 hr after the dosing, 3 dogs each were euthanized to collect ocular tissue samples. As the controls, 3 dogs administered vehicle were subjected to necropsy at 24 hr after dosing.

In the 14-day repeated-dose group, 3 dogs each per dose level were administered TP0446131 once daily at the dose level of 0 (vehicle), 30, or 50 mg/kg/day. The dogs were euthanized and subjected to necropsy at the end of the dosing period.

In the long-term repeated-dose group, 12 dogs were administered TP0446131 once daily for various dosing periods up to 13 weeks. The dose level was set at 30 mg/kg/day, which is the same dose level at which lenticular opacity was observed in a previous 13-week repeated-dose toxicity study. However, the dose level was transiently reduced to 20 mg/kg/day from Week 7 to Week 9 of dosing, because some dogs showed swelling of the distal portions of their limbs, a known toxicological effect of TP0446131, became notable in some dogs. Of the 12 dogs, 3 dogs were euthanized at Week 5 of dosing, another 3 dogs at Week 8 of dosing, at which lenticular opacity was initially detected by ophthalmologic examination. Of the remaining 6 dogs, 2 dogs were administered the drug for 13 weeks. The dosing in the remaining 4 dogs was discontinued at Week 8 or Week 9 of dosing, immediately upon detection of the first sign of lenticular opacity. These dogs were continuously examined for the subsequent 18 weeks of the recovery period. At the end of the dosing or the recovery period, the dogs were euthanized and subjected to necropsy. As controls, 9 dogs were administered the vehicle and subjected to necropsy at Weeks 8 or 13 of dosing, and at Week 13 of the recovery period following 13 weeks of dosing, at the same time as the necropsies were performed in the TP0446131-treated dogs (Table 1).

Table 1. List of studies and examination items.

Chemicals

Desmosterol, 7-dehydrocholesterol, and 5-α-cholesta-7,24-diene-3β-ol were purchased from Sigma-Aldrich (currently Merck, St. Louis, MO, USA). Cholesterol and cholecalciferol were obtained from Wako Pure Chemical Co. (currently FUJIFILM Wako Pure Chemical, Osaka, Japan), and 8-dehydrocholesterol, zymosterol, and desmosterol-d6 were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Cholesterol-d7 was obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Ophthalmologic examination

To monitor the occurrence and progression of the cataracts, ophthalmologic examinations were performed once a week in the long-term repeated-dose studies. The ophthalmologic examination was also conducted on Day 13 of dosing in the 14-day repeated-dose study. The anterior portions of the eye, optic media and ocular fundi were examined using a slit lamp and a binocular indirect ophthalmoscope. A mydriatic was instilled into the eyes of the dogs prior to examination of the optic media and ocular fundi (Mydrin P, Santen Pharmaceutical Co., Ltd., Osaka, Japan).

Blood sampling, blood chemistry and monitoring of the daily blood glucose profile

For the blood chemistry examinations, cephalic vein blood samples were collected from the animals, on the day of necropsy in the single-dose, 14-day repeated-dose, as well as long-term repeated-dose studies, into sampling tubes containing a coagulant (Venoject II autosep, VP-AS076KM, Terumo Corp., Tokyo, Japan). Serum was separated from the samples by centrifugation (4°C, approximately 1200 × g, 15 min) and stored in an ultra-low temperature freezer until the analyses. The total cholesterol, glucose, sodium, potassium, and chloride concentrations in the serum were measured by an automatic analyzer (7180, Hitachi High-Technologies Corp., Tokyo, Japan).

To monitor the daily blood glucose profile, a part of the plasma samples collected for the toxicokinetic analyses were used. The plasma glucose levels were measured by an automatic analyzer, and the maximum plasma concentration (Cmax) of glucose and the area under the plasma concentration time curve (AUC0-24hr) were calculated.

For the toxicokinetic analyses, cephalic vein blood samples were collected into sampling tubes containing EDTA-2K as the anticoagulant, prior to the start of dosing (pre-dose, except for Day 1 of dosing) and 0.5, 1, 2, 4, 8, and 24 hr post-dose on the day of dosing in the single-dose study, on Day 1 and Day 14 in the 14-day repeated-dose study, and on Day 1, Day 14, Week 5, Week 7, Week 9, and Week 12 in the long-term repeated-dose study. Plasma from the samples was separated by centrifugation (4°C, approximately 2,100 × g, 15 min), and stored in an ultra-low temperature freezer until the analyses.

Ocular tissue sampling

The dogs were anesthetized by intravenous injection of pentobarbital sodium (Somnopentyl, Kyoritsu Seiyaku Co., Ltd., Tokyo, Japan) and sacrificed by exsanguination from the femoral arteries and veins 4 or 24 hr after the dosing in the single-dose study, and at approximately 24 hr after the final dosing in the 14-day repeated-dose and long-term repeated-dose studies. The eyeballs were removed at necropsy. For analysis of the chemical constitutions of the ocular tissues and measurement of the tissue drug concentrations, the aqueous humor (approximately 0.6 mL per lateral eyeball), lens, and vitreous body (approximately 1 mL per lateral eyeball) were collected (except for the left eyeball in the long-term repeated-dose study). To sample the lenticular cortex, the lens was divided evenly, and the nucleus of the lens was removed. The collected tissues were placed in sampling tubes and weighed, then quickly frozen in liquid nitrogen, and stored in an ultra-low temperature freezer until the analyses.

Histopathological examination

At necropsy in the long-term repeated-dose study, the left eyeball was collected for histopathological examination. Davidson’s solution was prepared by mixing 99.5% ethanol (Kokusan Chemical Co., Ltd., Tokyo, Japan), acetic acid (Kokusan Chemical Co., Ltd.), 10% neutral buffered formalin (FUJIFILM Wako Pure Chemical Corp.) and distilled water at the ratio of 3:1:2:3. Aqueous humor was removed from the anterior chamber of the eyeball and an equal volume of the Davidson’s solution was injected into the anterior chamber. Then, the eyeball was immersed in Davidson’s solution for approximately 24 hr, and cut on a line containing the optic disc, vertically to the long posterior ciliary artery. The eyeball was further fixed by immersing in 10% neutral buffered formalin for longer than 6 days. Then, it was embedded in paraffin and sections were prepared and stained with hematoxylin-eosin.

Analyses of the chemical constituents of the lens cortex and aqueous humor

The lenticular cortex was homogenized with 0.1 mol/L sodium hydrate before the analysis. The concentrations of glucose, sodium, potassium, and chloride were measured using an automatic analyzer. The measurement value for sodium was corrected by subtracting the concentration of sodium contained in the homogenization solution of 0.1 mol/L sodium hydrate. Other chemical constituents were determined using commercially available kits, as follows: F-kit D-sorbitol/xylitol (J.K. International Inc., Tokyo, Japan) for measurement of sorbitol, Fructose colorimetric/fluorometric assay kit (BioVision Inc., Milpitas, CA, USA) for fructose, Glutathione fluorescent detection kit (Arbor Assays Inc., Ann Arbor, MI, USA) for glutathione (GSH) and glutathione-S-S-glutathione (GSSG), Superoxide dismutase assay kit (Cayman Chemical, Ann Arbor, MI, USA) for superoxide dismutase (SOD), Ascorbate assay kit (Cayman Chemical) for ascorbic acid, and Hydrogen peroxide fluorescent detection kit (Arbor Assays Inc.) for H2O2.

Quantitative measurement of TP0446131 in the plasma and lens cortex

For the quantification of TP0446131 in the ocular tissue, the lenticular cortex was homogenized with four times the volume of distilled water. The sample treatment was performed as follows: 10 µL of plasma or lenticular cortex homogenate was spiked with 10 µL of acetonitrile/water (1:1, v/v). Then, 200 µL of acetonitrile/methanol (9:1, v/v) containing deuterium-labeled TP0446131 as the internal standard was added to the solution and the mixture was centrifuged at 3,000 × g for 5 min at 4°C. Five microliters of the resulting supernatant was subjected to LC-MS/MS analysis, as follows. Chromatographic separation was performed using an LC-20AD or LC-30AD liquid chromatograph (Shimadzu, Kyoto, Japan) on a ZORBAX SB-C18 analytical column (4.6-mm i.d. 50 mm, 3.5-μm particle size; Agilent Technologies, Santa Clara, CA, USA). The gradient consisted of 0.1 w/v% ammonium acetate as solvent A and methanol as solvent B, starting at 55% B, holding for 0.5 min, ramping to 95% B over 2.5 min, holding for 0.5 min, decreasing to 55% B in 0.1 min, and then holding for 1.4 min. The flow rate was set at 1 mL/min, and the column temperature was maintained at 50°C. The sample measurement was performed using the API4000 or QTRAP6500 triple quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA) with the TurboIonspray ionization mode set in the positive ion detection mode for TP0446131 (m/z 394 → 160) and the internal standard (m/z 397 → 160). The lower limit of quantification (LLOQ) was 0.1 ng/mL for analysis of the Day-1 samples in the long-term repeated-dose study, and 1 ng/mL in the samples obtained in the other studies.

Toxicokinetic analysis

The toxicokinetic analysis was conducted using Phoenix WinNonlin ver. 6.2 (Certara, St. Louis, MO, USA). The maximum plasma concentration (Cmax), time to reach maximum plasma concentration (Tmax), and area under the plasma concentration time curve from time 0 to 24 hr post-dose (AUC0-24hr) were calculated for each dog, and the mean and standard deviation of each parameter for each group were calculated.

Quantitative measurement of cholesterol and desmosterol in the serum and lens cortex

For the quantification of cholesterol and desmosterol in the serum and lenticular cortex, the sample treatment was conducted as follows: 50 µL of serum or lenticular cortex homogenate was spiked with 20 µL of methanol and 20 µL of methanol containing an internal standard (cholesterol-d7). For the hydrolysis of cholesteryl ester, the sample was mixed with 450 µL of methanol and 200 µL of 10 mol/L potassium hydroxide, mixed thoroughly, and incubated at 40°C for 60 min. Following the incubation, 25 µL of 50 v/v% phosphoric acid, 500 µL of distilled water, and 1 mL of n-hexane were added to the sample and mixed well. The resulting sample was centrifuged at 2,095 × g for 5 min at 4°C, and the organic phase was transferred to a new tube and evaporated to dryness under a nitrogen gas stream at 40°C. The pellet was reconstituted with 500 µL of methanol, and subjected to LC-MS/MS analysis.

The cholesterol concentrations in the serum and lenticular cortex were determined as follows. Chromatographic separation was performed using an LC-20AD liquid chromatograph on a Hypersil Gold analytical column (2.1-mm i.d. × 150 mm, 3.0-μm particle size; Thermo Fisher Scientific, Waltham, MA, USA). The gradient consisted of 0.1 v/v% formic acid as solvent A and acetonitrile as solvent B, starting at 80% B, ramping to 98% B over 20 min, holding for 5 min, decreasing to 80% B in 0.01 min, and then holding for 10 min. The flow rate was set at 0.25 mL/min, and the column temperature was maintained at 40°C. The sample measurement was performed using the API4000 triple quadrupole mass spectrometer with the atmospheric pressure chemical ionization (APCI) mode set in the positive ion detection mode for cholesterol (m/z 369.3 → 147.3) and the internal standard (m/z 376.3 → 147.3).

The desmosterol concentrations in the serum and lenticular cortex were determined as follows. Chromatographic separation was performed using an LC-30AD liquid chromatograph on two Hypersil Gold analytical columns connected in series (2.1-mm i.d. × 150 mm × 2, 3.0-μm particle size). The gradient consisted of 0.1 v/v% formic acid as solvent A and acetonitrile as solvent B, starting at 70% B, ramping to 87% B over 102 min, stepping up to 98% in 0.01 min, holding for 20 min, decreasing to 70% B in 0.10 min, and then holding for 28 min. The flow rate was set at 0.25 mL/min, and the column temperature was maintained at 40°C. The sample measurement was performed using QTRAP6500 triple quadrupole mass spectrometer with the APCI ionization mode in the positive ion detection mode for desmosterol (m/z 367.3 → 147.3) and the internal standard (m/z 376.3 → 147.3). The LLOQ was 30 ng/mL for desmosterol and 3 µg/mL for cholesterol.

In all the selected reaction monitoring (SRM) transitions, the precursor ions of cholesterol and desmosterol correspond to each dehydrated protonated molecule, and their product ions correspond to the characteristic fragments which are formed by cleavage of the steroid C ring (Igarashi et al., 2011).

Qualitative measurement of unknown peaks and isomers of desmosterol

The extracted lenticular cortex and serum samples were also subjected to LC-APCI-MS/MS analysis to obtain product ion mass spectra for unknown peaks that had been detected during determination of desmosterol. For structural elucidation of the components in the unknown peaks, 5 authentic standards of desmosterol isomers, 7-dehydrocholesterol, 8-dehydrocholesterol, zymosterol, cholecalciferol, and 5-α-cholesta-7,24-diene-3β-ol, were dissolved separately in methanol (10,000 ng/mL) and subjected to analyses. In addition, 7-dehydrocholesterol and 8-dehydrocholesterol were separately added to the extracted lenticular cortex samples to compare the retention times between the standards and unknown peaks as follows: the lenticular cortex homogenate was treated by liquid-liquid extraction in the same manner as that for quantitative measurement of desmosterol, and the dried organic phase was reconstructed with 500 µL of methanol including 7-dehydrocholesterol (100 ng/mL) or 8-dehydrocholesterol (300 ng/mL) and subjected to the analysis. Chromatographic separation was performed under the same conditions as those used for the quantitative measurement of desmosterol.

RESULTS

Time of onset and development of lenticular opacity

In the long-term repeated-dose study, the dogs were administered TP0446131 at 30 mg/kg/day for dosing periods of up to 13 weeks, except for Week 7 to Week 9 of dosing, during which the dose level was decreased transiently to 20 mg/kg/day.

Ophthalmologic examination revealed the first sign of lenticular opacity at Week 8 or Week 9 of dosing (Fig. 1a and b). No lenticular opacity was observed until Week 7 of dosing in any of the dogs in this study. The lenticular opacities were initially detected around the equator of the lens. Subsequently, the area of the opacity gradually expanded along the suture line, and covered the whole area of the lens up to Week 11 or Week 12 of dosing. At the end of the 13-week dosing period, the ocular fundus could not be visualized due to the lenticular opacities (Fig. 1c).

Fig. 1

Ophthalmologic morphology of drug-induced cataract in dogs. In the long-term repeated-dose study, the dogs were administered TP0446131 at the dose of 30 mg/kg/day for a period of up to 13 weeks, except during Week 7 to Week 9 of dosing, during which time, the dose level was decreased transiently to 20 mg/kg/day. (a) Lens of a control dog. (b) Lens of a drug-treated dog at Week 8 of dosing; the first sign of lenticular opacity was found around the equator line (arrow heads); (c) Lens of a drug-treated dog at Week 13 of dosing: the area of the lenticular opacity had expanded to the whole area of the lens (arrow heads); (d) Lens of a drug-treated dog at Week 10 of the recovery period; in this dog, the drug administration had been discontinued even in the early stage after detection of the abnormal ophthalmologic finding (arrow heads); (e) and (f) Lens of a drug-treated dog at Week 1 (e) or Week 18 (f) of the recovery period; in this dog, the drug had been withdrawn in the very early phase after detection of the abnormal lenticular findings, and lenticular opacity did not progress to severe until Week 18 of the recovery period (arrow heads).

The area of opacity continued to expand even after withdrawal of the drug in the early stage after detection of the ophthalmologic finding. Administration of TP0446131 was discontinued in 4 dogs just after Week 8 or Week 9 of dosing when the first sign of lenticular opacity was detected. In 3 of the dogs, the lenticular opacities extended to the whole area of the lens during the recovery period, and their ocular fundus became unclear between Week 4 and Week 8 of the recovery period. Subsequently, in 2 of these 3 dogs, it became impossible to visualize the ocular fundus at Week 10 (Fig. 1d), and the remaining 1 of the 3 dogs was euthanized at Week 11 of the recovery period, because this dog had developed a mydriasis of both eyes. By contrast, in the remaining 1 dog, in which the very early phase of lenticular opacity was detected at Week 9 of dosing and TP0446131 was withdrawn immediately, the ocular fundus could be monitored from Week 1 (Fig. 1e) to Week 18 of the recovery period (Fig. 1f). In this dog, the lenticular opacity did not progress to become severe, even though the area of the opacity extended to the entire area of the lens.

Histopathology of the lenticular opacity

In the histopathological examination, the lenticular opacity observed by ophthalmologic examination was characterized by lens fiber degeneration. Swelling and disordered organization of the lens fibers were observed in the affected areas of lens. The severity of the lesion increased with increase in the dosing period (Table 2). At Week 5 of dosing, even prior to the manifestation of any abnormal findings on ophthalmologic examination, minimal degeneration of the lens fibers was observed in the equator (Fig. 2a and c) and posterior subcapsular region of the cortex (Fig. 2b and d). At Week 8 of dosing, the area of degeneration expanded from the surface to the deep layer of the lenticular cortex, and became more prominent around the equator (Fig. 2e) and posterior subcapsular cortex (Fig. 2f). By the end of the 13-week dosing period, the area of degeneration reached the deeper layers of the lenticular cortex (Fig. 2g and h).

Table 2. Results of ophthalmologic examination in the long-term repeated-dose study – time of onset and development of the lenticular opacity.
Fig. 2

Degeneration of the lens fibers during the dosing period. (a) and (b) Representative lens in a control dog; (c) and (d) Minimal degeneration of the lens fibers was observed at Week 5; (e) and (f) The area of degeneration expanded to the deeper layers of the cortex by Week 8, (g) and (h) by Week 13. The figures on the left show the equatorial segment and those on the right show the posterior subcapsular area of the lens. Scale bars represent 200 μm. *: The area of degenerations.

Even after the recovery period of 18 weeks, the degeneration of the lens fibers could still be observed in the dogs in which the drug had been discontinued at Week 8 or Week 9 of dosing. However, formation of normal lens fibers was noted in the subcapsular region of the cortex. The degenerated lens fibers were observed in the inner area of the lenticular cortex under the normal lens fibers (Fig. 3b). The severity of the degeneration varied from minimal to severe among the dogs (Table 3).

Fig. 3

Recovery of the lens fibers during the recovery period. The degenerated lens fibers were observed in the inner area of the lenticular cortex under the normal lens structure around the posterior subcapsular area of the lenses; (a) Representative lens in a control dog, (b) TP0446131 at recovery Week 18. Scale bars represent 100 μm. *: The area of degenerations.

Table 3. Results of histopathological examination after long-term repeated-dose administration and after the 18-week recovery period.

Chemical constituents of the lenticular cortex, serum, and aqueous humor

The chemical constituents known to be associated with generation of cataracts were quantitated in the lens of the dogs treated with TP0446131 for 1 day, 14 days, or 13 weeks. The constituents measured included sterols (cholesterol and desmosterol) (Zelenka, 1984), sugars (glucose, sorbitol, and fructose) (Ross et al., 1983), oxidative stress markers (ascorbic acid, GSH, GSSH, and SOD) (Wojnar et al., 2017; Zhang et al., 2017), and electrolytes (sodium, potassium, and chloride) (Rodríguez-Sargent et al., 1987). The concentrations of the sterols, glucose, and electrolytes were also examined in the serum, and the concentrations of glucose, oxidative stress markers (including H2O2), and electrolytes were measured in the aqueous humor.

As for the sterols, there were no differences in the cholesterol or desmosterol levels in the lenticular cortex or in the serum cholesterol levels in any of the dose groups as compared to the control group (Table 4 and Table 5). On the other hand, a 32% decrease of the serum desmosterol levels, but no change of the serum cholesterol level, was observed at 24 hr after a single dose of 100 mg/kg as compared to the control group (Table 5). In the repeated-dose groups also, a tendency towards decrease of the serum desmosterol levels was observed, with 52% and 48% decreases as compared to the control values in the dogs administered the drug at 30 and 50 mg/kg/day for 14 days, and a 34% decrease in the dogs administered 30/20 mg/kg/day for 13 weeks. Although a gradual recovery of the serum desmosterol levels was observed, a tendency towards a decrease was observed even at Week 18 of the recovery period in the dogs administered TP0446131 at 30/20 mg/kg/day for 13 weeks.

Table 4. Chemical constituents of the lens.
Table 5. Chemical constituents of the serum.

There were no obvious changes in the concentrations of sugars in the lenticular cortex (Table 4). However, as for glucose, the concentrations in the control dogs were lower than the detection limit at 24 hr after of dosing in the single-dose study and at Week 8 of dosing in the long-term repeated-dose study while glucose was detectable at the same time points in the lenticular cortex of the most dogs treated with TP0446131. In addition, there was a slight tendency towards increases in the sorbitol and fructose contents in the lenticular cortex in the dogs treated with TP0446131 for 14 days and 8 weeks, and 8 weeks, respectively.

A high glucose level was noted in both the serum (164 mg/dL in the treated dogs and 104 mg/dL in the control dogs) and aqueous humor (132 mg/dL in the treated dogs and 77 mg/dL in the control dogs) at 4 hr after administration of the drug at 100 mg/kg in the single-dose study (Table 5 and Table 6). On the one hand, the serum and aqueous humor glucose levels in the TP0446131 treated dogs examined at 24 hr after dosing were not significantly different from those in the control values in any of the single-dose, 14-day repeated-dose, and long-term repeated-dose studies. Therefore, there is a possibility that the daily blood glucose increased transiently after dosing throughout the dosing period in the repeated-dose studies. Thus, the daily blood glucose profile was examined in the plasma samples collected for the toxicokinetic analysis (Table 7). In the single-dose study, consistent with the high serum glucose level observed at 4 hr after dosing, the plasma glucose level increased and reached the Cmax value of 168.8 mg/dL at 2 hr after administration of the drug at 100 mg/kg. The transient increases in the plasma glucose levels were also observed in the 14-day and long-term repeated-dose studies. The Cmax value of the blood glucose increased up to 125.7 to 139.0 mg/dL in the dogs treated with the drug at 50 mg/kg/day. The increases in the blood glucose in the 30 and 20 mg/kg/day treatment groups were relatively slight; the Cmax values of the blood glucose levels ranged from 110.4 and 126.4 mg/dL between Day 1 to Week 12 of dosing. These values were slightly higher than those (104.7 to 111.0 mg/dL) observed in the control dogs.

Table 6. Chemical constituents of the aqueous humor.
Table 7. Blood glucose levels after repeated-dose administration in the dog plasma.

As for the levels of the oxidative stress markers and electrolytes, only slight changes were observed. In the lens cortex, a tendency towards a slight decrease in the SOD, GSH, and GSSG contents at 24 hr was noted in the dogs given the drug at 100 mg/kg in the single-dose study, and after 14 days in the dogs given repeated doses of 30 and 50 mg/kg/day (Table 4). In the aqueous humor, a tendency towards slight decrease in the ascorbic acid and chloride contents at 24 hr was noted in the animals dosed at 100 mg/kg and after 14 days in the animals administered repeated doses of 30 and 50 mg/kg/day (Table 6).

Changes in the unknown constituents in the lenticular cortex and serum

In the lenticular cortex and serum samples of the control and drug-treated groups, 3 unknown peaks (uk1, uk2, and uk3) were detected in the SRM chromatogram obtained for the detection of desmosterol (Fig. 4). Their peak responses exhibited dose- and time-dependent increases in the lenticular cortex (Fig. 5), not only in a relatively rapid manner, but also in a sustainable manner. Increase in the peak responses of uk1, uk2, and uk3 were already observed at 4 hr post-dose after single-dose administration of TP0446131 at 100 mg/kg, and the responses remained relatively high as compared to the control group even at Week 18 of recovery period in the animals dosed with TP0446131 at 30/20 mg/kg for 13 weeks. It is noteworthy that the same peaks were also found in the serum samples from the drug-treated dogs, and that the increases in the peak responses of uk1, uk2, and uk3 in both the lenticular cortex and serum were apparently observed prior to the onset of the cataract induced by TP0446131.

Fig. 4

SRM chromatogram of a sample of the lenticular cortex (m/z 367.3 → 147.3). Several peaks, including (a) desmosterol, unknown peaks (b) uk1, (c) uk2, and (d) uk3 were observed in SRM chromatogram for the detection of desmosterol (m/z 367.3 → 147.3).

Fig. 5

Peak areas of unknown peaks 1 to 3 after repeated dosing and recovery periods. (a) In the lenticular cortex, increases of the peak areas were observed after repeated-dose administration at 30 and 50 mg/kg/day. (b) In the serum, the increases in the peak areas observed after repeated-dose administration were similar to those observed in the lenticular cortex. However, the peak areas decreased transiently after the dose level was decreased transiently from 30 mg/kg/day to 20 mg/kg/day from Week 7 to Week 9. After the recovery period of 18 weeks, the unknown peak areas 1 to 3 in the serum decreased, becoming comparable to those in the control dogs.

Structural elucidation of the components in the unknown peaks found in the lenticular cortex and serum

The unknown peaks (uk1, uk2, and uk3) were observed in the same SRM chromatograms for desmosterol (m/z 367.3 → 147.3), indicating that their components had the same pair of precursor/product ions as desmosterol. In addition, these components were deemed to be highly lipophilic because of their strong retention on the lipophilic analytical column, similar to desmosterol, with a mobile phase which contained a high percentage of organic solvent (more than 70% methanol). These results suggested that the components in uk1, uk2, and uk3 were also sterol-related substances which have the same molecular weight as desmosterol. We, therefore, compared the chromatographic and mass spectral properties of uk1, uk2, and uk3 against those of 5 authentic standard desmosterol isomers: 7-dehydrocholesterol, 8-dehydrocholesterol, zymosterol, cholecalciferol, and 5-α-cholesta-7,24-diene-3β-ol. As a result, the retention time of cholecalciferol (68.3 min) was close to that of uk1 (68.5 min) and the times for 7-dehydrocholesterol (71.0 min) and 8-dehydrocholesterol (73.3 min) were close to that for uk2 (72.6 min), while the retention times of zymosterol (66.1 min) and 5-α-cholesta-7,24-diene-3β-ol (64.6 min) were not close to any of the unknown peaks. Subsequently, the product ion spectral patterns of cholecalciferol, 7-dehydrocholesterol, and 8-dehydrocholesterol were compared to those of uk1 and uk2, and the similarity among uk2, 7-dehydrocholesterol, and 8-dehydrocholesterol was confirmed (Fig. 6), suggesting that the component in uk2 was 7-dehydrocholesterol or 8-dehydrocholesterol. Then, we added 7-dehydrocholesterol or 8-dehydrocholesterol to the extracted lenticular cortex samples to precisely compare the retention times between the standards and uk2, and confirmed that the symmetry factor of uk2 did not differ before (1.15) and after (1.21) the addition of 7-dehydrocholesterol (Figs. 7a and 7b). On the other hand, peak broadening and splitting were observed in the samples in which 8-dehydrocholesterol had been added (Fig. 7c). Thus, there is the possibility that 7-dehydrocholesterol was a component of uk2.

Fig. 6

Product ion spectra of the protonated molecule (m/z 367.3) of (a) unknown peak (uk) 1, (b) cholecalciferol, (c) uk2, (d) 7-dehydrocholesterol, and (e) 8-dehydrocholesterol.

Fig. 7

SRM chromatograms of lenticular cortex samples to which 7- and 8-dehydrocholesterol (m/z 367.3 → 147.3) had been added. (a) Intact lenticular cortex sample; (b) lenticular cortex sample to which 7-dehydrocholesterol was added; (c) Lenticular cortex sample to which 8-dehydrocholesterol was added, were subjected to LC-MS/MS analysis.

Systemic exposure and concentrations of TP0446131 in the ocular tissues

The effects of systemic exposure to TP0446131 were examined by toxicokinetic analysis of the plasma, samples of which were collected periodically in the single-dose, 14-day repeated-dose, and long-term repeated-dose studies. In addition, the concentrations of TP0446131 in the ocular tissue were determined to investigate the relationship between the accumulations of the sterol-related substances and the onset of the cataract induced by TP0446131.

In the toxicokinetic analysis of the plasma obtained from the dogs after single-dose and repeated- dose administrations of TP0446131, dose-dependent increases in the Cmax and AUC0-24hr of TP0446131 were observed; however, no obvious accumulation of TP0446131 by repeated dosing was confirmed (Table 8). As for the tissue drug concentration, distribution of TP0446131 was observed in the ocular tissues collected at both 4 and 24 hours after the dosing, and the concentration of TP0446131 was higher in the lenticular cortex than in aqueous humor and vitreous body. There was no obvious accumulation of TP0446131 in the lenticular cortex by repeated dosing, as with the case of the plasma (Table 9).

Table 8. Systemic exposure of plasma to TP0446131.
Table 9. Drug concentrations in the ocular tissues.

DISCUSSION

TP0446131, a melanin-concentrating hormone receptor antagonist, induced the development of cataract in dogs, with the initial detection of a lenticular opacity by ophthalmologic examination at Week 8 to Week 9 of dosing in the long-term repeated-dose study, and the area of the opacity expanded with time during the dosing period, and did not reverse even after withdrawal of the drug in the early stage after detection of the ophthalmologic finding. Instead, the area of the opacity expanded even during the recovery period of 18 weeks.

Histopathologically, the lenticular opacity was characterized by degeneration of the lens fibers. The degenerated lens fibers caused irregularity in the ordered arrangement of the lens fibers, which is necessary to maintain the transparency of the lens, resulting in lenticular opacity. At Week 5 of dosing, degeneration of the lens fibers was observed in the subcapsular cortex around the equator of the lens. Then, histopathological examination revealed that the lesion area expanded from the surface to the deep layer of the lenticular cortex, as well as to the anterior and posterior subcapsular cortex by Week 9. It is known that new lens fibers differentiate from proliferating epithelial cells around the equator of the lens, then elongate toward both the anterior and posterior poles of the lens, and accumulate on the old lens fibers to sustain a clear lens (Song et al., 2009). The patterns of expression and progression of the pathological lesion suggested that the newly differentiated lens fibers were affected by the administration of TP0446131.

After the recovery period of 18 weeks, normal lens fibers were observed in the subcapsular region of the cortex, suggesting that the normal lens fibers had begun to regenerate. At the same time, a degenerated area was observed under the normal lens fibers in the cortex. Therefore, the degenerated lens fibers that had formed during the dosing period were not eliminated, but migrated into the deeper area of the lenticular cortex during the recovery period. This is considered to be the reason why the lenticular opacity seemed to have expanded to the whole area of the lens during the recovery period. In the dog in which the drug was discontinued immediately after detection of the first sign of lenticular opacity, the ocular fundus could be monitored throughout the recovery period of 18 weeks, even though the area of the opacity had expanded to the whole area of the lens. Therefore, it is considered that the visual acuity can be maintained at a minimum level even when the cortex is replaced by regenerated normal lens fibers, with the degenerated lens fibers becoming concentrated into the lenticular nucleus during the recovery period, if the drug is discontinued promptly upon detection of the abnormal ophthalmologic findings.

The chemical constituents known to be associated with the development of cataract were quantitated in the lenticular cortex, serum and aqueous humor of the dogs treated with TP0446131. It is noteworthy that the contents of cholesterol and its precursor, desmosterol, in the lens cortex of the dogs treated with TP0446131 did not differ from the control values. However, three unknown peaks (uk1, uk2, and uk3), which were assumed to correspond to sterol-related substances having the same molecular weight as desmosterol, based on the results of LC-MS/MS analysis, were found in the lenticular cortex. It should be noted that we did not examine whether TP0446131 altered the contents of sterol-related substances with other SRM transitions than desmosterol. It is well known that lens fibers need a large amount of cholesterol as a constituent of the cell membrane, because they have to elongate up to the thickness of the lens (Cenedella, 1996). However, the lens receives very little supply of cholesterol from the blood, and the lens fibers have to obtain all of their cholesterol by de novo synthesis (Pyrah et al., 2001). Thus, the lens is very susceptible to disturbances of the cholesterol biosynthetic pathway. Inhibitors of cholesterol biosynthesis, such as statins and triparanol, are well known to induce cataract in human and/or animals (Gerson et al., 1990; Kirby, 1967; Harris and Gruber, 1969; von Sallmann et al., 1963). As a general feature of cataract induced by these inhibitors, e.g., that seen in the drug-treated dogs, the opaque area of the lens expands from the equator to the central area of the cortex, since it is the newly differentiated lens fibers that are affected. In dogs treated with TP0446131, no decrease in the cholesterol content of the lens was noted as in the case of animals treated with (De Vries et al., 1993), although the possibility of decrease in the cholesterol levels occurring in local areas of the lens, such as the lenticular epithelial cells, cannot be excluded. In addition, of these sterol-related substances (uk1, uk2, and uk3), it appeared possible that uk2 contained 7-dehydrocholesterol; 7-dehydrocholesterol is well-known as a precursor of cholesterol in the cholesterol biosynthetic pathway. It is known that while substitution of cholesterol by 7-dehydrocholesterol may not change the membrane fluidity, such substitution could have profound effects on the activity of the membrane proteins (Xu et al., 2010). For example, substitutions of 7-dehydrocholesterol for cholesterol in the phospholipid vesicles has been reported to decrease the activity of the Na+ - Ca2+ exchanger by more than 70% (Vemuri and Philipson, 1989). It is well known that lack of cholesterol could induce cataract; however, in the present study, there is the possibility that abnormal accumulation of sterol-related substances affected the membrane function and was associated with the degeneration of the lens fibers in the dogs treated with TP0446131. Although further study is needed to clarify these points, the results suggested that TP0446131 possibly exerts some effects on the cholesterol biosynthesis pathway at the dose levels at which lenticular opacities developed in the dogs.

Interestingly, the increases of the sterol-related substances were also seen in the serum, even after a single-dose of TP0446131. In the 14-day and long-term repeated-dose studies, the peak areas of the sterol-related substances in the serum at 30 mg/kg/day were comparable between Day 14 of dosing and Week 13 of dosing. At 30 mg/kg/day, the sterol-related substances increased until 5 weeks of dosing, however the peak areas decreased transiently at Week 8 of dosing, consistent with the transient reduction of the dose levels (from 30 mg/kg/day to 20 mg/kg/day). After the recovery period of 18 weeks, the peak areas in the serum decreased to become comparable to those in the control dogs. These results were in contrast to those observed in the lenticular cortex, in which accumulation of the sterol-related substances was observed following repeated dosing. Since the lens fibers lost their nuclei during the differentiation process, it is likely that they also lost their metabolizing pathways for these sterol-related substances once accumulated. The increases in these sterol-related substances were detected in the serum before the onset of the cataract, suggesting the possibility of these sterol-related substances in the serum potentially serving as safety biomarkers for predicting the onset of cataract induced by TP0446131.

In regard to other chemical constituents than sterols, transient increase of the blood glucose was observed following administration of TP0446131. Increase of the glucose concentration in the aqueous humor, and a tendency towards increase in the glucose, sorbitol and fructose contents in the lens were also observed; these changes were considered to be secondary to the increase in the blood glucose levels. Except for the increase in the blood glucose level observed after single-dose administration, all changes were slight, and the Cmax values of blood glucose after repeated-dose (110.4 to 125.7 mg/dL) administration were within or only fell slightly outside the range of the historical data of the test facility. Cataract attributable to drug-induced hyperglycemia has been reported in diazoxide-treated dogs (Schiavo et al., 1975). Of the 6 dogs that developed cataract after oral dosing of diazoxide (50 to 200 mg/kg/day), 5 exhibited high blood glucose levels ranging from 124 to 425 mg/dL. Essentially, these blood glucose levels were higher than those observed in the drug-treated dogs in this study. In general, cataract associated with the accumulation of sugars shows swelling of the lens fibers due to the changes in the osmotic pressure, and the lenticular opacity diffuses throughout the whole area of the lenticular cortex (Kuwabara et al., 1969; Sato et al., 1991). In the case in which the drug was discontinued promptly upon detection of the lenticular opacity, this type of cataract is known to be reversible when the blood glucose level returns to the normal range (Kuwabara et al., 1969). However, no reversibility of the lenticular opacity was observed after withdrawal of TP0446131 in the long-term repeated-dose study by 18 weeks after the end of treatment. These results suggest that increased blood glucose may not have had much impact on the development of cataract in the drug-treated dogs.

Only slight changes were observed in the oxidative stress markers and electrolytes, including a tendency towards decrease of the SOD, GSH, and GSSG contents in the crystalline lens cortex, and a tendency towards decrease in the contents of ascorbic acid and chloride in the aqueous humor. These changes were not consistently observed throughout the dosing period. Therefore, the changes were not considered to be directly involved in the cataractogenesis, but to be secondary changes to the damage observed in the lens.

Of note, a relatively high concentration of TP0446131 was observed in the lenticular cortex even at 24 hr after a single dose. This manner of drug distribution might accelerate the development and progression of the cataract. However, repeated-dose administration had no obvious effect on the lens drug concentration, it was considered the drug did not accumulate permanently in the lenticular cortex. Consistently, formation of normal lens fibers was confirmed in the subcapsular region of the cortex after withdrawal of TP0446131 administration. Based on the above, the degenerations of the lens fibers were correlated with the transient increase of the TP0446131 concentrations in the lens.

In summary, administration of TP0446131 at 30/20 mg/kg/day induced the development of cataract in dogs. Ophthalmologically, the first sign of lenticular opacity was found at Week 8 or Week 9 of dosing. The lenticular opacity was characterized histopathologically by degeneration of the lens fibers. In the lens, although there was no change in the content of cholesterol, increases in the amounts of sterol-related substances were noted. One of these substances was considered to be 7-dehydrocholesterol, which is a precursor of cholesterol. These findings suggest that TP0446131 exerts certain effects on the cholesterol biosynthesis pathway, which might be involved in the development of the drug-induced cataracts. Increases in the sterol-related substances were also observed in the serum. Importantly, the increases in the serum were observed even before the onset of the cataract. Therefore, the serum levels of these substances may potentially serve as safety biomarkers to predict the onset of cataract induced by TP0446131.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
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