Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
JOURNAL OF OCULAR PHARMACOLOGY Volume 8, Number 1, 1992 Mary Ann Liebert, Inc., Publishers
The Efficacy of Aldose Reductase Inhibitors on Polyol Accumulation in Human Lens and Retinal Pigment Epithelium in Tissue Culture VENKAT N.
REDDY,'
LI-REN LIN,1 FRANK J. GIBLIN,1 MARJORIE PETER F. KADOR,3 and JIN H. KINOSHITA3
LOU,2
'Eye Research Institute of Oakland University, Rochester, Michigan; 2Alcon Research Laboratories, Fort
Worth, Texas; ^National Eye Institute, National Institutes of Health, Bethesda, Maryland
ABSTRACT The formation of excess sugar alcohol mediated by aldose reductase (AR) and its intracellular accumulation in lens with resultant hydration is thought to be the initiating mechanism in the pathogenesis of diabetic and galactosemic cataracts. AR is also involved in other diabetic complications including retinopathy and Therefore, there is heightened interest in developing effective AR neuropathy. inhibitors (ARIs) for possible clinical use in human diabetes. However, the evaluation of these drugs for potential clinical use requires that the compounds be evaluated in appropriate target tissues since AR from different tissues is known to exhibit differential susceptibility to ARIs. The relative efficacy of ARIs in human lens epithelium (HLE) and human retinal pigment epithelium (HRPE) was studied by measuring the degree of inhibition of galactitol formation at various concentrations of ARI following incubation of cells in high galactose media for 72 hrs. Regardless of the structural characteristics of the ARIs investigated, higher doses were required to inhibit polyol synthesis in HRPE as compared to HLE cells. Based on ED AL-3152 > AL-1576 > tolrcstat > statil > sorbinil. Since some ARIs are known to be bound to plasma proteins, it is conceivable that the observed differences in ED50 values could be due to differential binding to serum proteins in the culture medium. This possibility was examined by employing cultures of dog lens epithelium (DLE). These cells, which synthesize much higher levels of galactitol than HLE and HRPE, could be maintained in serum-free media for short periods (4 hrs) of time. The results, which demonstrate that the extent of polyol inhibition was the same in the presence or absence of serum, suggest that the differences in the potency of the inhibitors may reflect their inherent activity against AR in HLE and HRPE cells.
INTRODUCTION The polyol pathway of glucose metabolism contains two enzymes, aldose reductase (AR) and sorbitol dehydrogenasc. AR, in the presence of the coenzyme NADPH, catalyzes the reduction of aldose to its corresponding alcohol, sorbitol, which is further metabolized to fructose by sorbitol dehydrogenasc requiring the coenzyme NAD (Fig. 1). AR has a broad specificity so that many aldoses serve as substrates (1). Hexoses generally are poor substrates with high Kms for AR (2). However, when
43
Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
glucose
fructose
sorbitol
aldose reductase
galactose
sorbitol
galactitol
dehydrogenase
N.R.
FIGURE 1. Polyol Pathway are elevated as in diabetes or in galactosemia, significant levels of accumulate in tissues containing AR (3). The formation of excess sugar alcohol and its intracellular accumulation in lens epithelia or fiber cells, with the resultant hydration, is thought to initiate the pathogenesis of diabetic and galactosemic cataracts (4). The higher level of polyol accumulation in the lens of galactosemic than that of diabetic animals accounts for its more rapid development of cataract. The difference in the polyol levels is explained by the fact that galactose is a better substrate than glucose for AR and galactitol is not further metabolized by sorbitol dehydrogenase (3,4). The presence of AR can be demonstrated in a variety of cells (5-8) by the synthesis of sorbitol or galactitol upon exposure to glucose or galactose and by the abolishing of polyol synthesis by AR inhibitors (ARIs). We have recently shown that vacuole formation in human lens epithelial (HLE) and human retinal pigment epithelial (HRPE) cells, which are exposed to high sugar media, is the result of an osmotic effect of polyol formation (9,10) and that morphological and cell permeability changes are prevented by ARI in both cell types. Evidence is mounting that AR is involved in other diabetic complications including retinopathy, neuropathy and nephropathy (11). Therefore, there is heightened interest in developing effective inhibitors of AR for possible clinical use in human diabetes (12). However, the evaluation of these drugs for potential clinical use requires that the compounds be evaluated in appropriate target tissues since AR from tissues of different species as well as in the same animal is known exhibit differential susceptibility to ARIs (13,14). The cell culture system to which we have developed has proven useful in evaluating the effectiveness of ARIs. This study reports the evaluation of the potency of six ARIs of diverse in their ability to inhibit galactitol formation in cultured cells of structures HLE and HRPE exposed to 30 mM D-galactose. The possible effect of serum concentration in the culture medium on the inhibitory activity of ARIs on polyol accumulation was also investigated in both types of cells because differential The binding of the inhibitors to protein might influence their relative efficacy. results, which demonstrate that the extent of inhibition of galactitol formation was the same either in the presence or absence of serum, strongly suggest that the differences observed in the potency of the inhibitors may reflect their inherent activity against AR in the two tissues.
sugar
levels
polyol
can
MATERIALS AND METHODS
Cell Culture
a) Human
Lens
Epithelium (HLE)
Primary cultures of HLE were established from anterior capsule specimens obtained from 5-12 months old patients with retinopathy of prematurity as described previously (15). The cells were subcultured in Dulbecco's Modified Eagle's medium (DME) supplemented with 20% fetal bovine serum (FBS). Third passage
44
Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
FIGURE 2. Structural formulae of aldose reductase inhibitors
cells were then transferred to a 12 well plate (Falcon, Lincoln Park, N.J.) with a cell density of 200 K/well. After 24 hrs, when the cells attached to the plate became confluent, the medium was replaced with one containing 30 mM D-galactose in the presence or absence of ARIs and cultured for additional 72 hours.
b) Dog Lens Epithelium (DLE) Samples of
DLE were obtained from 6 month old golden retrievers which were after than Other experiments in an electrophysiology laboratory. anesthesia, the animals had received no medication and were considered normal. Primary cultures were obtained from expiants of anterior capsule using the same culture medium and conditions employed for HLE. The cells, which became confluent within one week, were serially passaged every three days. Fifth passage cells (400 K cell/well) were transferred to 6 well plates and after attachment to the culture dish, the initial medium was replaced with either serum-free or serum-containing 320 mOsm. media. Both media contained 30m D-galactose with an osmolarity of 310 ARI was added to the media when desired. The cells were cultured in the experimental media for 4 hours, and analyzed for polyols.
sacrified
-
c) Human Retinal Pigment Epithelium (HRPE) Donor eyes ranging in age from 30 50 years were obtained from the National Disease Research Interchange. The anterior 4 mm calotte was removed by a circumferential incision, and then vitreous body and retina were gently separated from the retinal pigment epithelial (RPE) cell layer. The eye cup was filled with 3 ml of saline containing 0.25% pronase (Sigma Chemical Co., St. Louis, MO) and incubated at 37°C for 5 mins. The enzyme solution was then replaced with Ca-Mg RPE cells were loosened with gentle trituration, free saline (CMFS) solution. collected and pelleted by centrifugation. They were resuspended in DME, supplemented with 15% FBS and plated on 60 mm Falcon dishes and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The confluent cells, after 2 3 weeks, were trypsinized and serially passaged every 3-5 days by splitting the cultures in a 1:3 ratio. Cells in the 5th passage, which showed no fibroblastic changes, were used in all of the experiments in this study. 400 K cells/well were transferred to 6 well -
-
45
plates and 24 hrs later, when the cells normal medium was substituted with high incubation was carried out for 72 hrs without
were
attached
to
galactose media with changing the media.
the or
culture dish, the without ARI. The
Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
Harvesting of Cells and Polyol Assay At the end of the incubation period the cells were rinsed quickly with CMFS solution warmed to 37°C. The procedure was repeated twice more. The cells were scraped and collected in 0.6 ml of 2% ZnSO^ and homogenized by sonication for 30 sees using a cell disrupter 200 (Branson Sonic Power Co. Danbury, CT; power level The homogenates were neutralized with an equivalent amount of 3, 70% pulse). at 3000 RPM for 10 mins. and centrifuged Aliquots of clear supernatant Ba(OH)2 were lyophilized and silylated with Tri-Sil Z (Pierce Chemical Co., Rockford, IL). Sample volumes ranging from 0.5 to 3.0 ul were analyzed using a Shimadzu GC-Mini 3, Gas Chromatograph (9). Freshly silylated mixtures of fructose, galactose, glucose, sorbitol and myoinositol were used as standards.
Media and Aldose Reductase Inhibitors 30 mM D-galactose was added to the normal medium containing FBS, the was adjusted to 310 320 mOsm by adding water and the Dulbecco's amino acid and Vitamin mixture (Gibco). ARIs were first dissolved in 0.25 N NaOH to make up a stock solution of 1 mM concentration and serially diluted with D-galactose containing medium to obtain the desired concentration. At the concentration of ARI used the pH of the medium was the same as control (pH. 7.2). ARI solutions were prepared fresh each time. The structures of the 6 compounds studied are shown in Fig. 2. AL-1576, AL-3152 and AL-4114 were provided by Alcon Laboratories, Ft. Worth, Texas and tolrestat, statil and sorbinil were gifts from Wyeth Ayerst, Princeton, N.J.; ICI, Americas, Wilmington, DE; and Pfizer Central Research When
osmolarity
-
Laboratory, Groton, CT, respectively.
RESULTS The presence of AR in a cell can be demonstrated by measuring the formation of polyol following incubation of the cells in media containing elevated levels of The extent of polyol formed is directly related to the glucose or galactose. activity of the enzyme in the tissue. Additional evidence that polyol formation is caused by AR is to abolish the synthesis with appropriate ARIs (9,10). Fig. 3 shows the inhibition of galactitol formation in HLE and HRPE cells cultured in a medium containing 30 mM D-galactose as a function of ARI In the absence of the inhibitor, HLE and HRPE cells concentration (AL 4114). accumulated approximately 320 and 370 nmoles galactitol/10" cells respectively. With increasing concentration of the ARI there was a progressive decrease in the amount of galactitol formed in both cell types. However, galactitol formation in HLE was inhibited to a greater extent than in HRPE at equivalent media concentration of the inhibitor. Thus, polyol formation in HLE was inhibited by uM whereas much nearly 70% when the ARI concentration in the medium was 10 higher concentration of the inhibitor was required to achieve the same degree of inhibition of galactitol formation in HRPE cells. The relative efficacy of ARI in HLE and HRPE was demonstrated by measuring the degree of inhibition of galactitol formation at various concentrations of ARI following incubation of the cells for 72 hrs. The dose required to inhibit polyol formation by 50% (ED™) was estimated from the regression lines to the data as shown in Fig. 4. ED5Q for AL-4114 in HRPE cells was found to be nearly 10 times higher than in HLE cells suggesting that the lens enzyme is more sensitive to inhibition than the retinal enzyme or a lower penetrability of ARIs into the RPE cells. Using similar procedures, the potency of six different ARIs (Fig. 2) in
inhibiting galactitol formation
in HLE and HRPE was determined. As summarized in characteristics of structural these compounds, were required to inhibit polyol formation in HRPE
regardless of the consistently higher doses of ARI Table
1,
46
450 «
Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
o
400
I
I 300 í 250 cd «
200
>
150
i
HRPE
f
-
•n
"o
100
i
50 0
10"
10_z
10"°
concentration(uM)
Al-4114
FIGURE 3. Dose response curves for the inhibition of galactitol formation in HLE and HRPE cells cultured for 72 hrs. Each data point represents three experiments, and error bars indicate two standard deviations.
3
HRPE
HLE
80
0 I...
10"
concentration(^iM)
AL-4114
Dncentration(/iM)
AL-4114
inhibition of FIGURE 4. Percent galactitol formation versus concentration of AL-4114 in HLE and HRPE cells cultured for 72 hrs. Triangles show the actual data points through which regression lines are drawn. Dotted lines intercepting X-axis give the ED™ values.
TABLE 1.
(ED50) of
Relative inhibitory activity cells. ARI concentrations (M).
ceir
AL4114
AL3152
HLE
3.6X10
7.0X10
3.6X10
HRPE
3.4X10
4.3X10
2.6X10
AL1576
-7
47
6 ARIs in HLE and HRPE
T0LRESTAT
STATIL
S0RBINIL
5.6X10
4.9X10
7.2X10
3 X10
3.2X10
4.8X10
350
Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
20%
serum
50
0.005
0.01
AI—3152
0.02
(uM)
FIGURE 5. Inhibition of aldose reductase by AL-3152 hrs in 2 different concentrations of serum.
HLE
cells
cultured
for 72
HLE cells. Under these experimental conditions, AL-4114 was the inhibitor while sorbinil showed the least activity. The order of potencies against both HLE and HRPE enzymes was AL-4114 > AL-3152 > AL-1576 > tolrestat > statil > sorbinil. These differences in ED™ values could be due to the inherent susceptibility of the enzyme from the two tissues or due to differences in the penetrability of the inhibitors into the cells. Another possibility is the differential behavior of the inhibitors bound to the serum proteins present in the culture medium. To test the influence of serum concentration on the ARI activity, galactitol levels were measured in HLE cells cultured in media containing either 5% or 20% serum with varying levels of AL-3152, but the extent of inhibition of polyol formation was unaffected by serum concentration in the media (Fig. 5). Attempts were also made to compare the effect of ARI in serum-free and serum-containing media. However, HLE and HRPE cells did not survive in serum-free media for 72 hrs. Shorter culture periods also proved unsatisfactory since the amount of galactitol formed could not be measured (Data not shown). To further examine the effects of ARI binding to serum proteins, DLE cells were Earlier reports (9,16) have shown that polyol levels in DLE could be employed. measured after a few hours of incubation. In contrast to the human cells, DLE cells synthesized much higher levels of galactitol when cultured for only 4 hours, probably due to a higher AR activity in this tissue. Furthermore, DLE cells when cultured in serum-free media maintained their normal morphology (as examined by phase contrast microscopy) over the 4 hour culture period. Therefore, the effect of ARIs on galactitol formation was examined in DLE cultured in the presence and absence of serum. A typical GLC profile of such an experiment with AL-3152 is shown in Fig. 6. It may be seen that the inhibition of polyol formation is essentially the same in both serum-free and serum-containing culture media suggesting that the ARI may not be bound to the serum protein/s. There are previous reports which indicate that some ARI are bound to plasma proteins. Over 85% of AL-1576 has been shown to be bound to serum albumin but the binding was independent of protein concentration (17). Tolrestat, which contains a carboxylic group, has been reported to be highly bound to plasma proteins. Following systemic administration of this compound, the percentage of free drug in the plasma varied from 0.7% in man to 4% in mouse (18). It was, therefore, of as
compared
most
to
potent
48
Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
FIGURE 6. The
effect of ARI (AL-3152) on galactitol formation in DLE cells cultured for 4 hrs in the presence and absence of serum. A) control media 30 mM galactose + 20% serum without ARI, B) 30 mM galactose + ARI without serum, C) 30 mM galactose + ARI + 20% serum. Peaks: a) alpha galactose; b) beta galactose; c) galactitol; d) myoinositol. -
to compare the inhibitory activity of tolrestat with other ARIs in inhibiting galactitol formation in DLE cells in the presence and absence of serum. Fig. 7 summarizes the data on the inhibition of galactitol formation in DLE cells at three different concentrations of each ARI in serum-free and serum-containing media. The results clearly demonstrate that the presence of 20% serum in the culture medium has no effect on the ability of any of the four ARIs studied to inhibit polyol formation. The fact that the inhibitory effect of tolrestat, which is known to bind plasma protein with greater affinity, was unaffected by the It is possible that there may presence at 20% serum in the medium was surprising.
interest
.25
.5
concentration Ui*/I)
concentration
JOURNAL OF OCULAR PHARMACOLOGY Volume 8, Number 1, 1992 Mary Ann Liebert, Inc., Publishers
The Efficacy of Aldose Reductase Inhibitors on Polyol Accumulation in Human Lens and Retinal Pigment Epithelium in Tissue Culture VENKAT N.
REDDY,'
LI-REN LIN,1 FRANK J. GIBLIN,1 MARJORIE PETER F. KADOR,3 and JIN H. KINOSHITA3
LOU,2
'Eye Research Institute of Oakland University, Rochester, Michigan; 2Alcon Research Laboratories, Fort
Worth, Texas; ^National Eye Institute, National Institutes of Health, Bethesda, Maryland
ABSTRACT The formation of excess sugar alcohol mediated by aldose reductase (AR) and its intracellular accumulation in lens with resultant hydration is thought to be the initiating mechanism in the pathogenesis of diabetic and galactosemic cataracts. AR is also involved in other diabetic complications including retinopathy and Therefore, there is heightened interest in developing effective AR neuropathy. inhibitors (ARIs) for possible clinical use in human diabetes. However, the evaluation of these drugs for potential clinical use requires that the compounds be evaluated in appropriate target tissues since AR from different tissues is known to exhibit differential susceptibility to ARIs. The relative efficacy of ARIs in human lens epithelium (HLE) and human retinal pigment epithelium (HRPE) was studied by measuring the degree of inhibition of galactitol formation at various concentrations of ARI following incubation of cells in high galactose media for 72 hrs. Regardless of the structural characteristics of the ARIs investigated, higher doses were required to inhibit polyol synthesis in HRPE as compared to HLE cells. Based on ED AL-3152 > AL-1576 > tolrcstat > statil > sorbinil. Since some ARIs are known to be bound to plasma proteins, it is conceivable that the observed differences in ED50 values could be due to differential binding to serum proteins in the culture medium. This possibility was examined by employing cultures of dog lens epithelium (DLE). These cells, which synthesize much higher levels of galactitol than HLE and HRPE, could be maintained in serum-free media for short periods (4 hrs) of time. The results, which demonstrate that the extent of polyol inhibition was the same in the presence or absence of serum, suggest that the differences in the potency of the inhibitors may reflect their inherent activity against AR in HLE and HRPE cells.
INTRODUCTION The polyol pathway of glucose metabolism contains two enzymes, aldose reductase (AR) and sorbitol dehydrogenasc. AR, in the presence of the coenzyme NADPH, catalyzes the reduction of aldose to its corresponding alcohol, sorbitol, which is further metabolized to fructose by sorbitol dehydrogenasc requiring the coenzyme NAD (Fig. 1). AR has a broad specificity so that many aldoses serve as substrates (1). Hexoses generally are poor substrates with high Kms for AR (2). However, when
43
Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
glucose
fructose
sorbitol
aldose reductase
galactose
sorbitol
galactitol
dehydrogenase
N.R.
FIGURE 1. Polyol Pathway are elevated as in diabetes or in galactosemia, significant levels of accumulate in tissues containing AR (3). The formation of excess sugar alcohol and its intracellular accumulation in lens epithelia or fiber cells, with the resultant hydration, is thought to initiate the pathogenesis of diabetic and galactosemic cataracts (4). The higher level of polyol accumulation in the lens of galactosemic than that of diabetic animals accounts for its more rapid development of cataract. The difference in the polyol levels is explained by the fact that galactose is a better substrate than glucose for AR and galactitol is not further metabolized by sorbitol dehydrogenase (3,4). The presence of AR can be demonstrated in a variety of cells (5-8) by the synthesis of sorbitol or galactitol upon exposure to glucose or galactose and by the abolishing of polyol synthesis by AR inhibitors (ARIs). We have recently shown that vacuole formation in human lens epithelial (HLE) and human retinal pigment epithelial (HRPE) cells, which are exposed to high sugar media, is the result of an osmotic effect of polyol formation (9,10) and that morphological and cell permeability changes are prevented by ARI in both cell types. Evidence is mounting that AR is involved in other diabetic complications including retinopathy, neuropathy and nephropathy (11). Therefore, there is heightened interest in developing effective inhibitors of AR for possible clinical use in human diabetes (12). However, the evaluation of these drugs for potential clinical use requires that the compounds be evaluated in appropriate target tissues since AR from tissues of different species as well as in the same animal is known exhibit differential susceptibility to ARIs (13,14). The cell culture system to which we have developed has proven useful in evaluating the effectiveness of ARIs. This study reports the evaluation of the potency of six ARIs of diverse in their ability to inhibit galactitol formation in cultured cells of structures HLE and HRPE exposed to 30 mM D-galactose. The possible effect of serum concentration in the culture medium on the inhibitory activity of ARIs on polyol accumulation was also investigated in both types of cells because differential The binding of the inhibitors to protein might influence their relative efficacy. results, which demonstrate that the extent of inhibition of galactitol formation was the same either in the presence or absence of serum, strongly suggest that the differences observed in the potency of the inhibitors may reflect their inherent activity against AR in the two tissues.
sugar
levels
polyol
can
MATERIALS AND METHODS
Cell Culture
a) Human
Lens
Epithelium (HLE)
Primary cultures of HLE were established from anterior capsule specimens obtained from 5-12 months old patients with retinopathy of prematurity as described previously (15). The cells were subcultured in Dulbecco's Modified Eagle's medium (DME) supplemented with 20% fetal bovine serum (FBS). Third passage
44
Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
FIGURE 2. Structural formulae of aldose reductase inhibitors
cells were then transferred to a 12 well plate (Falcon, Lincoln Park, N.J.) with a cell density of 200 K/well. After 24 hrs, when the cells attached to the plate became confluent, the medium was replaced with one containing 30 mM D-galactose in the presence or absence of ARIs and cultured for additional 72 hours.
b) Dog Lens Epithelium (DLE) Samples of
DLE were obtained from 6 month old golden retrievers which were after than Other experiments in an electrophysiology laboratory. anesthesia, the animals had received no medication and were considered normal. Primary cultures were obtained from expiants of anterior capsule using the same culture medium and conditions employed for HLE. The cells, which became confluent within one week, were serially passaged every three days. Fifth passage cells (400 K cell/well) were transferred to 6 well plates and after attachment to the culture dish, the initial medium was replaced with either serum-free or serum-containing 320 mOsm. media. Both media contained 30m D-galactose with an osmolarity of 310 ARI was added to the media when desired. The cells were cultured in the experimental media for 4 hours, and analyzed for polyols.
sacrified
-
c) Human Retinal Pigment Epithelium (HRPE) Donor eyes ranging in age from 30 50 years were obtained from the National Disease Research Interchange. The anterior 4 mm calotte was removed by a circumferential incision, and then vitreous body and retina were gently separated from the retinal pigment epithelial (RPE) cell layer. The eye cup was filled with 3 ml of saline containing 0.25% pronase (Sigma Chemical Co., St. Louis, MO) and incubated at 37°C for 5 mins. The enzyme solution was then replaced with Ca-Mg RPE cells were loosened with gentle trituration, free saline (CMFS) solution. collected and pelleted by centrifugation. They were resuspended in DME, supplemented with 15% FBS and plated on 60 mm Falcon dishes and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The confluent cells, after 2 3 weeks, were trypsinized and serially passaged every 3-5 days by splitting the cultures in a 1:3 ratio. Cells in the 5th passage, which showed no fibroblastic changes, were used in all of the experiments in this study. 400 K cells/well were transferred to 6 well -
-
45
plates and 24 hrs later, when the cells normal medium was substituted with high incubation was carried out for 72 hrs without
were
attached
to
galactose media with changing the media.
the or
culture dish, the without ARI. The
Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
Harvesting of Cells and Polyol Assay At the end of the incubation period the cells were rinsed quickly with CMFS solution warmed to 37°C. The procedure was repeated twice more. The cells were scraped and collected in 0.6 ml of 2% ZnSO^ and homogenized by sonication for 30 sees using a cell disrupter 200 (Branson Sonic Power Co. Danbury, CT; power level The homogenates were neutralized with an equivalent amount of 3, 70% pulse). at 3000 RPM for 10 mins. and centrifuged Aliquots of clear supernatant Ba(OH)2 were lyophilized and silylated with Tri-Sil Z (Pierce Chemical Co., Rockford, IL). Sample volumes ranging from 0.5 to 3.0 ul were analyzed using a Shimadzu GC-Mini 3, Gas Chromatograph (9). Freshly silylated mixtures of fructose, galactose, glucose, sorbitol and myoinositol were used as standards.
Media and Aldose Reductase Inhibitors 30 mM D-galactose was added to the normal medium containing FBS, the was adjusted to 310 320 mOsm by adding water and the Dulbecco's amino acid and Vitamin mixture (Gibco). ARIs were first dissolved in 0.25 N NaOH to make up a stock solution of 1 mM concentration and serially diluted with D-galactose containing medium to obtain the desired concentration. At the concentration of ARI used the pH of the medium was the same as control (pH. 7.2). ARI solutions were prepared fresh each time. The structures of the 6 compounds studied are shown in Fig. 2. AL-1576, AL-3152 and AL-4114 were provided by Alcon Laboratories, Ft. Worth, Texas and tolrestat, statil and sorbinil were gifts from Wyeth Ayerst, Princeton, N.J.; ICI, Americas, Wilmington, DE; and Pfizer Central Research When
osmolarity
-
Laboratory, Groton, CT, respectively.
RESULTS The presence of AR in a cell can be demonstrated by measuring the formation of polyol following incubation of the cells in media containing elevated levels of The extent of polyol formed is directly related to the glucose or galactose. activity of the enzyme in the tissue. Additional evidence that polyol formation is caused by AR is to abolish the synthesis with appropriate ARIs (9,10). Fig. 3 shows the inhibition of galactitol formation in HLE and HRPE cells cultured in a medium containing 30 mM D-galactose as a function of ARI In the absence of the inhibitor, HLE and HRPE cells concentration (AL 4114). accumulated approximately 320 and 370 nmoles galactitol/10" cells respectively. With increasing concentration of the ARI there was a progressive decrease in the amount of galactitol formed in both cell types. However, galactitol formation in HLE was inhibited to a greater extent than in HRPE at equivalent media concentration of the inhibitor. Thus, polyol formation in HLE was inhibited by uM whereas much nearly 70% when the ARI concentration in the medium was 10 higher concentration of the inhibitor was required to achieve the same degree of inhibition of galactitol formation in HRPE cells. The relative efficacy of ARI in HLE and HRPE was demonstrated by measuring the degree of inhibition of galactitol formation at various concentrations of ARI following incubation of the cells for 72 hrs. The dose required to inhibit polyol formation by 50% (ED™) was estimated from the regression lines to the data as shown in Fig. 4. ED5Q for AL-4114 in HRPE cells was found to be nearly 10 times higher than in HLE cells suggesting that the lens enzyme is more sensitive to inhibition than the retinal enzyme or a lower penetrability of ARIs into the RPE cells. Using similar procedures, the potency of six different ARIs (Fig. 2) in
inhibiting galactitol formation
in HLE and HRPE was determined. As summarized in characteristics of structural these compounds, were required to inhibit polyol formation in HRPE
regardless of the consistently higher doses of ARI Table
1,
46
450 «
Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
o
400
I
I 300 í 250 cd «
200
>
150
i
HRPE
f
-
•n
"o
100
i
50 0
10"
10_z
10"°
concentration(uM)
Al-4114
FIGURE 3. Dose response curves for the inhibition of galactitol formation in HLE and HRPE cells cultured for 72 hrs. Each data point represents three experiments, and error bars indicate two standard deviations.
3
HRPE
HLE
80
0 I...
10"
concentration(^iM)
AL-4114
Dncentration(/iM)
AL-4114
inhibition of FIGURE 4. Percent galactitol formation versus concentration of AL-4114 in HLE and HRPE cells cultured for 72 hrs. Triangles show the actual data points through which regression lines are drawn. Dotted lines intercepting X-axis give the ED™ values.
TABLE 1.
(ED50) of
Relative inhibitory activity cells. ARI concentrations (M).
ceir
AL4114
AL3152
HLE
3.6X10
7.0X10
3.6X10
HRPE
3.4X10
4.3X10
2.6X10
AL1576
-7
47
6 ARIs in HLE and HRPE
T0LRESTAT
STATIL
S0RBINIL
5.6X10
4.9X10
7.2X10
3 X10
3.2X10
4.8X10
350
Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
20%
serum
50
0.005
0.01
AI—3152
0.02
(uM)
FIGURE 5. Inhibition of aldose reductase by AL-3152 hrs in 2 different concentrations of serum.
HLE
cells
cultured
for 72
HLE cells. Under these experimental conditions, AL-4114 was the inhibitor while sorbinil showed the least activity. The order of potencies against both HLE and HRPE enzymes was AL-4114 > AL-3152 > AL-1576 > tolrestat > statil > sorbinil. These differences in ED™ values could be due to the inherent susceptibility of the enzyme from the two tissues or due to differences in the penetrability of the inhibitors into the cells. Another possibility is the differential behavior of the inhibitors bound to the serum proteins present in the culture medium. To test the influence of serum concentration on the ARI activity, galactitol levels were measured in HLE cells cultured in media containing either 5% or 20% serum with varying levels of AL-3152, but the extent of inhibition of polyol formation was unaffected by serum concentration in the media (Fig. 5). Attempts were also made to compare the effect of ARI in serum-free and serum-containing media. However, HLE and HRPE cells did not survive in serum-free media for 72 hrs. Shorter culture periods also proved unsatisfactory since the amount of galactitol formed could not be measured (Data not shown). To further examine the effects of ARI binding to serum proteins, DLE cells were Earlier reports (9,16) have shown that polyol levels in DLE could be employed. measured after a few hours of incubation. In contrast to the human cells, DLE cells synthesized much higher levels of galactitol when cultured for only 4 hours, probably due to a higher AR activity in this tissue. Furthermore, DLE cells when cultured in serum-free media maintained their normal morphology (as examined by phase contrast microscopy) over the 4 hour culture period. Therefore, the effect of ARIs on galactitol formation was examined in DLE cultured in the presence and absence of serum. A typical GLC profile of such an experiment with AL-3152 is shown in Fig. 6. It may be seen that the inhibition of polyol formation is essentially the same in both serum-free and serum-containing culture media suggesting that the ARI may not be bound to the serum protein/s. There are previous reports which indicate that some ARI are bound to plasma proteins. Over 85% of AL-1576 has been shown to be bound to serum albumin but the binding was independent of protein concentration (17). Tolrestat, which contains a carboxylic group, has been reported to be highly bound to plasma proteins. Following systemic administration of this compound, the percentage of free drug in the plasma varied from 0.7% in man to 4% in mouse (18). It was, therefore, of as
compared
most
to
potent
48
Journal of Ocular Pharmacology and Therapeutics 1992.8:43-52. Downloaded from online.liebertpub.com by Uc Davis Libraries University of California Davis on 01/02/15. For personal use only.
FIGURE 6. The
effect of ARI (AL-3152) on galactitol formation in DLE cells cultured for 4 hrs in the presence and absence of serum. A) control media 30 mM galactose + 20% serum without ARI, B) 30 mM galactose + ARI without serum, C) 30 mM galactose + ARI + 20% serum. Peaks: a) alpha galactose; b) beta galactose; c) galactitol; d) myoinositol. -
to compare the inhibitory activity of tolrestat with other ARIs in inhibiting galactitol formation in DLE cells in the presence and absence of serum. Fig. 7 summarizes the data on the inhibition of galactitol formation in DLE cells at three different concentrations of each ARI in serum-free and serum-containing media. The results clearly demonstrate that the presence of 20% serum in the culture medium has no effect on the ability of any of the four ARIs studied to inhibit polyol formation. The fact that the inhibitory effect of tolrestat, which is known to bind plasma protein with greater affinity, was unaffected by the It is possible that there may presence at 20% serum in the medium was surprising.
interest
.25
.5
concentration Ui*/I)
concentration
