aldose reductase inhibitors and diabetic complications

Aldose Reductase Inhibitors and Diabetic Complications

PHILIP RASKIN, M.D. JULIO ROSENSTOCK, M.D.

Dallas, Texas

From the Department of Medicine, University of Texas Health Science Center at Dallas, South- western Medical School, Dallas, Texas. Re- quests for repritns should be addressed to Dr. Philip Raskin, Department of Internal Medicine, University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Tex- as 75235. Manuscript submitted December 17, 1988, and accepted February 17, 1987.

A new series of compounds, the aldose reductase inhibitors, has recently been viewed as potentially useful in the area of medicine. These compounds, which are still only experimental and in various stages of development, may play an important role in the management of the complications related to diabetes mellitus. Furthermore, the financial impact of the possible widespread use of these agents in most diabetic subjects has prompted very active research by the drug industry. As of this date, there are at least five pharmaceutical companies in the process of investigating the efficacy and safety of different aldose reductase inhibitor drugs. The product that gets to the marketplace first will result in high profits for that company.

Although the idea of using aldose reductase inhibitors in patients with diabetes is a relatively recent one, interest in the polyol pathway is not. it was Mann [I] in 1946 who discovered that the main sugar in seminal fluid was fructose and that the fructose in seminal fluid was derived from blood glucose. Furthermore, there was a relationship between the level of blood glucose and the seminal fluid fructose concentration [2]. Hers [3] was the first to demonstrate that seminal fructose was formed via the polyol pathway.

Figure 1 shows the polyol pathway [4]. Aldose reductase (alditol: NADPH oxidoreductase, EC 1.1.1.2.1) is the rate-limiting enzyme of this pathway. It catalyzes the reduction of hexoses to their respective sugar alcohol (e.g., glucose to sorbitol and galactose to galactitol or its other name, dulcitol); sorbitol is then oxidized by the second enzyme of this pathway, polyol (sorbitol) dehydrogenase (i-iditol dehydrogenase EC 1-I. 1.14). Sorbitol dehydrogenase has broad substrate specificities for many sugar alcohols, additionally converting xylitol to ~xylulose and ribitol to D-ribulose. However, it has limited ability to further metabolize dulcitol, leading to enhanced accumulation of this sugar alcohol in galactosemia [5]. Generally, hexoses including glucose are poor sub- strates for aldose reductase. Aldose reductase has a high Michaelis constant (70 to 150 mM) for glucose and galactose; thus, when levels of the hexose are elevated, as in diabetes with hyperglycemia or in hyperga- lactosemia, significant polyol formation can occur [5].

Over the past decade, there has been considerable interest in the relationship between the polyol pathway and the small blood vessel complications of diabetes mellitus [5]. If this enzymatic pathway does serve as some pathogenic mechanism in all or some of the diabetic complications, then the inhibition of aldose reductase may represent a direct pharmacologic approach in the treatment of certain diabetic complications. This approach is distinct and separate from treatments designed to improve blood glucose levels [S].

298 August 1987 The American Journal of Medicine Volume 83

ALDOSE .-YuCTASE INHIBITORS AND DIABETIC COMPLICATIONS-RASKIN and ROSENSTOCK

Unregulated transpon in Lens. Schwenn Cell. RBC

Figure 1. The polyol pathway. Extracellular fluid (ECF). (Reproduced from [ 41.)

ALDOSE REDUCTASE IN TlSSUES

Aldose reductase has been demonstrated in many tissues in the body. Table I lists some of the tissues in which aldose reductase has been directly demonstrated [7-l 21.

In other studies, the presence of aldose reductase in various tissues has been implied by the demonstration that sorbitol was present and/or sorbitol levels were found to increase in the presence of diabetes or upon incubation of that tissue in a high-glucose medium in vitro. Thus, it is thought that aldose reductase is present in spinal cord [ 131 and in erythrocytes [ 14- 171. The finding that sorbitol levels can be measured in human erythrocytes and that ,they will change proportionally to plasma glucose levels is an important issue. This makes it possible to relate tissue sorbitol levels to those of plasma glucose and gives us a simple way to assess the effectiveness of the various aldose reductase inhibitors. Figure 2 shows the effect of increasing the glucose concentration on the production of sorbitol in intact human erythrocytes in vitro. Hubinont et al [ 151 have studied the effect of the in vivo administration of intravenous glucose and the hyperglycemia that results on erythrocyte sorbitol concentration. They showed that by raising the plasma glucose concentration to approximately 300 mg/dl by the infusion of glucose, erythrocyte sorbitol levels almost doubled within 45 minutes. When the glucose infusion was terminated and plasma glucose levels decreased, erythrocyte sorbitol levels returned to normal. Raskin et al [ 181 showed the relationship between fasting plasma glucose and erythrocyte sorbitol levels in patients with diabetes mellitus. A similar relationship has been observed by others as well [ 14,161. Thus, the use of erythrocyte sorbitol levels is, with some limitations, fairly

TABLE I Tissue Distribution of Aldose Reductase

Organ Tissue

Eye

Cornea Conjunctiva Retina

I Lens

Nerves Peripheral nerves Schwann cell Optic nerve Axons within myelin sheath Medulla Loop of Henle, conducting tu-

Kidney Glomeruli

Localization

Epithelium and endothelial Single layer of basal cells Ganglion, Mueller cells, peri-

cytes (mural cells) of retina capillaries

Epithelium

bule Podocytes

0 L ’ I I I E 100 300 500

Glucose (mg/dl)

Figure 2. Effect of in vitro glucose concentration on the production of sorbitol in intact human erythrocytes. (Repro duced from [ 141.)

well accepted by most investigators in the field as repre- sentative of sorbitol levels in other tissues. They also are used to measure the relative effectiveness of various aldose reductase inhibitor agents in vivo.

ALDOSE REDUCTASE IN DIABETIC CATARACT FORMATION

The formation of diabetic cataracts has been exclusively studied in terms of aldose reductase and the polyol pathway. If the polyol pathway is important in terms of the development of diabetic complications, then how does this occur? The pathogenesis of diabetic cataracts seem- ingly has been well worked out. Although the “osmotic

August 1987 The American Journal of Medicine Volume 83 299

ALDOSE REDUCTASE INHIBITORS AND DIABETIC COMPLICATIONS-RASKIN and ROSENSTOCK

Diabetic Cataract Development

Lenticular Changes

Normal Vacuolar Stage Cortical Cataract Nuclear Cataract

Biochemical Changes

Glucose K7

Glucose v

Glucose

Water-

Ygure 3. Schematic design of biochemical changes that occur during the development of a sugar cataract. Actual pictures of in vitro cataract formation. GSH = reduced glutathione; AA = amino acids. (Reproduced from [ 2 I] .)

theory” seems an appropriate explanation for the development of diabetic and galactosemic cataracts, we are not certain that it is operative in tissues other than the lens. The osmotic theory has been promulgated by Kino- shita and co-workers [19,20]. Stated simply, the theory suggests that the intracellular accumulation of polyols initiated by aldose reductase leads to an influx of water that results in permeability changes and eventually leads to a loss of cellular integrity. Figure 3 [21] shows a schematic of biochemical changes that occur during the development of a sugar cataract as well as pictures of actual in vitro cataract formation that correlate with the proposed biochemical changes. The increase in osmolali- ty caused by the accumulation of sorbitol and fructose draws water into the lens fibers, causing them to swell. The swelling has adverse effects since it increases the permeability to substances normally retained in the lens at concentrations higher than surrounding intraocular fluids. Thus, the concentrations of potassium, amino acids, glutathione, inositol, and ATP begin to decrease, and sodium and chlorine ions slowly begin to build up. As the process continues, a secondary osmotic change results from the electrolyte change of increased sodium and chlorine ions;

eventually the increases in these electrolytes become the predominant factor in lens swelling. The lens membranes become freely permeable to all substances other than the larger proteins. In this later stage, swelling is explained by the Donnan principle. It is accompanied by the appear- ance of the dense nuclear cataracts. In the formation of diabetic cataracts, the earliest visible change is the ap- pearance of swollen lens fibers caused by an increase in lens hydration. The swollen lens fibers eventually rupture with the liquification of the fibers, resulting in vacuole formation. In our judgment, the only tissue in which osmotic changes play any role is in the lens relative to cataract formation. In other tissues, the amount of water influx is insufficient to cause tissue damage. Other pathogenic mechanisms seem to be more important. These changes will be discussed subsequently.

The inhibition of in vitro cataract formation is a well- established method for evaluating the effectiveness and relative potency of aldose reductase inhibitors. There are considerable experimental data to support the effect of aldose reductase inhibitor on sugar cataracts. To begin with, Gonzalez et al [22] were able to measure the metabolic flux through the polyol pathway using nuclear


magnetic resonance in intact rabbit lens exposed to high concentrations of glucose in vitro. They concluded that one third of the glucose turnover in this model is via the polyol pathway. Hu et al [23] and Datiles et al [24] prevented cataract formation in the galactose-fed rat model with the aldose reductase inhibitor sorbinil. Beyer- Mears et al [25] were able to preserve lens growth, cell hydration, and protein components (alpha, beta, and gamma crystallins) with the topical administration of sorbinil to neonatal rats during galactose cataractogenesis. Gonzalez et al [26] studied the effect of sorbinil on the metabolic profile of the lens following induction of alloxan diabetes in the rat. The lens content of sorbitol and fructose was markedly increased with induction of diabetes. Administration of the aldose reductase agent, sorbinil, either normalized these abnormalities or partially corrected them. Other metabolic abnormalities in the lens in alloxan diabetes included diminished ATP, glutathione, and glutamate levels that were also corrected with sorbinil. Similar results have been obtained with Statil (ICt 128, 436), which is a potent non-spyrohydantoin-related aldose reductase inhibitor [27]. Treatment with this novel agent significantly reduced the increased lens sorbitol and fructose levels in streptozotocin diabetic rats. Statil at both 25 and 100 mg/kg prevented the development of any lenticular abnormalities for at least 74 days after the induction of diabetes.

ALDOSE REDUCTASE IN DtABETtC NEUROPATHY

Another mechanism by which aldose reductase and the accumulation of sorbitol may be implicated in the development of diabetic complications is best illustrated in terms of diabetic neuropathy. There is considerable evidence [28] that diabetic neuropathy may be related to changes in nerve myoinositol [29-321 content and the activity of sodium-potassium ATPase [33,34]. It is of interest that in both experimental and human diabetes, hyperglycemia causes increased activation of aldose reductase that generates accumulation of sorbitol in nerve tissue and a concomitant decrease in intraneuronal myoinositol levels. Both of these biochemical abnormalities are associated with a decreased nerve conduction velocity. These changes can be reversed by: (1) insulin treatment that prevents detectable hyperglycemia [29,35]; (2) supplementation of the diet with myoinositol [29,36,37]; and (3) administration of an akfose reductase inhibitor [27,38-411. It is important to note that three different aldose reductase inhibitors [Sorbinil,(CP-45634), ONO- 2235, and Statil(lCl-128,436)] have the identical effect on nerve conduction velocities. Furthermore, Statil has also recently been shown to prevent the defective axonal transport in streptozotocin-induced diabetic rats [42].

Hyperglycemia appears to lower intraneuronal myoinositol by at least three separate mechanisms. The first is a

Hyperglycemia ,

I \

\ Competitive Polyol

Inhibition

1

Pathway

\

\

4 Na+-Dependent I MI Uptake

i 4 Na+/K+ ATPase 4 Tissue

Activity MI

\ 4 Membrane Phosphoinositide

ALDOSE REDuCTASE INHIBITORS AND DIABETIC COMPLICATIONS-RASKIN and ROSENSTOCK

Figure 4. Proposed scheme of the interaction between hyperglycemia, sodium-potassium ATPase activity, and the polyolpathway. MI = myoinositol. (Reproduced from [ 441.)

competitive inhibition of the nerve cell uptake by glucose. Because glucose and myoinositol have nearly identical three-dimensional configuration, the two compete for the carrier system that mediates the active transport of myoinositol in peripheral nerve. The second mechanism is cyclic, arising from the fact that myoinositol uptake is both dependent upon and necessary for a normal transmem- brane sodium gradient. The third mechanism by which hyperglycemia blocks myoinositol uptake is through the accumulation of sorbitol via the increased activity of aldose reductase. Although the connection between the polyol pathway and the decrease in nerve myoinositol content is unclear at present, the use of an aldose reductase inhibitor will prevent the decrease in nerve myoinositol [42,43]. Figure 4 shows the proposed interaction of hyperglycemia on sodium-dependent myoinositol uptake, inositol phospholipid metabolism, and sodium-potassium ATPase activity, and the polyol pathway in the pathogenesis of diabetic neuropathy [44]. Of interest and impor- tance is that these same metabolic changes, i.e., accumulation of sorbitol with a subsequent reduction in tissue myoinositol and a reduction in sodium-potassium ATPase activity, have been demonstrated in both diabetic retina and glomeruli.

ALDOSE REDUCTASE IN DtABETtC RETtNOPATHY

The polyol pathway and aldose reductase have been implicated in two histologic features of diabetic retinopathy that involve retinal capillaries: (1) the selective loss in numbers of mural cells (pericytes); and (2) a thickening of the retinal capillary basement membrane. Mural cells contain aldose reductase [lo] and accumulate sorbitol. Of interest is the fact that retinopathy that appears to be


-- 28 weekr 44 week8

0 Control

m Gelectore

[PPI Gslsctoes l rorbinit I

Figure 5. Effect of sorbinil treatment on retinal capillary basement membrane (BMA) width in galactose-fed fats. (Reproduced from [ 4 73 .)

similar to diabetic retinopathy in humans develops in dogs [45] and rats [46,47] made galactosemic by the feeding of a high-galactose diet. The retinopathy in galactosemic dogs [45] is marked by saccular capillary aneurysms, hemorrhages, nonperfused or acellular vessels, tortuous hypertrophic capillaries, loss of capillary pericytes, and other lesions typical of diabetic patients. Furthermore, in both galactose-fed dogs [45] and rats [46,47] and in experimental diabetes in rats [48], retinal capillaries developed a thickening of the basement membrane. Admin- istration of sorbinil to galactose-fed rats [47] (Figure 5) or alconil [48] to diabetic rats prevents the thickening of retinal capillary basement membrane.

ALDOSE REDUCTASE IN DIABETIC NEPHROPATHY

The role of aldose reductase in the pathogenesis of experimental diabetic nephropathy has only recently been in- vestigated. Beyer-Mears [49] recently measured polyols in glomeruli isolated from control and streptozotocin- induced diabetic rats. Compared with control (nondiabet- ic) animals, the polyol content was increased lo-fold at six weeks following the induction of diabetes, and the myoinositol content was significantly reduced in diabetic glomeruli nine weeks following,the induction of diabetes. Administration of the aldose reductase inhibitor sorbinil prevented both the accumulation of polyol and the reduction of myoinositol. Sorbinil has also been shown to prevent renal hypettrophy in galactose-fed rats [50] and to diminish proteinuria in streptozotocin-induced diabetic rats [5 I].

TABLE II Development of Aldose Reductase Inhibitor Drugs

Drua Name Pharmaceutical

ComPanv Status


ICI-128,436 Statil Stuart Pharma- ceuticals, Im- perial Chemi- cal Industries (United King- dom)

Tolrestat Ayers&American Home Prod- ucts

Phase 213 studies (neuropathy) well underway

AY-27,773

CP-45,634 Sorbinil Pfizer Laborato- ries

ONO-2235 - ON0 Pharma- ceuticals (Ja- w)

Some phase 21 3 studies (neuropathy) have been completed; retinopathy trial ongoing

Some phase 21 3 studies (neuropathy) have been completed; retinopathy trial ongoing

Several open clinical trials (neuropathy) completed in Japan

ALDOSE REDUCTASE IN CLINICAL TRIALS

Clinical trials with the various aldose reductase inhibitor agents are now underway. Table II lists the various aldose reductase agents that are now in various stages of development.

The clinical trials involving the use of aldose reductase inhibitors have been limited to studies involving the use of these various agents for the treatment of diabetic neuropathy (Table Ill). Judzewitsch et al [52] conducted a double-masked, placebo crossover trial in 39 diabetic patients without clinical evidence of diabetic neuropathy. During the nine-week study period, each patient received either placebo or a single daily dose of 250 mg of sorbinil. During the nine weeks of treatment with sorbinil, nerve conduction velocity was greater than during a nine-week period of placebo therapy for all three nerves tested (Figure 6). There were no changes in glycemic control during the study. Nerve conduction velocity for all three nerves declined significantly within three weeks after cessation of the drug. As can be seen from Figure 6, the improvement in nerve conduction velocity was small, approximately 1 meter/second. Such small changes hardly warrant an enthusiastic response.

Jaspan et al [53] treated 11 patients with severe painful diabetic neuropathy with a single daily dose of 250 mg of sorbinil for a period of three to six weeks. Eight of the



TABLE III

Reference

Summary of Clinical Trials with Aldose Reductase Inhibitors

Duration of Number 01 Diabetic Treatment

Drug (Dose) Patients Neuropalhy (weeks) Placebo-Controlled Results

[521

1531

[541

[551

WI

t;i;

[591

Sorbinil (250 mglday)

Sorbinil (250 mg/day)



ONO-2235 (300 mg/ day) Tolrestat (200 mg/day) Sorbinil (250 mg/day)

Sorbinil(250 mg/day)

39 No

11 Yes

15 Yes

13 Yes

13 Yes

19 Yes 55 Yes

32 Yes 6

9

3-6

16

4

12

4-24 24

Yes

No

Yes

Yes

No

No Yes

Yes

Small improvement in motor nerve conduction velocity

Symptomatic improvement in eight of 11

improvement in pain and tendon reflex scores

No improvement in symptoms or electrophysiologic test results

Improvement in symptoms and motor nerve conduction velocity

Improvement in symptoms No symptomatic improvement; some

improvement in some neurophysiologic test results

Improvement in pain and neuropathy

+SEM

n- 20

Wash Placebo Sorbinil 1 Out

0 3 6 9 12 15 18 21

Time (weeks)

Figure 6. Effect of sorbinil treatment on peroneal nerve Figure 7. Effect of sorbinil treatment on pain score in conduction velocity (/VCVj in patients with diabetes (Repro patients with painful diabetic neuropathy. (Reproduced from duced from [ 5.21.) L531.1

11 patients also received placebo for a variable period of time ranging from several days to a few weeks. Response was assessed according to a 0 to 20 graphic scale for pain and by tests for motor and sensory nerve conduction velocities and cardiac autonomic nerve function. Eight patients had moderate to marked relief of pain, two had equivocal responses, and one had no change. Upon dis- continuation of the medication treatment, seven of the eight patients with response had a worsening of pain (Figure 7). During the course of treatment, autonomic nerve function improved significantly in six of seven pa-

A PAIN SCORE

-a

-16

Base1 Ine End 3 Weeks of Later

Sorbrnil

tients tested. Nerve conduction velocities improved in four of the seven tested.

Young et al [54] administered sorbinil(200 mg per day) to 15 patients with chronic painful diabetic neuropathy. A double-masked crossover study design was used. Treat- ment was evaluated by subjective pain responses, clinical examination, vibration perception threshold, nerve elec- trophysiology, and cardiovascular reflex tests. Among the many measurements, only the degree of pain, tendon reflex scores, and sural sensory potential amplitude improved. In four patients, an idiosyncratic reaction (rash)



developed that rapidly disappeared upon termination of the drug treatment. It is important to note that use of the aldose reductase inhibitor drug sorbinil has been associated with an alarmingly high incidence of side effects. These side effects are primarily skin lesions that are most likely related to the hydantoin structure of the drug. Stud- ies are now underway to evaluate if a lower initial dose of sorbinil will be effective in reducing the high incidence of side effects.

Lewin et al [55] administered sorbinil 200 mg daily for four weeks in 13 patients with symptomatic diabetic neuropathy. A double-masked placebo crossover study design was used. No beneficial effects of the sorbinil on nerve conduction velocities, abnormal autonomic nervous system function, or symptoms of painful neuropathy were observed. As pointed out by the authors, the population studied was older with longer duration of diabetes than in previous trials, and the study period was very short.

Two other studies, although quite preliminary, are worth mentioning. The first by Hotta et al [56] used a 300 mg daily dose of the aldose reductase inhibitor drug, ONO- 2235, in 13 patients with painful diabetic neuropathy. The drug was given for 12 weeks without any placebo control. Significant improvement in motor nerve conduction velocity in the ulnar nerve was observed over the 12-week treatment period. The improvement in electrophysiologic studies as well as those of subjective symptoms (pain and numbness) correlated with the reduction of erythrocyte sorbitol levels. Koglin et al [57] administered tolrestat in a dose of 200 mg per day to 19 patients with painful or paresthetic diabetic distal, symmetric peripheral neuropathy. They reported a marked improvement in symptoms in a large proportion of their patients. In fact, of the eight patients who had been treated for 24 weeks, seven reported improvement. Unfortunately, there was no placebo control in this study. Fagius et al [58] studied the effect of a 250 mg daily dose of sorbinil in patients with symptomatic diabetic polyneuropathy during a six-month

double-masked placebo-controlled trial. There was no improvement in the symptoms of diabetic neuropathy in those patients treated with sorbinil. There were, however, some improvements noted in the results of three of nine neurophysiologic tests and one of five tests of autonomic nerve function.

Finally, Rosenstock et al [59] have recently studied the short-term effect of sorbinil in 32 patients with severe painful diabetic neuropathy unresponsive to conventional analgesic and tricyclic therapy. A double-masked ran- domized study design was used. Patients received either placebo or 250 mg per day of sorbinil. Assessment was made using a 20-point self-administered neuropathy and pain symptom score. Pain scores and overall neuropathy improved after the six-week study in the sorbinil-treated group as compared with those patients who received placebo. They concluded, however, that longer studies will be required to assess the full effect of aldose reductase inhibitors, preferably in patients with less advanced diabetic neuropathy.

CONCLUSION

Aldose reductase inhibitors, from a theoretic perspective, seem to offer promise as possible agents for the treatment or even the prevention of the microvascular complications of diabetes. Although considerable theoretic data are available to support their effectiveness in experimental diabetes, there have been relatively few studies in humans. Those that have been performed often were poorly designed, i.e., lacked an appropriate control group, or were carried out in patients with markedly advanced disease. Longer prospective controlled trials are certainly required before these compounds can be advocated on a routine basis. Our bias is, however, that the real utility of these drugs will be as agents for the prevention of the complications of diabetes in some tissues (nerve and retina seem the most likely candidates) rather than as treatment for advanced disease.

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aldose reductase inhibitors and diabetic complications

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