INHIBITION OF RELEASE OR ACTION OF COUNTERREGULATORY HORMONES

New Pharmacological tes to Therapy

Glucagon analogues RUBIN BRESSLER, MD DAVID JOHNSON, MD

Currently available pharmacological agents have not been completely successful in restoring euglycemia in the non-insulin-dependent diabetes mellitus (NIDDM) patient. Several new approaches to the therapy of NIDDM have been formulated in recent years and are in various stages of laboratory or pharmaceutical development. Several of these agents are discussed in this article under categories relating to their mechanisms of lowering blood glucose: 1) inhibition of the release or action of counterregulatory hormones; 2) inhibition of postprandial glucose rise; 3) sensitization of tissues to insulin's actions; and 4) inhibition of gluconeogenesis, including inhibition of the long-chain acyl-CoA-carnitine acyltransferase I, the long-chain acylcarnitine translocase, and pyruvate carboxylase.

N

on-insulin-dependent diabetes mellitus (NIDDM) is a disease characterized by overproduction and underutilization of glucose. These defects derive from insulin resistance and impaired insulin secretion (1,2). Glucose overproduction is a result of an insensitivity to insulin of the enzymatic steps regulating hepatic glycogenesis, glycogenolysis, and gluconeogenesis. Peripheral insulin resistance is an underlying basis for underutilization of glucose by adipose tissue and muscle. The resultant hyperglycemia is a stimulant for more insulin secretion until the point where the (3-cells fail to keep pace with insulin needs. The currently available antidiabetic pharmacological agents have not

been totally successful in the amelioration of the pathophysiological abnormalities of NIDDM (3). We briefly discuss a number of new pharmacological approaches to the treatment of NIDDM. These potential drugs are in various stages of pharmaceutical development. The areas of our focus must be limited but include J) inhibition of the release or action of counterregulatory hormones; 2) inhibition of postprandial glucose rise; 3) sensitization of tissues to insulin's actions; and 4) inhibition of gluconeogenesis, including inhibition of the longchain acyl-CoA-carnitine acyltransferase I (LCAT I), the long-chain acylcarnitine translocase, and pyruvate carboxylase (PC).

FROM THE DEPARTMENTS OF INTERNAL MEDICINE AND PHARMACOLOGY, THE UNIVERSITY OF ARIZONA HEALTH SCIENCES CENTER, TUCSON, ARIZONA. ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO RUBIN BRESSLER, MD, PROFESSOR OF MEDICINE AND HEAD DEPT. OF INTERNAL MEDICINE, THE UNIVERSITY OF ARIZONA HEALTH SCIENCES CENTER, 1501 NORTH CAMPBELL AVENUE, TUCSON, AZ 85724.

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Glucagon, the polypeptide hormone secreted from pancreatic islet a-cells, plays an important role in the pathophysiology of diabetes mellitus (4,5). Whereas the relative or absolute deficiency of insulin causes glucose underutilization, glucagon secretion leads to overproduction of glucose by stimulating hepatic glycogenolysis and gluconeogenesis (6). This has led to a search for analogues of glucagon that could antagonize the effects of glucagon on target tissues (7—9). To be effective, these analogues must compete with glucagon for binding to the glucagon receptors but not activate the receptor to initiate target cell responses. As an additional complexity, there is increasing evidence that glucagon-receptor activation involves not only the adenylate cyclase system but the inositol phosphate pathway as well (9,10). Wakelam et al. (10) demonstrated that glucagon-receptor activation in hepatocyte membranes can stimulate phospholipase C activity, with production of the second messengers inositol trisphosphate and 1,2-diacylglycerol. Furthermore, the inositol and the adenylate cyclase systems interact with one another (11). It may be necessary to develop analogues that can inhibit both of these pathways either alone or in combination to block all of the effects of glucagon on hepatic glucose and fatty acid metabolism. During the past decade, considerable progress has been made in discovering the structural features of glucagon that are important for its binding to the glucagon receptor and conferring agonist or antagonist properties. Krstenansky et al. (12) drew attention to the importance of the COOH-terminal a-helical structure for binding affinity and activation of adenylate cyclase. Truncation of the NH2-terminal, e.g. des His1 glucagon, or

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Bressler and Johnson

introduction of hydrophobic groups and neutralization of the positive charges at the a- and e-NH2—positions results in glucagon antagonists (13). More recently, Unson et al. (9,14,15) showed that the substitution of glutamic acid for aspartic acid at residue 9 causes a large decrease in biological activity with retention of binding affinity. The two most potent glucagon antagonists that have been developed are (l-a-trinitrophenylhistidine,12-homoarginine)-glucagon (13) and des His (Glu9)-glucagon amide (9). Both the compounds cause rapid decreases in the hyperglycemia of streptozocin-induced diabetic (STZ-D) rats (8,9). The magnitude of the fall in blood glucose caused by these glucagon antagonists (~50%) is larger than that which somatostatin produces by inhibition of glucagon secretion (16,17). This may be due to the lack of suppression of insulin secretion with the glucagon receptor antagonists. In fact, des His [Glu9]-glucagon amide actually potentiates glucose-induced insulin release (18). Insulin in the peripheral plasma of STZ-D rats is undetectable by radioimmunoassay, but very low concentrations of insulin in the portal venous blood may normally counterbalance glucagon-stimulated hyperglycemia. Further research with glucagon antagonists must focus on several areas. First, more knowledge is needed regarding the mechanism of receptor activation and the interrelationship between the adenylate cyclase and the inositol systems. Second, development of more-potent and longer-lasting analogues would facilitate studies in larger diabetic animals such as the dog. Finally, the effects of glucagon antagonists on ketogenesis should be studied in diabetic animals prone to ketoacidosis. INHIBITION OF POSTPRANDIAL GLUCOSE RISE at-Glucosidase inhibitors Intestinal absorption of starch, dextrins, and disaccharides, such as sucrose, re-

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quires the action of intestinal brushborder a-glucosidases. Thus, inhibition of these digestive enzymes retards the absorption of carbohydrates, leading to an attenuated rise in postprandial blood glucose concentration. Over the past decade, several oligosaccharide inhibitors of intestinal a-glucosidases have been obtained from cultures of actinomycetes or produced by total chemical synthesis. When these agents are ingested before or during a meal, there is a slower and smaller rise in blood glucose in both control subjects and patients with diabetes mellitus.

Acarbose The most-studied drug of this type is acarbose, an inhibitor of intestinal a-glucosidase that is absorbed in only minimal amounts into the systemic circulation (19,20). Acarbose (Bayer, TeverkusenBayerwerk, Germany) is a competitive inhibitor of both glucoamylase and sucrase. It is a weak inhibitor of pancreatic a-amylase. During the past decade, numerous clinical investigations with acarbose demonstrated a decrease in postprandial blood glucose concentrations and glycosuria in both insulin-dependent diabetes mellitus (IDDM) and NIDDM patients (21,22). Modest improvement in glycosylated hemoglobin and fasting blood glucose (23) was also demonstrated in some studies. The dose of acarbose that will reduce postprandial hyperglycemia without significant malabsorption and intestinal side effects is usually 50-100 mg given with each large meal. Smaller dosages may be used with snacks. Measurement of breath hydrogen after ingestion of sucrose showed that 50 mg of acarbose decreased the glycemic response without appreciable malabsorption, whereas 100 mg caused 40% malabsorption, and 200 mg caused nearly complete malabsorption (24). Less malabsorption occurs after mixed meals containing starch (25). Acarbose is most effective when given with a starchy high-fiber diet

1992

with restricted amounts of sucrose and glucose (including fruit juices). The side effects of acarbose are due to the malabsorption of carbohydrate, with subsequent metabolism of the carbohydrate by colonic microflora. The most common side effects are increased flatulence and abdominal bloating. Some patients also experience diarrhea. Unfortunately, an increased number of kidney tumors in one strain of rats during chronic toxicity studies caused a temporary suspension of clinical investigations during the mid 1980s. Additional studies indicated that the tumors were not due to the drug itself, and recent clinical trials have confirmed the safety and efficacy of acarbose. Acarbose is available for clinical use in Germany and the Netherlands. Desoxynojirimycin inhibitors Several a-glucosidase inhibitors have been identified with chemical structure derived from nojirimycin and its reduced form, 1-desoxynojirimycin. The bestcharacterized member of this group is miglitol [N-(|3-hydroxymethyl)-l-des~ oxynojirimycin]. Like acarbose, miglitol (Bayer) is a potent competitive inhibitor of sucrase and glucoamylase (26). Unlike acarbose, miglitol is almost completely absorbed in the small intestine. Clinical trials with miglitol have shown a reduction in glycemic elevation in both IDDM (27) and NIDDM patients (28). The therapeutic dose is 50-100 mg three times daily with meals. Higher dosages produce malabsorption, with symptoms of flatulence, abdominal distention, and diarrhea. Despite differences in absorption and excretion between acarbose and miglitol, the overall clinical efficacies and side effect profiles are similar. Emiglitate is another derivative of desoxynojirimycin developed by Bayer. This compound is more potent and longer acting than acarbose and miglitol. However, clinical trials with emiglitate have not demonstrated as effective control of postprandial hyperglycemia as with acarbose and miglitol. Furthermore,

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New approaches to N1DDM therapy

side effects from malabsorption have been more common with emiglitate when used in comparably effective dosage. Therefore, emiglitate is no longer in development. Nippon Shinyaka (Kyoto, Japan) has also synthesized a series of N-substituted derivatives of 1-desoxynojirimycin that appear to be effective in early preclinical testing (29,30).

Other ct-glucosidase inhibitors Several additional classes of compounds have been identified with a-glucosidase inhibitory activity. A0—128 is a derivative of valiolamine developed by Takeda (Osaka, Japan) that has shown therapeutic efficacy in early phase II studies in diabetic subjects. Horii et al. (31) also synthesized a series of N-substituted valiolamines that appear to be more potent a-glucosidase inhibitors. Castanospermine (Merrell Dow) is an indolizidine alkaloid extracted from the seeds of the tree Castanospermum austral. It causes

potent and extremely long-lasting inhibition of intestinal a-glucosidase (32). When castanospermine was discovered to block the growth of human immunodeficiency virus, research into this aspect of its biological activity predominated over efforts to develop it as an antidiabetic drug (33). Merrell Dow has also synthesized a unique glucosidase inhibitor (MDL 25,637), which appears to be effective in early animal studies (34). Summary Numerous studies in animals and nondiabetic subjects have demonstrated the ability of a-glucosidase inhibitors to diminish postprandial glycemic excursion. Overdosage with any of these agents produces malabsorption, with associated flatulence, abdominal bloating, and diarrhea. The best response in diabetic subjects is obtained when the drugs are combined with diets high in starch and fiber with limited amounts of sucrose and glucose. Currently, only one compound, acarbose, has been marketed for clinical use in Germany and the Netherlands.

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2,4-dione; 35,36}. Ciglitazone has undergone various studies in several insulin-resistant animal models (36,37). These animal models include the obese yellow KK mouse, the diabetic db/db mouse, the obese ob/ob mouse, the fatty Zucker rat, and rats made insulin resistant by feeding a high-fat diet (36-42). The thiazolidinediones have demonstrated consistent antidiabetic effects when used in vivo to treat insulinNH EN6LITAZ0NE resistant animal models. Moreover, the in vivo treatment of these animal models has resulted in an augmentation of their Figure 1—Stmcturcs of thiazolidincdkmc de- adipose tissue, skeletal muscle, and liver rivatives. responses to in vitro insulin stimulation. A considerable body of data support the thiazolidinedione compounds' mechanism of action in insulin-resistant animal SENSITIZATION OF TISSUES TO models as being due to a sensitization of INSULIN'S ACTIONS: target tissues to insulin (36,37). THIAZOLIDINEDIONE Oral administration of ciglitazone DERIVATIVES— In the course of the to diabetic KK mice lowered blood glupast decade, a new class of antidiabetic cose in a dose-dependent manner and agents has been extensively studied, decreased plasma levels of insulin and which have specific efficacy in insulintriglycerides (39). The compound imresistant states. Many studies on the use proved glucose tolerance in the fatty of these agents have been carried out in insulin-resistant animal models, and the Zucker rat at lower plasma insulin conuse of one or more of these agents in centrations, suggesting an increased senhuman phase I and II studies is being sitivity to insulin (36,37,39). Studies in the ob/ob mouse confirmed the actions of pursued actively. ciglitazone and demonstrated its lack of At the time of this writing, the efficacy in insulinopenic animal models three thiazolidinedione derivatives like the STZ-D rat and the late-stage shown in Fig. 1 have been the most exdb/db mouse (36-38). Ciglitazone and tensively studied. The pharmacological other thiazolidinediones did not cause effects of the three drugs are similar in hypoglycemia in either treated insulinmost respects. The earliest member of this class resistant animal models or non-insulinof drugs and the most extensively resistant controls (36,37). Ciglitazone studied is ciglitazone {5-[4-(l-methylcy- decreased hyperglycemia, plasma insuclohexylmethoxy)benzyl]thiazolidine- lin, and blood lipids in several insulinIH

CIGLITAZONE

Table 1—Potentiation of in vitro actions of insulin on tissues of insulin-resistant rats and mice treated with pioglitazone (46,48,49) ADIPOSE TISSUE

MUSCLE

Ll\T.R

t

GLUCOSE UPTAKE

t

GLUCOSE UPTAKE

i

GLUCOSE OUTPUT

t

GLUCOSE OXIDATION

t

GLUCOSE OXIDATION

t

LiPin SYNTHESIS

t

LlPID SYNTHESIS

t

LlPID SYNTHESIS

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FFA

GLUCOSE FORMATION T .OAA

ATP

2-0XIRANECARB0XYLATES •asymmetric carbon

> Y ^ CoASH

STIMULATES

\

AcylCoA /

AcCOA + NADHLCFAO

I Outer Ml to ' Membrane

CPT 1 FEEDBACK PRODUCT INHIBITION

Pyruvlc

CH 3 (CH 2 ) 1 3 — C — C00CH3 —' CH2

Carnltlne

0

Acylcarnltlne Inner Mito Hewbrane —i

TRANSLOCASE JCPT II

METHYLPALMOXIRATE (2-Tetradecylglycidate, 2-TDGA)

AcCoA + NADH AcylCoA

CoASH

Energy

I ^-OXIDATION

Figure 2—Regulation of pathways ofpyruvate metabolism by long-chain fatty acid oxidation (LCFAO). OAA, oxaloacetic acid; AcCoA, acetyl-CoA.

resistant animal models after oral administration. The decrease in plasma insulin concentrations follow the fall in blood glucose and are thought to be a consequence of the thiazolidinedione's decreasing resistance to the actions of insulin (36,43). A consistent finding with administration of thiazolidinediones to insulin-resistant animal models has been the increase of insulin in the pancreatic islets (44,45). The mechanism for this finding is unknown. The oral administration of the several thiazolidinediones that have been studied results in a sensitization of adipose tissue, skeletal muscle, and liver to the actions of insulin. Epididymal fat pads from ob/ob mice treated with ciglitazone demonstrated increased insulinstimulated glucose oxidation and lipogenesis (41). The perfused hindquarter of ciglitazone-treated ob/ob mice showed augmented transport of 2-deoxy-Dglucose (42). Ciglitazone administration to rats made insulin resistant by a highfat diet potentiated the action of insulin on glucose transport in adipose tissue (40). The administration of thiazolidinediones to non-insulin-resistant animal models has resulted in no potentiation of insulin's actions (36,37). However, pio-

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AcetylCoA C07

Ketones

Regulatory Roles

Figure 3—Long-chain fatty acid metabolism. FFA, free fatty acids; CoASH, Coenzyme A; CPT, carnitine palmityltransferase.

CH2?0

ftftA CH 2 CH 2 CH 2 C00CH2CH3 CLOMOXIR

glitazone, an analogue of ciglitazone (Fig. 1), which decreases blood glucose and plasma insulin in insulin-resistant animal models, has been found to decrease plasma triglyceride levels in both nondiabetic and insulin-resistant diabetic animals (46). The stimulatory effect of the thiazolidinediones on insulin-induced glucose transport in tissues of treated insulin-resistant animal models has been found to be associated with an increase in the number of glucose transporters (36,37,47). The studies thus far published on the thiazolidinediones leave open the effect of these agents on insulin receptors and gluconeogenesis (36). Some studies have shown that thiazolidinedione therapy of insulin-resistant animal models is associated with an increase in receptor numbers without changes in receptor affinity (36,41). These changes, however, have not been of great magnitude, nor have they been confirmed in some studies (46). They could be the result of amelioration of the hyperinsulinemia, which downregulates insulin receptors (37,46).

1992

ftftYs CH 2

CH2

CH2

C00CH 2 CH 3

Figure 4—Structures of 2-oxiranecarboxylates.

Studies on tissues from insulinresistant animal models treated with pioglitazone suggested that the drug's effects are an augmentation of insulin's actions (37,46,48,49; Table 1). The in vivo antihyperglycemic and plasma lipid-lowering effects of pioglitazone and its potentiation of insulin actions on target tissues were thought to be effects of the thiazolidinedione on intracellular actions subsequent to postinsulin-receptor binding (37,46; Table 1). The hypothesis was consonant with the response of adipocytes isolated from pioglitazone-treated insulin-resistant animal models, whose glucose oxidation was stimulated by vitamin K or vanadate, glucose utilization stimulants that do not

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New approaches to NIDDM therapy

increased glucose transport and oxida• Controls IF1 Etomoxlr (10 mg/kg) tion. Moreover, the cells can be made 200Etomoxlr (50 mg/kg) • insulin resistant by exposure to dexa100methasone. Englitazone initiated a stimulatory effect on the uptake of 2- deoxyD-glucose at 30 min, which increased over 5 h and was maintained for 72 h. °o.. 10080The thiazolidinedione derivative overcame the insulin resistance created by 2 4 6 8 10 12 2 dexamethasone. The in vitro effect of enDays of Treatment Hours After Dosing with Drug (25 mg/kg, p.o.) glitazone on 2-deoxy-D-glucose transport was an increase in Vmax. This was Figure 6—Effect ofetomoxir in fasted diabetic Figure 5—E/fect of methy/-2-te£radecy/gyci- inhibited by treatment of the cells with mice (C57BUKSJ-db/db). From Wolf (67). date in diabetic fasted dog. FFA, free fatty acids. cycloheximide, a protein synthesis in® by Smith-Gordon. hibitor, or low incubation temperature (10°C), which inhibits translocation of glucose transporters to the plasma meminteract with membrane insulin binding brane (51). Several thiazolidinediones coneogenesis, and a basal rate of LCFAO are currently in phase I or II clinical is a critical feature of the gluconeogenetic sites (46). state (53-57). LCFAO provides ATP for The lowering of blood glucose by testing. energy, acetyl-CoA (AcCoA) for allosteric thiazolidinediones in insulin-resistant diactivation of PC (58), and NADH for abetic animal models could result from INHIBITION OF stimulation of glyceraldehyde-3-phosseveral pharmacological effects, which GLUCONEOGENESIS phate dehydrogenase activity (59,60). have been demonstrated. These include increased glucose oxidation by adipose LCAT inhibitors Uncontrolled diabetes mellitus tissue and muscle, increased glycogen Gluconeogenesis overactivity is an im- causes an increase in lipolysis and and lipid synthesis from glucose, and portant contributor to the hyperglycemia LCFAO. This results in a decrease in decreased glycogenolysis (36,37). Al- of uncontrolled diabetes mellitus. The glucose oxidation and an increase in gluthough decreased hepatic glucose output augmentation of gluconeogenesis is a coneogenesis due to a shift in pyruvate was shown to result from use of piogli- complex interplay of insulin insuffi- metabolism (54,61-63). Insulin defitazone in fatty rats, the role of glucone- ciency, glucagon excess, substrate avail- ciency decreases the activity of pyruvate ogenesis inhibition was not addressed ability, hepatic enzyme alterations, and dehydrogenase, whereas the AcCoA (48). Ciglitazone treatment of obi oh mice long-chain fatty acid oxidation (LCFAO; formed from LCFAO stimulates PC, augresulted in a decreased conversion of 52). LCFAO is an energy source for glu- menting the conversion of pyruvate to [14C]alanine, but not lactate, to glucose in the treated animal's perfused liver (42). Other investigators showed an in Table 2—Effect of etomoxir on fasted streptozocin-induced diabetic rats 4 h after vivo decrease of [14C] lactate to glucose in administration ciglitazone-treated ob/ob mice (50). In the studies where an inhibition of glucoC P T I ACTIVITY B neogenesis was found, the decreases 3-OHB + AcAc GLUCOSE (NMOL • MIN" 1 • MG~' 827-33 were small and unlikely to be the major PROT" 1 ) (MM) (MM) factor in the antihyperglycemic activity (MG/KG) of the drug (36). 2.07 ± 0.09 0.832 ± 0.100 6.93 ± 0.37 300-

1

A study has shown a direct effect of englitazone (Fig. 1) on 2-deoxy-Dglucose uptake by 3T3-L1 adipocytes in the absence of insulin (51). This adipose tissue cell line has been used to study insulin actions and factors that regulate glucose transport. The cells have insulin receptors and respond to insulin with

796

0.2 1.8 18.0

5.77 ± 0.13* (-17%) 5.07 ± 0.22* (-27%) 4.21 ± 0.16* (-39%)

0.423 ± 0.081t (-49%) 0.116 ± 0.019T (-86%) 0.112 ±0.038t (-87%)

0.86 ± 0.17T-

(-59%) 0.24 ± 0.07* (-88%) 0.03 ± O.Olt (-99%)

Values are means ± SE; n = 6. CPT I, carnitine palmityltransferase I. *P < 0.05, tp < 0.01, fP < 0.001 (Student/Welch). From Eistter and Wolf (72). ® by Drugs Future.

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We discuss three areas of pharmacological approach to the control of gluconeogenesis: 1) inhibition of LCAT I, 2) inhibition of long-chain acylcarnitine translocase, and 3) inhibition of PC by interfering with the stimulatory effect of the product of LCFAO, AcCoA, on the key gluconeogenesis enzyme.

Phenylethylhydrazine Phenelzlne CH,3 I CH2-CH2-NH-N .= C COO2-(Phenylethylhydrazono)propionate PEHP CH 3 CH = C-CH2-NH-NH2 Methylclnnamylhydrazine MCH

CH,

CH,

1

'

CH = C-CH2-NH-N = C

OO-

2-(3-Methylclnnamylhydrazono)proplonate MCHP

CH2-CH2-CH2-0N = C COOH 2-(3-Phenylpropoxylmlno)butyric acid PPIB Figure 7—Structures of some propionic acid derivatives and related hypoglycemic substances.

oxaloacetate, a glucose precursor (6163). The AcCoA and NADH formed during LCFAO act as feedback inhibitors of pyruvate dehydrogenase (53,63,64). These relationships are shown in Fig. 2. The effects of LCFAO on the stimulation of glucose production and the inhibition of glucose utilization make it an attractive pharmacological focus. The complex chain of biochemical events involved in LCFAO are well characterized, and drugs have been designed to decrease gluconeogenesis by either inhibiting LCFAO at some site or by trying to obviate the stimulatory effect of LCFAO at a target site like the PC.

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INHIBITION OF LCAT I— LCFAO is a complex system involving both cytoplasmic and mitochondrial enzyme systems (64,65). The relationships are shown in Fig. 3 (65). Long-chain fatty acids are taken up by liver cells and converted to long-chain fatty acyl-CoA (LCFACoA) derivatives by cytoplasmic enzymes. These LCFACoA metabolites cannot penetrate the mitochondrial membranes to mitochondrial sites of LCFAO. The LCFACoA and cytoplasmic carnitine are converted to a long-chain acylcarnitine (LCAC) catalyzed by LCAT I situated on the outer mitochondrial membrane. The LCAC can permeate the outer membrane. The LCAC penetrates the inner mitochondrial membrane catalyzed by a translocase enzyme of the membrane. Once inside the inner membrane, the LCAC is reconverted to a LCFACoA in the presence of CoA and LCAT II. The LCFACoA is subsequently oxidized to AcCoA, ketone bodies, and NADH. The shuttle system for transporting free fatty acids (FFAs) activated in the cytoplasm to mitochondrial sites of LCFAO is thought to be a rate-limiting step (64,66). Because of the anticipated ameliorating effect of decreasing LCFAO on the hyperglycemia and ketogenesis of diabetic patients, several fatty acid oxidation-inhibiting compounds were studied from the early 1970s (67,68). Although several of these proved to be effective LCFAO inhibitors, their in vivo toxicities precluded clinical use. Over the past decade, several compounds that inhibit LCFAO by an irreversible inhibition of camitine palmityltransferase (LCAT I; 67,69) have been extensively studied. The structures

1992

1004-

2 -

il

50--

PPIB 6mN

MCHP 2tlfl

Palraltnte |

| Capryllc Acid

Figure 8—Utilization of palmitate or caprylic acid in isolated rat hemidiaphragms in prcsencc and absence of 2-(3-methykinnamylhydrazono) propionate (MCHP) or 2-(3-phenylpropoxyimino)butyric acid (PPIB). From Haeckel et al. (83). w by Smith-Gordon.

are shown in Fig. 4. The compounds are substituted 2-oxirane carboxylic acids. The R isomers of these compounds have been shown to be the active isomer of the racemic compound (67,68). These compounds are first activated to CoA derivatives. It has been hypothesized that their inhibitory effect on LCAT I is due to an opening of the oxirane ring and an alkylation of a nucleophilic group at the active site of the LCAT I enzyme (68). The acyl-CoA derivatives of these drugs are not substrates for the LCAT I enzyme but may interfere with substrate binding of LCAT (68,69). These drugs are specific for LCAT I inhibition and do not inhibit LCAT II or other enzymes of (3- oxidation. The pharmacological effects of LCAT I inhibition include J) decreased LCFAO, 2) decreased AcCoA production, 3) decreased ketogenesis, 4) deactivation of PC due to decreased AcCoA production, 5) decreased gluconeogenesis due to deactivation of PC, and 6) increased glu-

797

New approaches to NIDDM therapy

0.6

6.0-,

a)

I B

I Control 2-(3-PhenylpropoxylmIno)-butyric acid (5) (5)

|f

0.2-

0-1 0.6400

500

600

|imol/kg -

0.2-

0

15-30

30-45

45-60

60-75

75-90

Minute Periuslon Time

Glucose 5 mmol/l

798

Pyruvate 2 mmol/1

Acetate 2 mmol/l

Figure 10—Influence of 2-(phenylethylhydraFigure 11—Utilization of glucose, lactate, zono)propionate (PEHP), 2-(3-methylcinnamylpyruvate, and acetate in isolated rat hcmidiahydrazono)pwpionate (MCHP), and their corphragms in presence and absence of 0.5 mM responding hydrazines on blood glucose 2-(3-phenylpropoxyimino)hutyric acid. From concentration in fasted guinea pig 3 h after inHaeckel et al. (83). ® by Smith-Gordon. traperitoneal injection. Mean values of 5 experiments (controls from 32 animals). From Haeckel et al. (83). © by Smith-Gordon.

Figure 9—Influence of 2-(3-methylcinnamylhydrazono)propionate (MCHP; 0.04 mM) on coneogenesis from pyruvate. Studies glucose formation from pyruvate (a) and lactate with the rat hemidiaphragm demon(b) in perfused liversfromguinea pigs fasted 48 strated that methyl-2-TDGA inhibited h. MCHP was added 45 min (0) after substrates LCFAO in this tissue, with a concomitant (15 mM). From Haeckel et al. (83). « by Smith- increase in glucose oxidation to CO2 Gordon. (70). These observations lend consider-

cose oxidation due to disinhibition of pyruvate dehydrogenase due to decreases in AcCoA and NADH, which are feedback inhibitors (Fig. 2), or disinhibition of phosphofructokinase due to decreased citrate production from LCFAO (67,69). In vitro effects of LCAT I inhibitors have been demonstrated in liver, diaphragm, and cardiac muscle, and in vivo effects have been demonstrated in several species of nondiabetic, fasted, and diabetic animals. Experiments with isolated rat hepatocytes indicate that by inhibiting LCAT I, methyl-2-tetradecylglycidate (methyl-2-TDGA) causes a decrease in long-chain acylcarnitine formation (57). Inhibition of LCFAO in the hepatocytes was accompanied by a decrease in glu-

Lactate 1 mmol/1

able support to the concept of the interrelation of LCFAO and glucose metabolism discussed previously. As shown in Fig. 5, methyl-2TDGA lowered the plasma glucose and P-hydroxybutyrate levels in a diabetic dog without affecting either insulin output or FFA concentrations in plasma (71). Similar results have been obtained in alloxan-induced diabetic rats. Both acute (Table 2) and chronic studies (Fig. 6) with etomoxir in diabetic animals have shown its antidiabetic efficacy (67,68). The animals used in the studies in Fig. 6 were extremely insulinresistant diabetic mice. Studies with etomoxir-CoA in perfused rat livers showed it to be > 100 times as effective as the physiological regulator of LCAT I, malonyl-CoA (67,72). Organ-specific differences have been ascertained in LCAT I inhibition

and resynthesis after administration of these agents (67,73). The half-maximal inhibiting concentration for inhibition of LCAT 1 in rats given oral etomoxir is 100 times lower in liver than in heart or skeletal muscle (67). However, chronic administration of both 2-TDGA and etomoxir has produced some degree of cardiac hypertrophy in rats and mice (67,74). The effects of these agents on cardiac structure and function in animals is complex and is discussed in more detail elsewhere (67,75). A few clinical studies have been conducted with racemates of 2-TDGA and etomoxir. Although neither compound displayed significant toxicity, the efficacy was modest compared with the results achieved in animal studies. However, the dosages used in clinical studies were low compared with the 5 - 2 0 mg/kg used in animal studies. Doses of the LCAT I inhibitors ranged from 50 to 200 mg/day, with most patients receiving doses at the lower end of the range. Reported studies have been conducted in small numbers of patients. The efficacy of subacute administration of etomoxir was tested in extremely insulin-resistant diabetic mice.

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INHIBITORS OF ACYLCARNITINE TRANSLOCASE: HYDROZONOPROPIONIC ACIDS— The use of monoamine oxidase inhibitors (MAOIs) of the hydrazine type for the treatment of depressive I illness resulted in hypoglycemic episodes in both nondiabetic and diabetic subjects (77-80). Phenelzine was tried as therapy for diabetic patients and was successful in lowering blood glucose levels. However, the potent MAOI caused several 0.01 0.1 1 Figure 12—Formation of phenylacetyl-CoA severe adverse reactions, which discourPhenylacetyl CoA mM by isolated rat liver mitochondria. Rat liver miaged pursuit of this mode of therapy tochondria from 1.7 g original liver were incu(81). bated at ambient temperature for 10 min with 10Figure 13—Phenylacetyl-CoA inhibition of It was later found that hydrazine mM lactate and 1 mM pyruvate. Mitochondrial purified pyruvate carboxylase activity was linked and MAOIs of the hydrazine type were extracts were assayed by reverse-phase high- to malate dehydrogenase decrease in absorbance converted to hydrazones in the perfused performance liquid chromatography: 30 or 70 ofNADH at 340 nm during 15-min incubation. guinea pig liver (82). The hypoglycemic mM methanol/50 mM KH2PO4, pH 5.3; detec- Enzymes were incubated in buffer containing 50 action of perfused phenelzine was intion at 254 nm. Quantitation by standard addi- mM Tris-HCl (pH 7.75), 100 mM KCl, 5 mM creased by perfusion with pyruvate, alMgCl with 5.0 mM pyruvate, 0.03 mM acetyl2 tion method with phenylacetyl-CoA (Sigma, St. though the MAOI effect of phenelzine CoA, 2 mM ATP, 0.2 mM NADH, 50 mM Louis, MO) as reference. KHCOj, and concentrations of phenylacetyl-CoAwas reduced (82). The studies with shown. From Thampy et al. (99). & by Arch phenelzine demonstrating that hydraBiochem Biophys. zones formed in the course of its hepatic metabolism were the active hypoglyceThese animals were treated daily with 0, mic species led to the synthesis of a num10, or 50 mg/kg etomoxir from the age of ber of active hydrazones (83). The sev4 mo onward. The fasting blood glucose of the control mice rose over the course and not at 18 h. Studies in N1DDM pa- eral hydrazones formed from hydrazines of the experiment. Administration of 10 tients treated with increasing dosages of and pyruvate or synthesized chemically and 50 mg/kg etomoxir led to a 43 and etomoxir (20, 50, and 100 mg) every 7 are shown in Fig. 7. These compounds 55% decrease in fasting blood levels 2 h days effected a blood glucose fall (8.7 ± were ascertained to be potent blood after administration compared with the 0.4 to 6.8 ± 0.5 mM [157.3 ± 6.4 to glucose-lowering agents after oral administration to nondiabetic and diabetic control mice (72). 122.4 ± 8.5 mg/dl]). The fall in blood 2-TDGA (methylpalmoxirate) glucose was accompanied by an antike- animals (82,83). The search for new was administered to six IDDM patients tonemic and hypotriglyceridemic effect. compounds was directed toward candidates with little to no MAOI activity. for 11 days at a fixed dose of 50 mg/day. Euglycemic clamp studies on NIDDM Several promising compounds were proThis regimen was partly effective in depatients showed that 50 mg of etomoxir duced, including 2-(3-methylcinnamylcreasing ketonemia and fasting and postcaused a 33% increase of insulin-medi- hydrazono)propionate (MCHP) and prandial hyperglycemia in three of the ated glucose uptake, increasing mean 2-(3-phenylpropoxyimino)butyric acid patients, which allowed for a reduction clearance rate from 4.1 ± 0.9 to 5.4 ± (PPIB; Fig. 7). in insulin dose (76). 1.2 mg • kg" 1 • min" 1 . Several other Several clinical studies on the use 2- (Phenyle thylhydrazono) propistudies on NIDDM patients treated for of etomoxir in nondiabetic subjects and onate (PEHP) and MCHP were potent diabetic patients have been conducted several days to 2 wk with dosages of hypoglycemic agents in fasted guinea and reported in abstract form (67). Sin- etomoxir ranging from 25 to 100 mg/day pigs, whereas MAOI activity was minigle or multiple dosage administration of showed decreases in fasting blood glu- mal. PEHP reduced the glucosuria of etomoxir to nondiabetic fasting subjects cose, plasma triglycerides, cholesterol, STZ-D rats (82,83). demonstrated the therapy to be anti- and blood ketones. Studies on the hypoglycemic acketonemic and to lower plasma triglycFurther large-scale clinical stud- tivity of the hydrazonopropionic acid deerides. However, a blood glucose fall ies are awaited on the efficacy and toxic- rivatives revealed a biochemical template (40%) was only seen at 36 h of fasting ity of these LCAT I-inhibiting drugs. akin to that of the inhibitors of LCAT I Mitochondria . subttrate

1

MBochondrta • 1.0 mM 0 CH 2 CO 8 H • substrate

0 CH 2 COSCoA 13.6 mln, 0.07 mM

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Table 3—Effect

of to-phenylalkanoic acids on glucose production SUBSTRATES (10 MM)

P H E N Y L - ( C H 2 ) N - C O O H (4 M M )

PYRUVATE

L-LACTATE

L-ALANINE

GLYCEROL

D-Fructose

CONTROL

(0.83) -22 ± 2 -42 ± 3 -60 ± 3 -94 ± 4

(0.91) -15 ± 1 -30 ± 2 -63 ± 3 -81 ± 3

(0.37)

-30 -47 -63 -93

(1.20) -25 ± 2 -23 ± 2 -28 ± 2 -24 ± 2

(190) -8± 2 -23 ± 2 -23 ± 2 -21 ± 2

1 N = 2 N= 3 N = 4 N=

±2 ±3 ±3 ±3

Relative inhibitions of gluconeogenesis in perfused 48-h-starved rat livers from various substrates by series of co-phenyl-substituted fatty acids. Values arc means ± SE from 3 to 5 livers. Control rates (in n,mol glucose • min~' • g~ l liver [wet wt]) are in parentheses; all other values show percentage of change of these

(83-85). In addition, these compounds were shown to inhibit glucose uptake from the jejunum of rats and guinea pigs (86). The studies on MCHP have shown that its in vitro and in vivo effects are similar to the LCAT I inhibitors with specific regard to 1) decreased LCFAO but not medium-chain fatty acid oxidation (83,87; Fig. 8), 2) MCHP effected a decrease in hepatic gluconeogenesis associated with a fall in hepatic AcCoA and a subsequent decreased activity of PC (83; Fig. 9), 3) a fall in blood glucose (83; Fig. 10), and 4) stimulation of peripheral glucose utilization (83; Fig. 11). These features of drug action made it likely that the locus of action was akin to that of the LCAT I inhibitors.

itory of palmitate oxidation than the LCAT I inhibitors (85). Both MCHP and PPIB have passed toxicological tests in animals, and PPIB is in early clinical trials (83).

Inhibition of hepatic gluconeogenesis is the primary site of action of the hydrazonopropionates (84). Several studies have found that the hydrazonopropionates inhibit the LCAT (88,89). This site of inhibition could account for all of the experimental data on these agents. The low degree of toxicity of the hydrazonopropionates and PPIB makes them promising compounds for clinical testing. These compounds are less inhib-

INHIBITION OF PC— PC is a biotincontaining enzyme that catalyzes the synthesis of oxaloacetate from pyruvate in the presence of ATP and bicarbonate:

12000 1000-

1000080006000-

500-

Table 4—O 2 consumption by perfused liver SUBSTRATES (10 MM) EFFECTORS CONTROL + 4 MM OCTANOIC

PYRUVATE

L-LACTATE

3.1 ±0.1 4.2 ± 0.2

3.4 ±0.1 4.5 ± 0.2

2.9 ±0.1

3.3 ±0.1

ACID 4-4 MM PHENYLBUTYRIC ACID

O 2 consumption of 48-h-starved perfused rat livers as influenced by representative of straight-chain even fatty acids and co-phenyl-substituted fatty acids. Values (in fimol O 2 • min"' • g~' liver [wet wt]) are means ± SE from 5 to 10 livers.

800

6

§ z

Control

Phanylproptontc AcW2mM

Control

Figure 14—Production of ^CO^ by 2.5 x 10b isolated hepatocytes from L-l'4C]palmitate in presence and absence of 2 mM phenylpropionic acid. Hepatocytes were incubated at 37°C in phosphate buffer (pH 7 A) with 5 mM glucose and 0.15 mM l'4C]palmitate bound to 0.075 mM bovine serum albumin and '4CO2 collected for 30 min. Data from Brendel and Meezan (100). ® by Annal Biochem.

Phonyt* propionic Add2mM

F i g u r e 1 5 — P r o d u c t i o n of I4CO2 by 2.5 X JO'1 isolated hepatocytes from [U-'4C]glucose in presence and absence of 2 mM phenylpropionic acid. Hepatocytes were incubated at 37°C in phosphate buffer (pH 7A) with 5 mM lHC]glucose and 0.15 mM palmitate hound to 0.075 mM bovine serum albumin and HCO2 collected for 30 min. Data from Brendel and Meezan (100). m by Annal Biochem.

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of PC and stabilizes the enzyme in aqueous solutions (92). Under conditions where fatty acid oxidation is significant, such as fasting or diabetes mellitus, AcCoA concentrations increase, and PC is 0.5activated. This stimulates gluconeogenesis. Interest in pharmacological con30 45 trol of PC over the years has not been rewarded by a clinically useful PC inhibGroup II (n - 4) itor. The classes of inhibitors studied include 1) nucleotides that are competitive inhibitors of ATP, 2) acyl-CoA derivatives that might be competitive with AcCoA and prevent allosteric activation of PC, and 3) pyruvate analogues and dicarboxylic acids that inhibit the transcarboxylation step and decrease enzyme 15 30 45 Time (days) activity by interaction with bound magnesium (66). These studies have not proFigure 16—Urinary glucose in dh/dh (diabetgressed to any in vivo work in gluconeic) mice with and without phenylacetic acid. ogenesis inhibition. However, several Crossover study of dh/db diabetic mice on phe- acyl-CoA derivatives, including butyrylnylacetic acid. CoA, isovaleryl-CoA, isobutyryl-CoA and propionyl-CoA, have been shown to inhibit gluconeogenesis or PC in rat and pig livers (93,94). Inhibition of PC in enzyme-biotin + ATP + HC0 3 — vitro by means of CoA-sequestering aroenzyme-biotin - CO2~ + A DP + P h matic acids has been reported (95,96). In enzyme-biotin — CO2~ + pyruvate — a study utilizing benzoic acid as a CoAsequestering agent, it was concluded that enzyme-biotin + oxaloacetate, competition between AcCoA and benATP + HCO 3 " + pyruvate — zoyl-CoA for the activator site on PC was oxaloacetate + A DP + Pj. insignificant and that depletion of CoA, and subsequent failure to form adequate The vertebrate PC is a tetramer of amounts of AcCoA, was responsible for four identical subunits (90,91). Each the inhibition (96). The sensitive role of subunit is a multifunctional protein con- PC in regulating both the citric acid cycle taining the two catalytic activities shown and gluconeogenetic activity in the liver in the above equations: the biotin car- makes for possible toxicity in attempting boxylase and the transcarboxylase. Each PC inhibition. subunit contains a site for biotin binding In 1973, studies from our laboand a center for allosteric regulation by AcCoA (90,91). PC is a mitochondrial ratory (97) demonstrated that a series of enzyme that plays an important meta- phenylalkanoic acids were gluconeogenbolic role in the regulation of oxaloace- esis inhibitors in the isolated perfused rat tate necessary for the function of the cit- liver (97). The inhibition of glucose forric acid cycle and as a substrate for mation from a variety of precursors was glucose production. Enzyme activity of found (Table 3). There was no decresignificance is found in the liver and kid- ment in O2 consumption by the livers ney, which are gluconeogenetic sites. Ac- perfused with these agents (Table 4; 97). CoA is required for allosteric activation These compounds have been studied in Qroup I (n - 4)

•

Not Treated

O

Treated, 2.0/L drinking water

I

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regard to both their mechanism of action and their in vivo activity. It was clear that the phenylalkanoyl-CoAs were capable of directly inhibiting PC, but the amounts of compound needed to inhibit gluconeogenesis were such that they could act to sequester CoA and inhibit LCFAO and the citric acid cycle. Studies were carried out on the utilization of glucose and palmitate by phenylalkanoic acid-treated liver cells. Figures 14 and 15 (100) show that phenylpropionic acid did not inhibit the oxidation of either palmitate or glucose by rat liver cells. These studies support the intact nature of LCFAO, glycolysis, and the citric acid cycle with use of the phenylalkanoic acid in liver cells at concentrations that inhibit gluconeogenesis. They show that the degree of sequestration of CoA effected by the formation of the phenylalkanoyl-CoA derivatives was not sufficient to interfere with cell metabolism. It further suggests that the inhibition of PC by the phenylalkanoylCoAs plays a significant role in the inhibition of gluconeogenesis in vitro. Preliminary in vivo studies have also shown blood glucose-lowering activity with these compounds. Studies of urine glucose output in dh/db diabetic mice treated with phenylacetic acid (2 g/L in drinking water) resulted in a fall in glucose output. These results are encouraging, because the amount of phenylacetic acid used was very low. These data are shown in Fig. 16. Further animal studies are in progress. The action and potential toxicity may be localized to the liver, because it is the primary site of formation of acyl-CoA esters of these compounds. Phenylalkanoic acids are not activated to acylCoAs in cardiac or skeletal muscle, which lack the necessary activating enzymes. References 1. DeFronzo RA: The triumvirate: (3-cell, muscle, liver. A collusion responsible for N1DDM. Diabetes 37:667-87, 1988

801

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2. Reaven GM: Role of insulin resistance in human disease. Diabetes 37:1595607, 1988 3. RosenstockJ, Raskin P: Diabetes and its complications: blood glucose control vs. genetic susceptibility. Diabetes Metab Rev 4:417-35, 1988 4. Unger RH, Orci L: The essential role of glucagon in the pathogenesis of diabetes mellitus. Lancet 1:14-16, 1975 5. Unger RH: Role of glucagon in the pathogenesis of diabetes: the status of the controversy. Metabolism 27:1691709, 1978 6. Johnson MEM, Das NM, Butcher FR, Fain JN: The regulation of gluconeogenesis in isolated rat liver cells by glucagon, insulin, dibutyryl cyclic adenosine monophosphate, and fatty acids. J Biol Chem 247:3229-35, 1972 7. Khan BA, Bregman MD, Nugent CA, Hruby VJ, Brendel K: (Des-histidine) (N- e - phenylthiocarbamoyllysine' 2 )glucagon: effects on glycogenolysis in perfused rat liver. Biochem Biophys Res Commun 93:729-36, 1980 8. Johnson DG, Goebel CV, Hruby VJ, Bregman MD, Trivedi D: Decrease in hyperglycemia of diabetic rats by a glucagon receptor antagonist. Science 215: 1115-16, 1981 9. Unson CG, Gurzenda EM, Merrifield RB: Biological activities of des His [Glug] glucagon amide, a glucagon antagonist. Peptides 10:1171-77, 1989 10. Wakelam MJO, Murphy GJ, Hruby VJ, Houslay MD: Activation of two signaltransduction systems in hepatocytes by glucagon. Nature (Lond) 323:68-71, 1986 11. Houslay MD, Anderson N, Murphy GJ, Pyne NJ, Wakelam MJO, Wilson A: Glucagon desensitization identifies "cross-talk" between two distinct receptor-signaling pathways. In Current Com-

Glucagon amino groups: evaluation of modifications leading to antagonism and agonism. J Biol Chem 255:1172531, 1980 14. Unson CG, Gurzenda EM, lwasa K, Merrifield RB: Glucagon antagonists: contribution to binding and activity of the amino-terminal sequence 1-5, position 12, and the putative alpha-helical segment 19-27. J Biol Chem 264:78994, 1989

15. Unson CG, MacDonald D, Ray K, Durrah TL, Merrifield RB: Position 9 replacement analogs of glucagon uncouple biological activity and receptor binding. J Biol Chem 266:2763-66, 1991 16. Sakurai H, Dobbs R, Unger RH: Somatostatin-induced changes in insulin and glucagon secretion in normal and diabetic dogs. J Clin Invest 54:1395-1402, 1974 17. Gerich JE, Lorenzi M, Schneider V, Karam JH, Rivier J, Guillemn R, Forsham PH: Effects of somatostatin on plasma glucose and glucagon levels in human diabetes mellitus. New Engl J Med 291:544-47, 1974 18. Kofod H, Unson CG, Merrifield RB: Potentiation of glucose-induced insulin release in islets by des His [Glug] glucagon amide. Int J Pept Protein Res 32: 436-43, 1988 19. Schmidt DD, Frommer W, Junge B, Muller L, Wingender W, Truischeit E: a-Glucosidase inhibitors. Naturwissenschaften 64:535-36, 1977 20. Puls W, Keup V, Krause HP, Thomas G, Hoffmeister F: Glucosidase inhibition. Naturwissenschaften 64:536-37, 1977 21. Vierhapper H, Bratusch-Marrain A, Waldhause W: a-Glucoside hydrolase inhibition in diabetes. Lancet 2:1386, 1978 22. Walton RJ, Sherif IT, Noy GA, Alberti KGMM: Improved metabolic profiles in munications in Molecular Biology. Cold insulin-treated diabetic patients given Spring Harbor, NY, Cold Spring Haran alpha-glucosidehydrolase inhibitor. bor, 1987, p. 99-105 BrMedJ 1:220-21, 1979 12. Krstenansky JL, Zechel C, Trivedi D, 23. Johnson DG, Bressler R: Short-term efHruby VJ: Importance of the C-termi ficacy of acarbose in obese patients with alpha-helical structure for glucagon's non-insulin-dependent diabetes mellibiological activity. Int J Pept Protein Res tus. In Acarbose. Effects on Carbohydrate 32:468-75, 1988 and Fat Metabolism. Creutzfeldt W, Ed. 13. Bregman MD, Trivedi D, Hruby VJ:

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W, Ed. Amsterdam, Excerpta Med., 1982, p. 86-96 Lembke B, Foelsch VR, Creutzfeldt W: Effect of 1-desoxynojirimycin derivatives on small intestinal disaccharidase activities and on active transport in vitro. Digeston 31:120-27, 1985 Dimitriadis G, Hatziagelaki E, Ladas S, Linos A, Hillebrand 1, Raptis S: Effects of prolonged administration of two new a-glucosidase inhibitors on blood glucose control, insulin requirements and breath hydrogen excretion in patients with insulin dependent diabetes mellitus. EurJ Clin Invest 18:33-38, 1988 Katsilambros N, Philippides P, Toskas A, ProtopapasJ, Frangaki D, Marangos M, Siskoudis K, Anastasopulou K, Xefteri H, Hillebrand 1: A double-blind study on the efficacy and tolerance of a new alpha-glucosidase inhibitor in type 2 diabetics. Arzneim Forsch 36:113638, 1986 Yoshikuni Y, Ezure Y, Aoyagi Y, Enomoto H: Inhibition of intestinal aglucosidase and postprandial hyperglycaemia by N-substituted moranoline derivatives. J Pharmacobio-Dyn 11: 356-62, 1988 Yoshikuni Y, Ezure Y, Seto T, Mori K, Watanabe M, Enomoto H: Synthesis and a-glucosidase-inhibiting activity of a new alpha-glucosidase inhibitor, 4-O-a-D-glucopyranosylmoranoline and its N-substituted derivatives. Chem Pharm Bull 37:106-109, 1989 Horii S, Fukase H, Matsuo T, Kameda Y, Asano N, Matsui K: Synthesis and a-D-glucosidase inhibitory activity of N-substituted valiolamine derivatives as potential oral antidiabetic agents. J Med Chem 29:1038-46, 1986

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32. Danzin C, Ehrhard A: Time-dependent inhibition of sucrase and isomaltase from rat small intestine by castanospermine. Arch Biochem Biophys 257:47275, 1987 33. Tyms AS, Berne EM, Ryder TA, Nash RJ, Hegarty MP, Taylor DL, Mobberley MA, DavisJM, Bell EA, Jeffries DJ, Taylor-Robinson D, Fellows LE: Castanospermine and other plant alkaloid inhibitors of glucosidase activity block the growth of HIV. lancet 2:1025-26, 1987 34. Rhinehart BL, Robinson KM, Liu PS, Payne AJ, Wheatley ME, Wagner SR: Inhibition of intestinal disaccharidases and suppression of blood glucose by a new a-glucohydrolase inhibitor-MDL 25,637. J Pharmacol Exp Ther 241:91520, 1987 35. Sohda T, Mizuno K, Imamiya E, Sugiyama Y, Fujita T, Kawamatsu Y: Studies on antidiabetic agents. 11. Synthesis of 5-(4-(l-methylcyclohexylmethoxy)benzyl)thiazolidine-2,4-dione and its derivatives. Chem Pharm Bull 30:35803600, 1982 36. Steiner KE, Lien EL: Hypoglycemic agents which do not release insulin. Prog Med Chem 24:209-48, 1987 37. Colca JR, Morton DR: Antihyperglycemic thiazolidinediones. In Antidiabetic Drugs. Bailey CJ, Flatt PR, Eds. London, Smith-Gordon, 1990, p. 255-61 38. Chang AY, Wyse BM, Gilchrist BJ, Peterson T, Diani AR: Ciglitazone, a new hypoglycemic agent. 1. Studies in oh/oh and db/db mice, diabetic Chinese hamsters, and normal and streptozotocin-diabetic rats. Diabetes 32:830-38, 1983 39. Fujita T, Sugiyama Y, Taketomi S, Sohda T, Kawamatsu Y, lwatsuka H, Suzuoki Z: Reduction of insulin-resistance in obese and/or diabetic animals by 5-[4-(l-methylcyclohexylmethoxy)benzyl]thiazolidine-2,4-dione (ADD3878, U-63287, Ciglitazone), a new antidiabetic agent. Diabetes 32:804-10, 1983 40. Kraegen EW, James DE, Jenkins DJ, Chisholm DJ, Storlien LH: A potent in vivo effect of ciglitazone on muscle insulin-resistance induced by high fat

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