Cliapter 1

Current Scenario of (})run (})evefopment for the treatment strateoies of (})ia6etes

Chapter 1. Current Scenario of Drug Development

for the treatment of Diabetes 1.1 Introduction

Diabetes mellitus is a major and growing public health problem throughout the world,

with an estimated worldwide prevalence in 2000 of 150 million people, expected to increase

to 220 million people by 2010. 1•2 Pronounced changes in human behaviour and lifestyle over

the last century have resulted in a dramatic increase in the incidence of diabetes worldwide.

Presently, diabetes is the 41h leading cause of death in developed countries and its

management exerts a vast economic and social burden. 3.4

Many people also have other abnormalities of glucose (sometimes called

"prediabetes") manifest either as impaired fasting glucose (IFG) levels or as impaired

glucose tolerance (IGT). The criteria for diagnosis of diabetes and prediabetes are

summarized in Table 1.5 Collectively, diabetes, IFG, and IGT have been dubbed

"dysglycemia".6 The combination of dysglycemia, obesity, dyslipidemia, and blood pressure

elevation is known as the "metabolic syndrome" or the "dysmetabolic syndrome" or

"diabesity". A number of metabolic and clinical abnormalities are included in the

dysmetabolic syndrome, as summarized in Table 2.

Table 1. Diagnostic Criteria for Diabetes Mellitus and prediabetes

normal

Prediabetes a

fasting plasma glucose mgldL (mmol/L) <100 (5.6)

impaired fasting glucose 100-125 (5.6-6.9)

2 h c

plasma glucose mgldL (mmol/L) < 140(7.8)

impaired glucose tolerance 140-199 (7.8-11.0)

diabetes mellitus ~126 (7.0) or ~200 (11.1)

0 Diabetes also may be diagnosed with plasma glucose greater than 200 mg/dL (11.1 mmol/L) and unequivocal symptoms (polyuria, polydipsia, unexplained weight loss). Diagnostic criteria for diabetes mellitus includes confirmation on a subsequent day. ~asting = no caloric intake for at least 8 h. c2 h following a 75g oral glucose load, i.e., oral glucose tolerance test (OGTT).

Table 2. Components of the Dysmetabolic syndrome

• insulin resistances hyperinsulinemia relative to glucose levels

1

Chapter 1

• acanthosis nigricans

• central obesity

• glucose intolerance or type 2 diabetes

• blood pressure elevation or hypertension

• dyslipidemiashypertriglyceridemia and decreased HDL cholesterol, small, dense LDL cholesterol

particles

• increased plasma uric acid

• Hypercoagulability-increased plasminogen activator inhibitor

• vascular endothelial dysfunction

• coronary artery disease

Diabetes mellitus is a group of metabolic disorders, characterized by hyperglycemia

arising as a consequence of a relative or severe deficiency of insulin secretion, or its

resistance or both along with the metabolic derangements of carbohydrates, fats and

proteins.7'8 Diabetes mellitus is commonly classified into two broad types:

· 1. Insulin Dependent Diabetes Mellitus (IDDM) or type 1 diabetes characterized by severe

deficiency of insulin production, usually due to autoimmune destruction of [3-cells of

pancreas.9

2. Non-Insulin Dependent Diabetes Mellitus (NIDDM) or type 2 diabetes, the more common

type, is usually due to resistance to insulin action in the setting of inadequate .

1. 1 s compensatory msu m secretory response. '

Type 2 Diabetes Mellitus {T2DM) is the most important type of diabetes because

more than 90% of diabetics are of this type. Further, 80 percent of T2DM patients are obese

and most of them hyperinsulinaemic and insulin-resistant.10 TIDM is the result of impaired

insulin stimulated glucose uptake and utilization in liver, skeletal muscle, adipose tissue and

impaired suppression ofhepatic glucose output.

5%

o Type 2 Diabetes

• Other types of diabetes

95%

Figure 1. Type 2 diabetes accounts for up to 95% of all cases of diabetes.

Insulin resistance is very common because of obesity, a sedentary life style and aging

(Figure 2) resulting in high blood pressure, dyslipidemia and diabetes. Type 2 diabetes does

not always occur with insulin resistance but defect in insulin secretion is associated with

2

microvascular complications viz. blindness, neuropathy and nephropathy11•12 and

macrovascular complications leading to atherosclerosis, limb amputation. 13 In this situation

presence of C-peptide and absence of markers of autoimmunity such as antibodies to

glutamic acid decarboxylase may help to diagnose T20.14 In view of rapid increase in

diabetes cases WHO and American Diabetes Association have reduced the figure of the

blood glucose level from 140mg/dl to 126mg/dl for the risk of diabetes. 15•16 Estimation of

glycosylated haemoglobin (HbA1c), which is a marker of average plasma glucose

concentration, is still valid for the diagnosis ofT2D. 17•18

Figure 2. Causes and consequences of insulin resistance.

Hyperglycemia occurs in the basal or fasting state because of increased hepatic

glucose production as a consequence of abnormality in insulin secretion and hepatic insulin

resistance. In contrast, hyperglycemia in prandial state arises due to increased absorption of

glucose from the gastrointestinal track and its reduced disposal because of deficient insulin

secretion to compensate insulin resistance Figure 3.

PANCREAS

MUSCLE

1NTESTIN : GLUCOSE ABSORPTION

NERVOUS SYSTEM FAT

Figure 3. Regulation of blood glucose (J. Med. Chern. 2004, 47, 41 13-41 I 7).

Current therapies for the management of T2D include suitably balanced diet, exercise,

and variety of pharmacological agents including insulin, sulfonylureas, biguanides and

3

thiazolidinediones (TZDs). These agents act by different mechanisms to normalize blood

glucose levels and avoid serious complications that affect the kidney, cardiovascular,

ophthalmic and nervous systems. 19 There are mainly five categories of orally active

antidiabetic drugs on the market: sulfonylureas (SU) and non-sulfonylurea (non-SU) insulin

secretagogues that stimulate insulin secretion by pancreatic cells; with increasing severity of

diabetes, insulin administration is prescribed, metformin, a biguanide for type 2 diabetes,

thiazolidinedione class of Peroxisome Proliferator-Activated Receptor Gamma (PP AR-y)

activators such as pioglitazone and rosiglitazone and the a-glycosidase inhibitors that delay

intestinal carbohydrate absorption and blunt postprandial glucose excursions. In 1999, the

discovery was announced that a PTP 1 B knockout mouse displays enhanced insulin

sensitivity,20 and resistant to weight gain.21 These findings generated a vivid interest in

PTP1B and other PTPs22•23 as drug targets for diabetes and, possibly, obesity. Our objective

in this review is to project the current scenario of treatment strategies for T2DM.

1.2 Insulin or Insulin Secretagogues

Oral agents that increase pancreatic insulin secretion are called secretagogues. This

class of therapeutics can be divided into sulfonylureas and non-sulfonylureas.

1.2.1 Insulin

Insulin is a peptide hormone secreted from ~-cells of pancreas, which mediates glucose

transport from blood to the cells (figure 4). For the past several years, insulin is being also

used for the treatment of T2D. 19 It regulates the blood glucose primarily by stimulating

translocation of glucose transporter GLUT 4 from intracellular sites to the membrane. It also

controls the blood sugar level by inhibiting gluconeogenesis, glycogenolysis24 and

metabolism of free fatty acid.25 Insulin acts through signal transduction mechanism by

interacting with insulin receptor present on the cell membrane of all cell types mostly liver

and fats. Insulin receptor is tetrameric glycoprotein consisting of 2a and 2~ subunits, linked

by disulfide bond and spread across the membrane. The a- and ~-subunits function as

allosteric enzymes in which a-subunit inhibits the tyrosine kinase activity of ~-subunit.

Binding of insulin to the binding site present in a-subunit induces aggregation and

internalization of receptors along with the bound insulin molecule leading to the stimulation

of kinase activity in ~-subunit followed by transphosphorylation and a conformational

change of receptor that further increases kinase activity of ~-subunit26 which ultimately leads

to the stimulation of insulin metabolizing enzymes. Insulin action is also mediated by certain

second messengers like phosphatidylinositol glycan (PIG) and diacylglycerol (DAG) formed

by specific phospholipase C.

4

Chapter 1

Very recently, a new chemical substance, hepatic insulin sensttlzmg substance

(HISS) has been discovered which is yet to be identified chemically.27 HISS is secreted from

liver in response to the injection of insulin and brings about 50-60% of glucose disposal in a

dose dependent manner of insulin for a wide range ( 5-l 00 mL/Kg).

Insulin is administered by subcutaneous injection, which is not always convenient

mode of drug administration. Moreover, exogenous insulin does not replicate the normal

pattern of the nutrient related basal insulin secretion. The difference in the action of injected

and endogenously secreted insulin is due to difference in their pharmacokinetic

pathways.28.29 These shortcomings have been minimized by developing rapid acting insulin

analogs, insulin lyspro and insulin aspartate.30 Long acting insulin analogs, insulin glargine

and insulin detemir31•32 are available for the treatment of type 2 diabetes. Another analog of

insulin, glulisine, is currently in advance clinical trials. 33•34

1.2.2 Sulfonylureas as Oral Antidiabetic Agents

Sulfonylurea receptor represents one of the most important classes of KA TP

channels, found in ~-cells of pancreas. Sulfonylurea insulin secretagogues act through these

receptors, which exert their actions in response to the cytosolic concentration of A TP. The

increase in A TP concentration on glucose metabolism in normal conditions closes the KA TP

channels leading to membrane depolarization of the ~-cells of pancreas. The depolarized

membrane opens intracellular calcium ion channel, resulting Ca2+ entry into the cell. The

increase in intracellular calcium ion concentration triggers insulin exocytosis.

1.2.2.1 Sulfonylureas as Insulin Secretagogues

Sulfonylurea insulin secretagogues, being the first line of oral antidiabetic agents,

have been studied extensively. Carbutamide, the first sulfonylurea was initially discovered

serendipitously, followed by other members of this series. These compounds have been

classified as first and second-generation of sulfonylureas. The important first generation

sulfonylureas are shown in Table 1. Refractory failures, hypoglycemia and weight gain are

the major side effects of these agents. 35

1.2.3 Non-sulfonylureas as Insulin Secretagogues

These compounds have rapid onset but short duration of action that makes them

suitable agents for the treatment of hyperglycemia as well as hyperinsulinemia. Initially,

meglitinide the first member of this series, an analog of glibenclamide was studied for its

antidiabetic and insulinotropic activity more than two decades ago. 36 Structural

5

Chaptu 1

manipulations of this compound led to the development of other members. 37 Repaglinide

(prandin), nateglinide, mitiglinide and an interesting morpholinoguanidine derivative BTS

67582 are new insulinotropic agents. Non-sulfonylurea compounds are superior drugs with

respect to first and second generation of sulfonylureas because they do not cause

hypoglycemia and weight gain. Repaglinide is more active compared to nateglinide.38

Table 1: First generation of sulfonylureas.

S.No N arne of the drug R• R2

Carbutamide NH2 C4H9

2 Tolbutamide CH3 C4H9

3 Chlorpropamide Cl C3H1

4 Tolazamide CH3 Azepan-1-yl

5 Acetohexamide H3CCO Cyclohexyl

Cl

j--uN~S!)2 0

"'==< ~ ~HN~-o OCH3 HN

Glyburide

HC-o-~ SO 0 3 ' 2 // - HN\

HN-NJ)

Glidazide

Figure 4: Second generation of sulfonylureas

c1th __rQ-cooH 0~~ - HN H I ~o ~O)()&N ~

0 I 0 CH OCH3 HOOC

0 3

Meglitinide

Q )····Q-coNH+COOH

H

Nateglinide

Figure 5. Non-sulfonylureas secretagogues.

Repaglinide CH3

Mitiglinide

BTS67 582

6

1.3 Glucose Mediated Insulin Secretion Enhancers

1.3.1 Glucagon like peptides and Dipeptidylpeptidase-IV

The two major adverse side effects of weight gain and loss of ~-cell function

associated with current regime of drugs have shifted the focus on glucagon like peptide

hormones as new potential drugs for the treatment of type 2 diabetes. 39

It has long been known that oral administration of glucose results in increased insulin

secretion relative to intravenous glucose administration. The increased insulin secretion in

response to oral nutrients is known as the "incretin" effect. In recent years, the incretin

hormone glucagon-like peptide 1 (GLP-1) has been the subject of intense research efforts

related to the treatment of type 2 diabetes.40'41 GLP-1 is a 30 amino acid hormone produced

by L-cells of the intestinal mucosa that results from tissue-specific processing of the

proglucagon gene.42 This is the same gene that is present in a cell of the endocrine pancreas.

Posttranslational processing of the proglucagon gene transcript in a cell produces the 29

amino acid hormone glucagon as the primary bioactive product, along with other

proglucagonderived fragments. The primary products of the L-cell, GLP-1 and GLP-2 are

-50% homologous to glucagon. GLP-2 regulates gastric acid secretion and intestinal

motility, possesses intestinal trophic activity.

GLP-1 and GLP-2 induce insulin secretion to transport glucose across plasma

membrane in response to food intake. The other major physiological functions of GLPs are

inhibition of glucagon secretion, gastric emptying,43 and reduction of appetite.44 The

insulinotropic function of GLP-1 and GLP-2 are mediated by glucagon like peptide-1 and

peptide-2 receptors. The structures of these receptors are quite similar with each other and

possess significant homology with other endocrine hormone glucagon, and glucose

dependent insulinotropic polypeptide (GIP) receptors. These are G-protein linked receptors

localized mainly in ~-cells. GLP-1 and GLP-2 activate second messenger cAMP to induce

their insulinotropic activity.

It is also predicted that GLP-2 mediates its biological activity via activating Ca2+

inositol triphosphate pathways45 The GLP therapy is preferred over sulfonylurea because

they affect insulinotropic properties only in presence of glucose and do not cause

hypoglycemia, which is generally associated with sulfonylurea. The basic limitation of this

therapy is the low bioavailability of these peptides due to short half-life as they are rapidly

degraded by enzyme dipeptidylpeptidase IV (DPP IV), a serine protease which cleaves a

dipeptide from the N-terminus to give the inactive GLP-1 [9-36] amide.46 These limitations

7

have been minimized either by synthesizing inhibitors for dipeptidylpeptidase enzyme47 or

by developing analogs ofGLPs which are resistant to DPP-IV.

1.3.2 DPP-IV Characteristics & Mechanism:

X-ray structures of DPP-N that have been published since 2003 give rather detailed

information about the structural characteristics of the binding site. Many structurally diverse

DPP-N inhibitors have been discovered considering the properties of the binding site: 48

1. A deep lipophilic pocket combined with several exposed aromatic side chains for

achieving high affinity small molecule binding.

2. A significant solvent access which makes it possible to tune the physico-chemical

properties of the inhibitors that leads to better pharmacokinetic behavior.

DPP-N is a 766 amino acid transmembrane glycoprotein which belongs to the

prolyloligopeptidase family. It consists of three parts; a cytoplasmic tail, a transmembrane

region and an extracellular part. The extracellular part is divided into a catalytic domain and

an eight-bladed P-propeller domain. The latter contributes to the inhibitor binding site. The

catalytic domain shows an a/(3-hydrolase fold and contains the catalytic triad Ser630 -

Asp708- His740. The Sl-pocket is very hydrophobic and is composed of the side chains:

Tyr631, Va1656, Trp662, Tyr666 and Val711 (figure 6a). Existing X-ray structures show that

there is not much difference in size and shape of the pocket that indicates that the S 1-pocket

has high specificity for proline residues.49

Li!and

NH2 ~ S1- pocket

S2-podurt(~P1 Figure 6a.

tm=('*"' 'Vo./0 A--····/···~~1:-·~T-

CliiBS 'I ~ \ '·.,_ Slt511

- .\ ·•······•... 0

I .... ··· ..... -\_,_

.. J-~ -.us I Ligand and DPP-4 binding site

Figure 6b.

Figure 6: (a) A generic structure of a substrate like inhibitors. (b) The key interactions between the ligand and DPP-N complex. The ligand's basic amine forms a hydrogen bonding network. The nitrile reacts with the catalytic active serine and forms an imidate adduct.

8

1.3.3 Distribution and Mechanism:

During a meal the incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent

gastric inhibitory polypeptide (GIP) are released from the small intestine into the

vasculature. The hormones regulate insulin secretion in a glucose-dependent manner. GLP-1

has many roles in the human body; it stimulates insulin biosynthesis, inhibits glucagon

secretion, slows gastric emptying, reduces appetite and stimulates regeneration of islet ~­

cells. GIP and GLP-1 have extremely short plasma half-lives due to a very rapid inactivation.

The enzyme responsible for the metabolism is DPP-IV. Inhibition of DPP-IV leads to

potentiation of endogenous GIP and GLP-1 and hence improves treatment of type 2 diabetes

(Figure 7a).50

a) Stimulate insulin release

b) lnhib~ glucagon release

l

1 DDP-IV

123

c

30

Active

c:::J llncretin hormone 0 12 3 30

Inactive

Figure 7. (a) DPP-IV inhibitors inhibit DPP-IV and thus prolong the duration of GLP-1 and GIP activity, resulting in lower blood glucose level. (b) DPP-N cleaves two amino acids from the N-terminal end of peptides.

DPP-IV selectively cleaves two amino acids from peptides, such as GLP-1 and GIP

which have proline or alanine in the second position (Figure 7b). At the active site of the

protease, there is a characteristic motif of three amino acids, Asp-His-Ser. DPP-IV is the

CD26 T -cell activating antigen which is widely distributed in human organs and tissue.

Tissues which strongly express DPP-IV include the exocrine pancreas, sweat glands, salivary

and mammary glands, thymus, lymph nodes, biliary tract, kidney, liver, placenta, uterus,

prostate, -skin and the capillary bed of the gut mucosa where most GLP-1 is inactivated

locally. DPP-IV is attached to the plasma membrane of the endothelia of almost all organs in

the body. It is also present in body fluids, such as blood plasma and cerebrospinal fluid, in a

soluble form. DPP-IV inactivates GLP-1 and GIP very rapidly. Regarding GIP and GLP-1,

alanine and proline are crucial for biological activity, so elimination of these amino acids

leads to formation of metabolites that are inactive. Thus, preventing the degradation of the

incretin hormones GIP and GLP-1 by inhibition ofDPP-4 is an exciting therapeutic strategy.

9

1.3.4 Discovery and Development of DPP-IV inhibitors:

It is important to find a fast and accurate system to discover new DPP-IV inhibitors

with ideal therapeutic profiles. Three-dimensional models can provide a useful tool for

designing novel DPP-IV inhibitors. Pharmacophore models have been made based on key

chemical features of compounds with DPP-IV inhibitory activity. The first DPP-IV

inhibitors were reversible inhibitors and came with adverse side effects because of low

selectivity. Researchers suspected that inhibitors with short half-lives would be preferred in

order to minimize possible side effects. However, since clinical trials showed the opposite,

the latest DPP-IV inhibitors have a long-lasting effect. One of the first reported DPP-IV

inhibitor was P32/98 from Merck. It used thiazolidide as the PI-substitute (figure 6a) and

was the first DPP-IV inhibitor that showed effects in both animals and humans but it was not

marketed due to side effects. Another old inhibitor is DPP-728 from Novartis, where 2-

cyanopyrrolidine is used as the PI-substitute. The addition of the cyano group generally

increases the potency.

:~ff:,~ HN

N~ f

1

~)r--NH

NC-:.

}(J

O NC

Saxagliptin NC~ NVP-DPP728

. (IC50 = 22 nM)

Vildagliptin (IC50 = 3.5 nM)

H,£q 0 CN

Cyclohexylglycine-(2S)­cyanopyrrolidine

Figure 8. Inhibitors of dipeptidylpeptidase-IV enzymes.

F NH2 0

F~N~N,N y ~N-1 F CF3

Sitagliptin (MK-0431 -januvia) IC50 = 18nM

lle-thiazolidine Val-pyrrolidide

A natural peptide agonist, exendin-4 has been isolated from lizard venom with

increased half-life in plasma and a prolonged antidiabetic effect. Liraglutide (NN 22I ),

exenatide and ZPIOA51 (analog of exendin-4) have been developed as potent DPP-4

resistant. Liraglutide (NN221), as DPP-IV resistant analog ofGLP-I which has the potential

for regeneration of beta cells and successfully entered in Phase 2 of clinical trials. Liraglutide

plus metformin combination successfully improves glycemic control and lowers body weight

subjects with type 2 diabetes.52 Vildagliptin (LAF 237),53 saxagliptin (BMS-477118), ile-

IO

thiazolidine, NVP-DPP728,54 pyrazolidine55 derivatives, several nitrile class of compounds

like cyclohexyl glycine -(2S)-cyanopyridine56 and sitagliptin57 are reported as inhibitors of

dipeptidyl peptidase-IV enzyme. Vildagliptin was the first in a new class of oral antidiabetic

agents known as DPP-IV inhibitors or "incretin enhancers" and now in advanced-stage of

development for the treatment of type 2 diabetes. Sitagliptin (MK-0431-januvia), beta amino

acid derived specific DPP-IV inhibitor has recently been developed by MERCK. Sitagliptin

plus metformin combination successfully completed clinical trials and finally approved by

FDA as a drug. The several DPP- IV inhibitors are shown in figure 7.

1.4 Inhibitors of Hepatic Glucose Production

1.4.1 Glucagon Antagonist

Glucagon a 29 amino acids peptide hormone secreted from a-cells of pancreas

catalyzes both gluconeogenesis as well as glycogenolysis. Liver being a metabolic organ

maintains normal blood glucose level by regulating production of endogenous glucose

through gluconeogenesis and glycogenolysis in diabetics. 58 Thus glucagon antagonists are

highly desirable to suppress the formation of glucose by the liver. Hepatic glucose

production occurs due to insufficient insulin secretion or development of insulin resistance in

the liver. Several enzymes that regulate rate-controlling steps in gluconeogenesis and

glycogenolysis are obvious targets to inhibit hepatic glucose output. Glucagon is a seven

transmembrane G-protein coupled receptor, which mediates its effect via stimulation of

cAMP. Numerous glucagon antagonists have been reported from natural and synthetic

sources.59•60 Styrylquinoxazoline CP-99711 (I) has been reported for glucagon antagonistic

activity in rats.61 Pyrrolo[1, 2-a]quinoxaline62 (II), thiophene derivative63 (III) showed high

affinity for glucagon receptor. The representative examples of each of these classes are

shown in Figure 8.

Q N:-.. N

~

N I '>--!\

N/ ~ H

VI

11

F F

Ar

Cl

VII

VIII IX

Figure 9: Glucagon receptor antagonists.

Mercaptobenzimidazole NNC-92-168764 (IV) the first non-peptide competitive

human glucagon receptor antagonist has been reported by Novo-Nordisk. Other human

glucagon receptor antagonists include alkylidine hydrazide (V), biaryl am ides, 65 triaryl

imidazoles66 (VI) and triarylpyrroles67 (VII).

Recently, an entirely new class of compounds, substituted biaryls has been identified

through high-throughput screening as glucagon receptor antagonist. These compounds act by

inhibiting cAMP, stimulated by glucagon. Out of many derivatives the compounds (VIII)

and (IX) were selected for clinical trial but due to undesirable side effects and low

bioavailability (40%), further development of these compounds has been discontinued.

Glucagon receptor antagonists have been extensively reviewed recently.68

1.4.2 Biguanides

Biguanides are basically the guanidine derivatives constitute a well-known class of

antimalarials. The antidiabetic activity of guanidine derivatives was initially discovered as a side effect of proguanil, an antimalarial drug69 that led to the synthesis of potent antidiabetic

drugs like metformin, phenformin and buformin. Biguanides, including phenformin and

metformin, were introduced in 1957 as oral antidiabetic drugs.70 Phenformin was later

withdrawn in many countries due to its side effect of lactic acidosis. Metformin is now a

widely used biguanide for the therapy of type 2 diabetes. Metformin lowers blood glucose

level without causing overt hypoglycemia or stimulating insulin secretion.

NH NH~I NH NH II II ~ H N)l_N)l_N~CH

H N./'...N./'...N 2 H H 3 2 H H

Phenformin Buformin

Figure 10. Antidiabetic biguanides.

12

The molecular targets of these agents have not yet been identified. Their primary

mode of action seems to be inhibition of hepatic gluconeogenesis. 71 The main side effect of

metformin monotherapy is gastrointestinal (GI) symptoms. At 2550 mg daily dosage,

patients experienced abdominal discomfort, bloating, and metallic taste. Metformin

monotherapy is considered to be the first line treatment for patients prone to weight gain

and/or dyslipidemic who have failed to achieve adequate glycemic control on dietary

management. Metformin is also used in combination with other antihyperglycemic agents

and insulin. Metformin at higher doses of 0.5g to 1.50g has an absolute oral bioavailability72

of 50-60%. The bioavailability of the drug declines at higher oral doses. 73

1.5 Insulin Sensitizers

Compounds which decrease insulin resistance are called insulin sensitizers or

enhancer of insulin action. Thiazolidinediones (TZDs) are chemical compounds that reduce

insulin resistance, increase insulin-stimulated glucose disposal and improve glycemic control

by improving peripheral consumption and suppressing hepatic glucose production. TZDs act

by binding to peroxisome proliferator activated receptor-y (PPARy) which regulate the

transcription of a number of insulin responsive genes intimately involved in the control of

glucose and lipid metabolism.74'75 PPARy is a nuclear receptor expressed in adipocyte and

interacts with retinoid X receptor and form heterodimer RXR. 76 Interaction of TZDs with

PP ARy brings about conformational change in PPARy-RXR complex, which induce

expression of various genes, involved in fatty acid uptake in adipose tissues. 77 Ciglitazone,

the first member of this class of compounds was discovered serendipitously. Troglitazone

(Rezuli), pioglitazone (Actos), and rosiglitazone (Avandia) are the other important agents of

this series. After the approval of troglitazone in 1997, it was withdrawn due to a rare but

clinically serious incidence of hepatotoxicity. 78 The mechanism of troglitazone-induced

hepato toxicity is not clear but it is likely related to inflammation via an immunological

mechanism.75 The toxicity does not appear to be associated with the other TZDs approved

later, pioglitazone and rosiglitazone, both of which proved to be efficacious in clinical trials

in lowering fasting plasma glucose, HbAlc, and triglycerides and increasing HDL. 79

The major side effects of this class of compounds are edema and weight gain,

possibly due to their ability to stimulate adipocyte differentiations. The mechanism of

development of edema is still unclear. Current status of therapeutic importance of

peroxisome proliferator activated receptor in diabetes has been recently reviewed

extensively. 80

13

Chapter 1

HO

Ciglitazone Troglitazone

0

Pioglitazone

Rosiglitazone

Figure 11. Potent insulin sensitizers.

1.6 a-glucosidase inhibitors

a-glucosidase inhibitors are oral anti-diabetic drugs used for T2DM that work by

preventing the digestion of carbohydrates (such as starch and table sugar). Carbohydrates are

normally converted into simple sugars (monosaccharides), which can be absorbed through

the intestine. Hence, a-glucosidase inhibitors reduces the impact of carbohydrates on blood

sugar, therefore they are generally used to establish greater glycemic control over

hyperglycemia in T2DM, particularly with regard to postprandial hyperglycemia. They may

be used as monotherapy in conjunction with an appropriate diabetic diet and exercise, or they

may be used in conjunction with other anti-diabetic drugs.

The a-glucosidase inhibitors (AGis) are a class of nonsystemic drugs that do not

target specific pathophysiological defects in type 2 diabetes. The enzyme is located in the

brush border of the small intestine and is required for the final step in the breakdown of

carbohydrates such as starch, dextrins, and maltose to absorbable monosaccharides.81 The

important members of this class of compounds includes Acarbose (Precose),82 miglitol

(Glyset), 83 vogalibose. 84 The a-glucosidase inhibitors act as competitive inhibitors of a­

glucosidase enzyme, which retards the process of carbohydrate absorption and delays meal

derived glucose in diabetic as well as in normal subjects.81 These drugs are taken before the

meal. The striking side effects of these agents are gastrointestinal, abdominal discomfort, and

diarrhoea. 85

14

HO O HO~_O"'-

OH ;;!l'c{ ~O~OH O

-:;;;-- NH 'OH

HO OH OH OH OH Acarbose OH

OH

~ HO~NI

Ho'··y··'oH

OH

Miglitol

Figure 14. a-Glucosidase inhibitors.

1. 7 Targetting Insulin Signaling Pathways

1.7.1 Protein Tyrosine Phosphatase 1B Inhibitors

Vogalibose

Protein tyrosine phosphatase-IS (PTPIB) emerged only 9 years ago as a new drug

target for the treatment of diabetes and obesity. The enzyme belongs to the family of tyrosine

phosphatases, which consists of around 90 members. 86'87 Interestingly, this target had a very

long gestation period, starting with 19th century observations that vanadium salts are of

therapeutic utility in diabetes, followed by the biochemical discovery that vanadate is a

potent, nonselective inhibitor of phosphatases. By the mid-1980s, it was understood that

blocking one or more phosphatases could enhance the phosphorylation state of the insulin

receptor kinase ~ subunit and/or its downstream signaling partners and revert insulin

resistance, which is a characteristic of type 2 diabetes. Experiments based on PTPIB

knockout mice showed enhanced IR and IRS-I phosphorylation in the liver and skeletal

muscle and also mice were found healthy, lean and obesity resistant with low level of plasma

glucose and insulin. 88'89 These findings generated a vivid interest in PTP 1 B and other PTPs

as drug targets for diabetes and obesity.

1.7.2 Validation ofPTP1B as a drug target for diabetes and obesity

PTPIB is localized to the cytoplasmic face of the endoplasmic reticulum and is

expressed ubiquitously, including in the classical insulin-targeted tissues such as liver,

muscle and fat. 90 Mounting evidence from biochemical, genetic and pharmacological studies

support a role for PTP1B as a negative regulator in both insulin and leptin signaling, which

can associate with and dephosphorylate activated insulin receptor (IR) or insulin receptor

substrates (IRS) (Figure 12).91'92 Overexpression ofPTPIB in cell cultures decreases insulin­

stimulated phosphorylation of IR and/ or IRS-1, whereas reduction in the level of PTP 1 B, by

15

Chapter 1

antisense oligonucleotides or neutralizing antibodies, augments insulin initiated

signaling.93•94 Analyses of quantitative trait loci and mutations in the gene encoding PTPIB

in humans support the notion that aberrant expression of PTPIB can contribute to diabetes

and obesity.95 Mice that lack PTPIB display enhanced sensitivity to insulin, with increased

or prolonged tyrosine phosphorylation of IR in muscle and liver.96 Interestingly, PTPIB_1_

mice are protected against weight gain and have significantly lower triglyceride levels when

placed on a high-fat diet. This is unexpected because insulin is also an anabolic factor, and

increased insulin sensitivity can result in increased weight gain. PTPIB was subsequently

shown to bind and dephosphorylate JAK2, which is downstream of leptin receptor.97 Thus,

the resistance to diet-induced obesity observed in PTPIB-t- mice is likely to be associated

with increased energy expenditure owing to enhanced leptin sensitivity. Recent tissue­

specific knockout results indicate that body weight, adiposity and leptin action can be

regulated by neuronal PTP1B.98 Inhibiting neuronal PTPIB would require drugs that

penetrate the blood-brain barrier. Consistent with the above results, antisense-based

oligonucleotides that target PTPIB have shown efficacy in type 2 diabetes and have entered

Figure 11. Proposed mode of action of PTPlB: IR. insulin receptor R and a subunits; IRS-I, insulin receptor substrate-I; Ie(R), leptin(receptor); Jak; Janus kinase; STAT signal transducer and activator of transcription; PI3K., phosphatidylinositol-3-kinase; Akt, protein kinase B. (Drug Dicovery Today 1007, 12, 373-381)

phase 2 clinical trials.99 In addition, small-molecule inhibitors of PTPIB can work.

synergistically with insulin to increase insulin signalling and augment insulin-stimulated

glucose uptake.100 Moreover, pretreatment of leptin-resistant rats with a potent and selective

PTPIB inhibitor results in a marked improvement in leptin-dependent suppression of food

intake.101 Collectively, these biochemical, genetic and pharmacological studies provide

strong proof-of-concept, validating the notion that inhibition of PTPIB could address both

diabetes and obesity and making PTPIB an exciting target for drug development.

16

I Food uptake and energy homeostasis

Figure 13. Role ofPTPIB in insulin and leptin signaling (Drug Dicovery Today 2007, 12, 373- 381 )

1.7.3 Challenges in developing PTPlB-based small molecule therapeutics:

Selectivity is one of the major issues in the development of PTPIB inhibitors as

drugs. Because all PTPs share a high degree of structural conservation in the active site, the

pTyr (phosphotyrosine)-binding pocket, designing inhibitors with both high affinity and

selectivity for PTPIB poses a challenge. Fortunately, PTP substrate specificity studies have

shown that pTyr alone is not sufficient for high-affmity binding and residues flanking the

pTyr are important for PTP substrate recognition. 102

Bioavailability is another important issue in the development of PTPIB-based small­

molecule therapeutics. The active sites of PTPs have evolved to accommodate pTyr, which

contains two negative charges at physiological pH. Consequently, most active-site-directed

PTP inhibitors (non-hydrolyzable pTyr mimetics) reported to date possess a high charge

density to serve as competitive inhibitors. Such molecules are generally not drug-like, with

limited cell membrane permeability. Several strategies have been applied to improve the cell

permeability and/or bioavailability ofPTPlB inhibitors.

1.7.4 Development of Potent and selective PTPlB inhibitors

With the advent of PTPIB as a new target for T2DM various novel and selective

PTPIB inhibitors have been developed. Numerous literatures are available covering several

classes of molecules as potent PTPIB inhibitors.103 The development of various potent

molecules for PTPlB inhibitory activity can be categories on the basis of following four

approaches:

(a) The Library Approach (X & XI). 104

(b) The 'Linked-fragment' Approach (Xll-XIV).105

17

(c) The confonnation assisted Approach (XV-XVI). 106

(d) Targeting allosteric sites for improved selectivity and bioavailabilty (XVII-XIX). 107

1.7.4.1 The Library Approach

A focused library approach was used to identify highly potent and selective PTP 1 B

inhibitors that are capable of bridging and simultaneously associating with both the active

site and an adjacent peripheral site. 104 The library contains (i) a biasing pTyr to ensure

association with the active site and (ii) a structurally diverse set of 23 linkers that tether the

pTyr moiety to (iii) a structurally diverse set of eight aryl acids, which were designed to

associate with the peripheral subsite, positioned near the active site.

1.7.4.2 The 'Linked-fragment' Approach

In addition to the proximal non-catalytic site defined by Lys41, Arg47 and Asp48, a

second aryl phosphate-binding site, adjacent to the PTP 1 B active site, was identified from

crystal structures of the protein in complex with pTyr and a small aryl phosphate 49. 108 This

second aryl phosphate-binding site lies within a region (Arg24 and Arg254) that is not

conserved among the PTPs. A 'linked fragment' approach was employed to develop potent

and selective PTP1B inhibitors that can engage both the active site and the second aryl

phosphate-binding site. 105

1.7.4.3 The conformation assisted Approach

Structure-based modelling has been used to target unique PTP1B conformations for

inhibitor development with both high affinity and selectivity. 106 A series of benzotriazole

phenyldifluoromethylphosphonic acids were synthesized as non-peptidic PTP1B inhibitors.

Many of these compounds showed good inhibitory activity, at the sub-mM level, for PTP 1 B

but none of them had selectivity compared with TC-PTP.

1.7.4.4 Targeting allosteric sites for improved selectivity and bioavailabilty

A secondary allosteric site has recently been described for PTPIB, and several small­

molecule inhibitors that occupy this site stabilize an inactive conformation ofPTPlB. Unlike

the pTyr binding active site, the allosteric site is not well conserved and possesses is

substantially less polar. Thus, targeting the allosteric site might present an alternative

strategy for developing selective inhibitors with acceptable pharmacological properties. 107

Figure 14a showing the potent and selective PTPlB inhibitors based on the

approaches. One of the earliest strategies for designing PTPlB inhibitors has focused on the

incorporation of tyrosine phosphate mimetics into peptidomimetic backbones. The

18

phosphotyrosine moiety which penetrates the catalytic 9A-deep cleft has been successfully

mimicked by three different negatively charged chemical groups: difluorophosphonomethyl

aryl, oxalylaminobenzoic acid and carbomethoxybenzoic acid. Among these,

difluoromethylene phosphonates have been shown to yield potent phosphatase inhibitors.

The bis-difluorophosphonate-phenylanaline (X), one of the most potent inhibitor series of

the target to date (Ki) 2.4 nM, has good selectivity over most phosphatases, including T -cell

PTP (TC-PTP). Another phosphatase, PTEN/NMAC (phosphatase and tensin homolog) is

part of this pathway as phospholipid phosphatase that indirectly stimulates phospho-inositol

3-kinase (PI3K). 109 PTEN has been recently identified as a significant antidiabetic drug

target. 110 Recently ottana et al. reported 5-Arylidene-2-phenylimino-4-thiazolidinones (XX)

as potent PTP 1 B and LMW-PTP inhibitors. 111

F

XV

0 OHOEt

~CX(-NH 0

COOEt XVIII

Figure 14a. Potent PTPlB inhibitors

F

Br

HO

XIX

0 R1 II ' S-N II , 2 0 R

Br XVII

R1 = R2 =CH3

R1 = H, R2 = Vso2NH2

19

Pei et a1. 112 have reported several alpha-haloacetophenone derivatives (XXI) as potent

neutral protein tyrosine phosphatase inhibitors, which covalently alkylate the conserved

catalytic cysteine residue in the PTP active site. Malamas et al. 113 reported two novel series

ofbenzofuran/benzothiophene- biphenyl oxo-acetic acids and sulfonylsalicylic acid as potent

inhibitors of PTP-1B with good antihyperglycemic activity. They found that compound

XXII normalizes plasma glucose level at the 25 mglkg dose (po) and 1 mg/kg dose (ip).

Wrobel and co-workers 114 reported a series of 11-arylbenzo[ b ]naphtho[2,3-d]furanlthiophene

derivatives XXIII, which selectively inhibit PTP-1 B over other PTPs and lowered the blood

glucose level in ob/ob mice. Hu et a1. 115 reported several flavonoids including a flavonol

XXIV, which showed potent inhibitory activity against PTP-1B enzyme. Won et al. isolated

12 new flavonones (XXVa & XXVb) from the stem bark of Erythrima abyssinica as potent

PTP1B inhibitors. Recently, Goel et al. reported 116'117 several functionalized acetophenones

(XXVI), benzofurans (XXVII), naphthofurans (XXVIII), and dibenzofurans (XXIX) as

novel protein tyrosine phosphatase-1B inhibitors and various nature mimicking furanyl-2-

pyranones 118 (XXX) and 1 ,3-teraryls 119 (XXXI) as potent antihyperglycemic agents.

o r-0---cooH ____;='\____J x

r~O R~b ) XX XXI

Ar

XXII

0<COOH

0

HO'f'O ~

~ 0

HO Br XXIII

R1 rt

HOI(YO,xx:, "" I yY HOI(YO'(' ~ OH 0 XXVa yy ~ OH 0

XXIV OH 0 XXVb OH 0 OH

OH SMe COOMe

~h OCOOCH, 0 RO XVII

XXVI (0) XXVIII

N (lCOOH

~CN N~OH R1

0 NH2 :60 XXX

XXXII

Figure 14b. Natural and synthetic PTPlB inhibitors.

20

Chcqrte¥ 1

Several PTP 1 B inhibitors are in clinical development. 120 Recently, diaryloxamic

acid105 (XXXII) has been identified as selective and potent PTP1B inhibitor for using a

linked-fragment strategy. Recently a newer approach to inhibit PTP1B through peroxides

have been reported by several workers Davis et al. 121 reported the inactivation ofPTP1B by

amino acid hydroperoxides. Gates et al. 122 reported that organic peroxides have the ability to

cause the thio-reversible, inactivation of PTP 1 B (figure 14 shows the variable organic

peroxides).

0 0 0 CI~0,0H ~0,0H _)l

0_.0H

'h- co2-A B c

0 ~0/0H do'OH (jOOH

0 D E F

Figure 15. Showing variable organic peroxides ' \ ·.,\~-~~~7~7:'})

\~~=/' "'~>--. _:·~~ TH-171'13 1.8 PPARa/y dual and Pan-agonist

The nuclear receptor PP ARa is the legitimate target for fibrate class of lipid lowering

agents and PPARy is the validated target for glitazone class of insulin sensitizers. Thus there

is a resurgence of interest in the development of new anti-diabetic drugs that combine the

insulin-sensitizing effects of PPARy together with the additional lipid lowering activity of

PP ARa. A pan-agonist, capable of stimulating all three peroxisome proliferater-activated

receptors (PPARs) as a group, expected to be useful for treatment of T2DM. Clinical

evidences obtained from benzafibrate-based trials are in support of the pan-PPAR agonist

concept as therapeutic approach. Numerous chemicals have been employed to design new

class of dual agonists. One strategy was to cyclize the fibrate structure mimick in order to

fibrate and glitazone structures in one chemical entity. 123 The glitazars emerged as first

example of dual PP ARa/y class. Muraglitazar was the first dual-PPARa/y agonist approved

by US Food and Drug Administration (FDA). In obese diabetic db/db mice, muraglitazar has

efficacious antidiabetic effects and improves metabolic abnormalities. 124 Recently, Artis

et.al.125 reported a novel pan-agonist, indeglitazar, which gives partial response against

PP ARy and full response against PP AR(), is know progressed to phase II clinical trials.

Recently discovered some dual PPARa/y activators have been depicted in Figure 16. Using

carbazol-derived dual PP ARaly agonist as a structural template, a novel pan-agonist has

been designed, 126 and benzofibrate has been approved as belongs to this class. New

21

compounds with improved pan-agonist activity compaired to benzofibrate have been also

developed, such as L Y -465608, DRF-11605 and CS-204. 127 Among these, indole-based

compounds such as BPR1H036 display insulin sensitizing effects and in-vitro glucose uptake

improvement. 128 Moreover, a number of pan-agonists, with insulin sensitizing and

antihyperglycemic actions, are currently tested in clinical studies. PLX-204 is in pre-clinical

phase whereas netoglitazone in phase II clinical trial.

Muraglitazar

0 0

~~~NH F3C~ MeO)l) ~-{

0

MK-0767 I KRP-297

ceo~" F Netoglitazone

pan

Figure 16. PP ARo./y dual and Pan-agonist.

1.9 Antiobesity Agents

~ ryo,~~ ~6 llA0~ o

Tesaglitazar I AZ-242

Ragaglitazar IDRF2725

vi:' wo~o ( ~

}-- BPR1H036 0 pan

cf~ J-oH ~

N 1,0

r('YS:..b lndeglitazar

"o~ In Phase II Clinical Trial

Obesity is a multigenic disease associated with several metabolic abnormalities such

as insulin resistance, cardiovascular morbidity and cancer. 129 This has established obesity as

therapeutic targets for the management of type 2 diabetes. Basically, following three

therapeutic approaches are currently being pursued.

1. Agents reducing energy intake or appetite suppressants

2. Agents affecting energy expenditure.

3. Agents affecting dietary absorption of fats.

22

1.9.1 Agents Reducing Energy Intake or Appetite Suppressants

Agents, which reduce energy, are called appetite suppressants. These compounds

affect secretion of various biogenic amines. Based on the nature of the amines

(neurotransmitters) secreted, these agents have been classified into following two categories.

1.9.1.1 Noradrenergic Agents

These agents induce release of noradrenalin from nerves endings in hypothalamus.

Noradrenalin release activates postsynaptic a 1- and l31-adrenoceptor that suppress appetite.

Phentermine, 130 phenylpropanolamine, 131 diethylpropion and mazindol 132 are the drugs of

this class for the treatment of obesity. Mazindol is the only drug approved for clinical use.

Phentermine phenylpropanolamine Diethylpropion Mazindol Cl

Figure 17: Noradrenergic agonists.

1.9.1.2 Serotonergic Agents

This group of appetite suppressants acts by inhibiting reuptake of serotonin (5-

hydroxytryptamine ). Dexfenfluramine, serteraline and fluoxetine are drugs of this class but

they are no longer in clinical use for the treatment of obesity due to intolerable side effects

and have been reviewed extensively. 133•134

1.10 I33-Adrenoceptor Agonists

133-Adrenoreceptor is a member of G-protein coupled adrenoreceptor family. The

agonists of this receptor are very effective for thermogenic antiobesity, lipolysis and insulin

sensitizing activity in rodents. Their main sites of action are white and brown adipose tissue

and muscle. The 133-adrenoceptor mRNA levels are lower in human than in rodent adipose

tissue.

BRL 37344, R = H BRL 35135 R =Me

CGP 12177

Figure 18: fl3.Adrenoceptor Agonists as antidiabetic agents.

THIQ

23

p3-Adrenoreceptor agonists fall into three main chemical classes: arylethanolamines

(BRL 37344, BRL 35135}, aryloxypropanolamines (CGP 12177}, and trimetoquinols (CL-

316243).135 The above compounds are in advanced clinical trials. Oral bioavailability and

tissue selectivity are striking problems of this compounds. 136

1.11 Miscellaneous

1.11.1 Tumor Necrosis Factor Alpha (TNF-a.)

The mechanisms of insulin resistance caused by obesity are not so far clearly

understood. According to first hypothesis it is due to increased flux of substrates into the

adipose tissues resulting in elevated levels of secreted factors responsible for insulin

resistance but according to second hypothesis the levels of TNF-a. mRNA protein is

increased. It has been observed that TNF-a. inhibits signaling events mediated by insulin

resistance. TNF-a. is a proinflammatory cytokines responsible for pathogenesis of a spectrum

of diseases. 137 The overproduction of these cytokines (TNF-a.) also leads to insulin

resistance that has been established by null mutation in obese. 138 This notion was further

supported by significant increase in peripheral insulin stimulated glucose uptake139 on

neutralization ofTNF-a. in obese fa/fa rats improving insulin sensitivity in peripheral tissues.

1.11.2 Preparation of P38 MAP Kinase Inhibitors

P38-Mitogen-activated kinase plays a central role in the synthesis of TNF-a.. This

protein belongs to a group of serine /threonine kinases that include c-Jun N-terminal kinase

(JNK) and extracellular signal-regulated kinase (ERK). The compounds RWJ67657 and VX-

702 are potent inhibitors of P38 MAP Kinase140 and are in advanced clinical stage. Another

compound pyrazole derivative141 and BIRB 796 a potent selective inhibitor of P38a. is in

clinical trial for treatment of autoimmune diseases.

RWJ67657 BIRB 796

Figure 19. Inhibitors ofP38 MAP Kinase.

24

1.11.3 Glucokinase Activators

The pathogenesis of type 2 diabetes is not only obesity or insulin resistance but also

mutations in the genes of regulatory enzymes of glucose. This was established with the

discovery of a mutated gene142 encoding glucokinase (GK) in maturity onset diabetes of the

young type 2 (MODY2). GK is an enzyme responsible for the phosphorylation of glucose in

pancreatic P-cell and hepatocyte leading to glycogen synthesis and decrease in hepatic

glucose production. In MODY2 patients, there is a net reduction in the formation of

glycogen and increase in the production of the glucose in liver. 143 Based on these

observations numerous compounds have been synthesized to obtain potent GK activators.

Screening of 120,000 synthetic organic compounds of diverse chemical structures led

to the discovery of a compound R00281675. 144 It has been found to activate recombinant

human glucokinase potentially in a dose dependent manner.

R00281675

Figure 20. Glucokinase activators.

1.12 Summary

The current clinical scenano on the managment of diabetes and associated

complicatons have highlighted the importance of antihyperglycemic agents for the

management of diabetes mellitus and associated microvascular /macrovascular

complications. Majority of the earlier therapeutic agents for the management of diabetes had

been developed without defined molecular targets or on the basis of understanding of disease

pathogenesis. Advances in molecular pharmacology and medicinal chemistry have given a

new insight of molecular pathways of diabetes and its complications. These targets have

been identified on the basis of predicted roles in modulating one or more aspects of

pathogenesis of diabetes. Important strategies for management of diabetes are: 1)

augmenting glucose mediated insulin secretion and insulin sensitivity, 2) inhibiting hepatic

glucose production and its absorption by the cells, 3) targeting specific molecular targets in

insulin signaling pathways, 4) control of obesity and altered lipid metabolism to improve

insulin sensitivity.

25

ChcqJ"ter 1

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28

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