Paul D. van Poelje

Targeting thyroid hormone receptor-beta agonists to the liver reduces cholesterol and triglycerides and improves the therapeutic index.

Despite efforts spanning four decades, the therapeutic potential of thyroid hormone receptor (TR) agonists as lipid-lowering and anti-obesity agents remains largely unexplored in humans because of dose-limiting cardiac effects and effects on the thyroid hormone axis (THA), muscle metabolism, and bone turnover. TR agonists selective for the TRβ isoform exhibit modest cardiac sparing in rodents and primates but are unable to lower lipids without inducing TRβ-mediated suppression of the THA. Herein, we describe a cytochrome P450-activated prodrug of a phosphonate-containing TR agonist that exhibits increased TR activation in the liver relative to extrahepatic tissues and an improved therapeutic index. Pharmacokinetic studies in rats demonstrated that the prodrug (2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)phenoxy)methyl]-2-oxido-[1,3,2]-dioxaphosphonane (MB07811) undergoes first-pass hepatic extraction and that cleavage of the prodrug generates the negatively charged TR agonist (3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)phenoxy)methylphosphonic acid (MB07344), which distributes poorly into most tissues and is rapidly eliminated in the bile. Enhanced liver targeting was further demonstrated by comparing the effects of MB07811 with 3,5,3′-triiodo-l-thyronine (T3) and a non-liver-targeted TR agonist, 3,5-dichloro-4-(4-hydroxy-3-isopropylphenoxy)phenylacetic acid (KB-141) on the expression of TR agonist-responsive genes in the liver and six extrahepatic tissues. The pharmacologic effects of liver targeting were evident in the normal rat, where MB07811 exhibited increased cardiac sparing, and in the diet-induced obese mouse, where, unlike KB-141, MB07811 reduced cholesterol and both serum and hepatic triglycerides at doses devoid of effects on body weight, glycemia, and the THA. These results indicate that targeting TR agonists to the liver has the potential to lower both cholesterol and triglyceride levels with an acceptable safety profile.

MB06322 (CS-917): A potent and selective inhibitor of fructose 1,6-bisphosphatase for controlling gluconeogenesis in type 2 diabetes.

In type 2 diabetes, the liver produces excessive amounts of glucose through the gluconeogenesis (GNG) pathway and consequently is partly responsible for the elevated glucose levels characteristic of the disease. In an effort to find safe and efficacious GNG inhibitors, we targeted the AMP binding site of fructose 1,6-bisphosphatase (FBPase). The hydrophilic nature of AMP binding sites and their widespread use for allosteric regulation of enzymes in metabolic pathways has historically made discovery of AMP mimetics suitable for drug development difficult. By using a structure-based drug design strategy, we discovered a series of compounds that mimic AMP but bear little structural resemblance. The lead compound, MB05032, exhibited high potency and specificity for human FBPase. Oral delivery of MB05032 was achieved by using the bisamidate prodrug MB06322 (CS-917), which is converted to MB05032 in two steps through the action of an esterase and a phosphoramidase. MB06322 inhibited glucose production from a variety of GNG substrates in rat hepatocytes and from bicarbonate in male Zucker diabetic fatty rats. Analysis of liver GNG pathway intermediates confirmed FBPase as the site of action. Oral administration of MB06322 to Zucker diabetic fatty rats led to a dose-dependent decrease in plasma glucose levels independent of insulin levels and nutritional status. Glucose lowering occurred without signs of hypoglycemia or significant elevations in plasma lactate or triglyceride levels. The findings suggest that potent and specific FBPase inhibitors represent a drug class with potential to treat type 2 diabetes through inhibition of GNG.

Liver-Targeted Drug Delivery Using HepDirect Prodrugs

Targeting drugs to specific organs, tissues, or cells is an attractive strategy for enhancing drug efficacy and reducing side effects. Drug carriers such as antibodies, natural and manmade polymers, and labeled liposomes are capable of targeting drugs to blood vessels of individual tissues but often fail to deliver drugs to extravascular sites. An alternative strategy is to use low molecular weight prodrugs that distribute throughout the body but cleave intracellularly to the active drug by an organ-specific enzyme. Here we show that a series of phosphate and phosphonate prodrugs, called HepDirect prodrugs, results in liver-targeted drug delivery following a cytochrome P450-catalyzed oxidative cleavage reaction inside hepatocytes. Liver targeting was demonstrated in rodents for MB06866 [(2R,4S)-9-[2-[4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphorinan-2-yl]methoxyethyl]adenine (remofovir)], a Hep-Direct prodrug of the nucleotide analog adefovir (PMEA), and MB07133 [(2R,4S)-4-amino-1-[5-O-[2-oxo-4-(4-pyridyl)-1,3,2-dioxaphosphorinan-2-yl]-β-d-arabinofuranosyl]-2(1H)-pyrimidinone], a HepDirect prodrug of cytarabine (araC) 5′-monophosphate. Liver targeting led to higher levels of the biologically active form of PMEA and araC in the liver and to lower levels in the most toxicologically sensitive organs. Liver targeting also confined production of the prodrug byproduct, an aryl vinyl ketone, to hepatocytes. Glutathione within the hepatocytes rapidly reacted with the byproduct to form a glutathione conjugate. No byproduct-related toxicity was observed in hepatocytes or animals treated with HepDirect prodrugs. A 5-day safety study in mice demonstrated the toxicological benefits of liver targeting. These findings suggest that HepDirect prodrugs represent a potential strategy for targeting drugs to the liver and achieving more effective therapies against chronic liver diseases such as hepatitis B, hepatitis C, and hepatocellular carcinoma.

Reduction of hepatic steatosis in rats and mice after treatment with a liver‐targeted thyroid hormone receptor agonist

Non‐alcoholic fatty liver disease (NAFLD) is one of the most common forms of chronic liver disease, with a prevalence ranging from 10% to 30%. The use of thyroid hormone receptor (TR) agonists for the treatment of NAFLD has not been considered viable because thyroid hormones increase free fatty acid (FFA) flux from the periphery to the liver, induce hepatic lipogenesis, and therefore could potentially cause steatosis. MB07811 is an orally active HepDirect prodrug of MB07344, a liver‐targeted TR‐β agonist. The purpose of these studies was to assess the effects of MB07811 on whole body and liver lipid metabolism of normal rodents and rodent models of hepatic steatosis. In the current studies, MB07811 markedly reduced hepatic steatosis as well as reduced plasma FFA and triglycerides. In contrast to MB07811, T3 induced adipocyte lipolysis in vitro and in vivo and had a diminished ability to decrease hepatic steatosis. This suggests the influx of FFA from the periphery to the liver may partially counteract the antisteatotic activity of T3. Clearance of liver lipids by MB07811 results from accelerated hepatic fatty acid oxidation, a known consequence of hepatic TR activation, as reflected by increased hepatic mitochondrial respiration rates, changes in hepatic gene expression, and increased plasma acyl‐carnitine levels. Transaminase levels remained unchanged, or were reduced, and no evidence for liver fibrosis or other histological liver damage was observed after treatment with MB07811 for up to 10 weeks. Additionally, MB07811, unlike T3, did not increase heart weight or decrease pituitary thyroid‐stimulating hormone beta (TSHβ) expression. Conclusion: MB07811 represents a novel class of liver‐targeted TR agonists with beneficial low‐density lipoprotein cholesterol‐lowering properties that may provide additional therapeutic benefit to hyperlipidemic patients with concomitant NAFLD. (HEPATOLOGY 2009.)

Inhibition of Fructose 1,6-Bisphosphatase Reduces Excessive Endogenous Glucose Production and Attenuates Hyperglycemia in Zucker Diabetic Fatty Rats

Gluconeogenesis is increased in type 2 diabetes and contributes significantly to fasting and postprandial hyperglycemia. We recently reported the discovery of the first potent and selective inhibitors of fructose 1,6-bisphosphatase (FBPase), a rate-controlling enzyme of gluconeogenesis. Herein we describe acute and chronic effects of the lead inhibitor, MB06322 (CS-917), in rodent models of type 2 diabetes. In fasting male ZDF rats with overt diabetes, a single dose of MB06322 inhibited gluconeogenesis by 70% and overall endogenous glucose production by 46%, leading to a reduction in blood glucose of >200 mg/dl. Chronic treatment of freely feeding 6-week-old male Zucker diabetic fatty (ZDF) rats delayed the development of hyperglycemia and preserved pancreatic function. Elevation of lactate (∼1.5-fold) occurred after 4 weeks of treatment, as did the apparent shunting of precursors into triglycerides. Profound glucose lowering (∼44%) and similar metabolic ramifications were associated with 2-week intervention therapy of 10-week-old male ZDF rats. In high-fat diet–fed female ZDF rats, MB06322 treatment for 2 weeks fully attenuated hyperglycemia without evidence of metabolic perturbation other than a modest reduction in glycogen stores (∼20%). The studies confirm that excessive gluconeogenesis plays an integral role in the pathophysiology of type 2 diabetes and suggest that FBPase inhibitors may provide a future treatment option.

A Potent and Selective AMPK Activator That Inhibits de Novo Lipogenesis

AMP-activated protein kinase (AMPK) is a heterotrimeric kinase that regulates cellular energy metabolism by affecting energy-consuming pathways such as de novo lipid biosynthesis and glucose production as well as energy-producing pathways such as lipid oxidation and glucose uptake. Accordingly, compounds that activate AMPK represent potential drug candidates for the treatment of hyperlipidemia and type 2 diabetes. Screening of a proprietary library of AMP mimetics identified the phosphonic acid 2 that bears little structural resemblance to AMP but is capable of activating AMPK with high potency (EC50 = 6 nM vs AMP EC50 = 6 μM) and specificity. Phosphonate prodrugs of 2 inhibited de novo lipogenesis in cellular and animal models of hyperlipidemia.

Fructose-1,6-bisphosphatase Inhibitors. 2. Design, Synthesis, and Structure−Activity Relationship of a Series of Phosphonic Acid Containing Benzimidazoles that Function as 5′-Adenosinemonophosphate (AMP) Mimics

Efforts to enhance the inhibitory potency of the initial purine series of fructose-1,6-bisphosphatase (FBPase) inhibitors led to the discovery of a series of benzimidazole analogues with human FBPase IC(50)s < 100 nM. Inhibitor 4.4 emerged as a lead compound based on its potent inhibition of human liver FBPase (IC(50) = 55 nM) and significant glucose lowering in normal fasted rats. Intravenous administration of 4.4 to Zucker diabetic fatty rats led to rapid and robust glucose lowering, thereby providing the first evidence that FBPase inhibitors could improve glycemia in animal models of type 2 diabetes.

Pradefovir: a prodrug that targets adefovir to the liver for the treatment of hepatitis B.

Adefovir dipivoxil, a marketed drug for the treatment of hepatitis B, is dosed at submaximally efficacious doses because of renal toxicity. In an effort to improve the therapeutic index of adefovir, 1-aryl-1,3-propanyl prodrugs were synthesized with the rationale that this selectively liver-activated prodrug class would enhance liver levels of the active metabolite adefovir diphosphate (ADV-DP) and/or decrease kidney exposure. The lead prodrug (14, MB06866, pradefovir), identified from a variety of in vitro and in vivo assays, exhibited good oral bioavailability (F = 42%, mesylate salt, rat) and rate of prodrug conversion to ADV-DP. Tissue distribution studies in the rat using radiolabeled materials showed that cyclic 1-aryl-1,3-propanyl prodrugs enhance the delivery of adefovir and its metabolites to the liver, with pradefovir exhibiting a 12-fold improvement in the liver/kidney ratio over adefovir dipivoxil.

Discovery of Phosphonic Diamide Prodrugs and Their Use for the Oral Delivery of a Series of Fructose 1,6-Bisphosphatase Inhibitors

Like most phosphonic acids, the recently discovered potent and selective thiazole phosphonic acid inhibitors of fructose 1,6-bisphosphatase (FBPase) exhibited low oral bioavailability (OBAV) and therefore required a prodrug to achieve oral efficacy. Syntheses of known phosphonate prodrugs did not afford the desired OBAV; hence, a new class of prodrugs was sought. Phosphonic diamides derived from amino acid esters were discovered as viable prodrugs, which met our preset goals: excellent aqueous stability over a wide pH range, benign byproducts (amino acids and low molecular weight alcohols), and most importantly good OBAV leading to robust oral glucose lowering effects. These desirable properties of phosphonic diamides represent significant improvements over existing prodrug classes. Optimization of the diamide prodrugs of phosphonic acid 2a (MB05032) led to the identification of diamide 8 (MB06322), the first reported orally efficacious FBPase inhibitor.

Fructose-1, 6-Bisphosphatase Inhibitors for Reducing Excessive Endogenous Glucose Production in Type 2 Diabetes

Fructose-1,6-bisphosphatase (FBPase), a rate-controlling enzyme of gluconeogenesis, has emerged as an important target for the treatment of type 2 diabetes due to the well-recognized role of excessive endogenous glucose production (EGP) in the hyperglycemia characteristic of the disease. Inhibitors of FBPase are expected to fulfill an unmet medical need because the majority of current antidiabetic medications act primarily on insulin resistance or insulin insufficiency and do not reduce gluconeogenesis effectively or in a direct manner. Despite significant challenges, potent and selective inhibitors of FBPase targeting the allosteric site of the enzyme were identified by means of a structure-guided design strategy that used the natural inhibitor, adenosine monophosphate (AMP), as the starting point. Oral delivery of these anionic FBPase inhibitors was enabled by a novel diamide prodrug class. Treatment of diabetic rodents with CS-917, the best characterized of these prodrugs, resulted in a reduced rate of gluconeogenesis and EGP. Of note, inhibition of gluconeogenesis by CS-917 led to the amelioration of both fasting and postprandial hyperglycemia without weight gain, incidence of hypoglycemia, or major perturbation of lactate or lipid homeostasis. Furthermore, the combination of CS-917 with representatives of the insulin sensitizer or insulin secretagogue drug classes provided enhanced glycemic control. Subsequent clinical evaluations of CS-917 revealed a favorable safety profile as well as clinically meaningful reductions in fasting glucose levels in patients with T2DM. Future trials of MB07803, a second generation FBPase inhibitor with improved pharmacokinetics, will address whether this novel class of antidiabetic agents can provide safe and long-term glycemic control.