Polymer–phloridzin conjugates as an anti-diabetic drug that Inhibits glucose absorption through the Na⁺/glucose cotransporter (SGLT1) in the small intestine

Abstract

Poly(γ-glutamic acid)s (γ-PGA) modified with phloridzin, which is an inhibitor of the Na⁺/glucose cotransporter (SGLT1), via a ω-amino triethylene glycol linker were synthesized. The potential of γ-PGA–phloridzin conjugates (PGA-PRZs) obtained as a novel oral anti-diabetic drug was examined by in vitro and in vivo experiments. A PGA-PRZ with a 15% phloridzin content inhibited glucose transport from mucosal to serosal sides of the everted rat's small intestine, and its inhibitory effect was as strong as that of intact phloridzin. When the PGA-PRZ was given orally to rats before glucose administration, the glucose-induced hyperglycemic effect was significantly suppressed. On the other hand, reduction of an increase in the blood glucose concentration was scarcely observed when the PGA-PRZ was substituted with a double amount of intact phloridzin. This difference in the biological activity between PGA-PRZ and intact phloridzin might have resulted from the improved stability of a glucoside bond of phloridzin through the conjugation with γ-PGA. These results suggest that the γ-PGA–phloridzin conjugates have potential as oral anti-diabetic drugs.

Introduction

Diabetes is a metabolic disease characterized by a congenital (type I insulin-dependent diabetes mellitus) or an acquired (type II non-insulin-dependent diabetes mellitus) inability to transport glucose from the blood to the cells. Hyperglycemia results from a deficiency of insulin secretion or insulin resistance. About 90% of diabetic patients suffer from type II diabetes, and the prevalence of diabetes worldwide will double over the next 25 years [1]. Therapy for diabetes focuses on retarding the progress of disease, which induces complications such as retinopathy, nephropathy, and neuropathy. Clinical data shows that it is essential for the therapy to maintain an appropriate blood glucose level strictly [2]. Current therapies include not only medication, but also caloric restriction. Many anti-diabetic drugs such as insulin, sulfonylurea derivatives [3], [4], biguanide and thiazolidine derivatives [5], [6], [7], [8], [9], and α-glucosidase inhibitors [2] have been used clinically.

Carbohydrates in the diet are hydrolyzed by digestive enzymes. α-Glucosidase cleaves the α-glucoside bonds of disaccharides such as maltose to yield monosaccharides. The resulting monosaccharides are then absorbed from the small intestine actively through the Na⁺/glucose cotransporter (SGLT1) located on the mucosal side of epithelial cells. It is known that the SGLT1 possesses high substrate specificity. Glucose and galactose are absorbed from the small intestine through the SGLT1, but disaccharides are not substrates against this transporter. Since the recognition of glucose by the SGLT1 is the first step of glucose absorption [10], its inhibition can be one of the most effective approaches to suppress an increase in blood glucose levels after a meal [11], [12], [13], [14].

Phloridzin (Fig. 1), which is found in the bark and stems of apple trees, is known to inhibit glucose transport competitively through the binding of the glucose moiety to the SGLT1 [10], [15], [16]. However, phloridzin has not been used as an oral anti-diabetic drug because toxic phloretin is released through the hydrolysis of a glucoside bond of phloridzin by intestinal β-glucosidase [17], [18], [19]. This hydrolysis also results in a reduction of phloridzin activity because of the lack of intramolecular glucose residues. Although low molecular weight-phloridzin analogues and phloretin derivatives have been examined, promising results have not been obtained yet [18], [20], [21].

We designed a novel polymer–phloridzin conjugate to overcome the above-mentioned problems. If the phloretin part is chemically bound to polymers via non-biodegradable linkers, then the resulting polymer–phloridzin conjugates will not be absorbed from the small intestine because of their high molecular weight. No release of phloretin from the conjugates in the gastrointestinal tract will assure the safety of the conjugates. Kopeček et al. also reported that steric hindrance of the polymer chain contributed to the stability of enzyme-susceptible chemical bonds [22], [23], [24]. It is expected that the efficacy of the conjugates will be higher than that of free phloridzin because the polymers will prevent the glucoside bond of phloridzin from cleaving in the gastrointestinal tract. Kopeček and his coworkers have been investigating the usefulness of polymer–drug conjugates as a parenteral delivery system of anti-cancer drugs on the basis of enhanced permeability and retention [25]. They also reported that conjugates could deliver anti-cancer drugs directly into the colorectal cancer after their oral administration [22], [26]. Sakuma and Lu recently found that conjugates could be used as carriers for the oral absorption enhancement of poorly absorptive alendronic acid [27]. In these studies, drugs were bound to the polymer backbone via biodegradable linkers such as peptides, and the linkers were designed so as to release drugs after the delivery of conjugates to targeting tissues. Contrary to them, the polymer–phloridzin conjugate was designed as a drug itself, not as a prodrug of phloridzin. Here, poly(γ-glutamic acid) (γ-PGA) was chosen as the polymer backbone. γ-PGA is a bioproduct secreted by Bacillus subtilis strain F-2-01, and has been studied as a biocompatible polymer [28], [29], [30], [31]. Recently, Akashi et al. demonstrated that γ-PGA could be used as a biocompatible drug carrier for vaccines [30]. In this paper, a γ-PGA–phloridzin conjugate (PGA-PRZ) was designed and synthesized, and its in vitro/in vivo performance was examined.