Exploiting the peptide uptake transporters PEPT1 and PEPT2 to improve drug absorption and targeting

Glucagon like Peptide-1 (GLP-1) is a gut hormone released after food consumption to stimulate insulin secretion. GLP-1 has a very short half-life due to its degradation by the proteolytic enzyme dipeptidyl peptidase-IV (DPP-IV; CD26) which cleaves peptides after proline residues. Therefore, specific inhibition of DPP-IV is an attractive therapeutic approach to stimulate glucose dependent insulin secretion. In addition to its role in glucose homeostasis, DPP-IV has been implicated in immune disorders, HIV-1 infection and tumor progression and hence indications for its inhibitors extend past the treatment of diabetes. One of the most advanced DPP-IV inhibitors is isoleucine thiazolidide (Ile-thiazolidide, P32/98) which was in phase II evaluation by Probiodrug. Researchers at the Technical University of Munich have recently shown that the oral activity of this drug results from its ability to bind at the intestinal PEPT1 transporter and thereby allowing to be transported across the gut wall. This finding was exploited, using Ile-thiazolidide and its analogues to characterize assays able to quantify PEPT1 binding and transport. These tools open the way for the rational design of peptide linked drugs with improved intestinal absorption. A second similar protein PEPT2 transports peptides across the respiratory tract epithelium and another assay was developed for this transporter. This assay could allow the targeting of thiazolidides to the lung. The selective inhibition of DPP-IV within the airway may be of use in the treatment of lung cancer. More generally, harnessing peptide transporters to allow the improved uptake and targeting of therapeutics can be extended to many other drug classes and indications.

According to WHO, there are some 130 million diagnosed diabetics in the world, a figure that is predicted to increase to 300 million by 2025. The market for diabetes therapeutics is also rising with global sales reportedly topping $8.1 billion for the 12 months to September 2000, a 19% increase over the previous 12 months. The sale of insulin accounts for 30% of the market and much emphasis is currently being placed on the development of various non-injectable insulin (click here for a review of this field). Oral antidiabetic drugs are however the leading class of drugs used to treat the disease, accounting for almost 63% of sales.

Oral antidiabetic drugs have traditionally focussed on metformin and sulphonylurea. Until 1995, the sulfonylurea class of drugs which act by increasing insulin secretion was the only choice in the United States other than insulin for treating type 2 diabetes. The explosion of drugs available for controlling blood glucose began when Glucophage (metformin) became available in 1995, quickly followed by the approval of the insulinotropic agent Repaglinide in 1997 and the thiazolidinedione insulin sensitizers such as Avandia and Actos, which were both launched in 1999.

GLP-1 has attracted attention of researchers as a new approach to treat diabetes. GLP-1 is a gut hormone released after food consumption to stimulate insulin secretion. GLP-1 has a very short half-life due to its degradation by the proteolytic enzyme dipeptidyl peptidase-IV (DPP-IV; CD26) which cleaves peptides after proline residues. Therefore, specific inhibition of DPP-IV is an attractive therapeutic approach to stimulate glucose dependent insulin secretion. Indeed Novartis’ LAF 237A is currently in Phase 3 of clinical development (see today's press release evaluating the efficacy of LAF 237 in combination with metformin), while a second of the company’s DPP-IV has been shown in a double-blind, multicenter study, of type 2 diabetics to reduce glucose levels without causing significant hypoglycemia. In addition to its role in glucose homeostasis, DPP-IV has been implicated in immune disorders, HIV-1 infection and tumor progression and hence its inhibitors are of immense potential value.

As well as LAF 237A, P32/98 has also been shown to dramatically improve glucose tolerance in animal models of hyperglycemia and is also now in phase II evaluation in humans. In December 2003, Probiodrug announced preliminary data from the first phase IIa trial of its second development candidate P93/01 in diabetics. The double-blind, placebo-controlled, cross-over trial, performed in Germany with 16 drug-naïve diabetes patients, showed a reduction of meal-induced glucose excursions. In a recent study researchers at the Technical University of Munich have demonstrated that the oral activity of such DPP-IV inhibitors is due in part to their specific absorption by the small intestinal di- and tri-peptide uptake transporter, PEPT1. Furthermore this group has developed a series of assays capable of rapidly screening for other peptidic molecules able to bind to and traverse the intestinal wall on PEPT1 thereby establishing a system for optimizing the oral activity of peptidomimetic drugs.

The screening system consisted of a primary assay that determined the apparent affinity of test compounds for interaction with PEPT1 based on competition with the uptake of the radiolabelled dipeptide D-Phe-Ala in Pichia pastoris yeast cells expressing the transporter. A secondary functional screen then evaluated electrogenic transport of the test compound by recording inward currents induced by the compounds in Xenopus laevis oocytes expressing PEPT1 thereby distinguishing molecules that simply bind the protein from those that are actively transported by it.

The Munich group screened a wide range of different dipeptides and structurally related compounds. In the primary screen, dipeptides and tripeptides as well as Ile-thiazolidide and His-thiazolidide were able to reduce the uptake of D-Phe-Ala. The EC50 for Ile-thiazolidide (500nM) was of a similar order of magnitude to that of the natural substrate gly-gly. Di- and tri-peptidyl thiazolidides displayed considerably reduced EC50 values. Hence using this assay it is possible to optimize the peptide moiety linked to the thiazolidides with respect to PEPT1 binding. Using the secondary screen it was shown that Ile-thiazolidide stimulates PEPT1 transport activity with an EC50 similar to that in the primary screen. In contrast other petidyl thiazolidides such as glu-gly-thiazolidide bound to PEPT1 but did not stimulate transport activity

In addition to PEPT1, a second transporter, PEPT2 is responsible for reabsorption of peptides after glomerular filtration in the kidney and also transport within tissues including the lung and glia cells of the central nervous system. Although the thiazolidides, notably Ile-thiazolidide and Val-thiazolidide, bound to PEPT2 this did not result in transport activity. Hence by modeling the peptide group of the aminoacyl thiazolidide a molecule can be designed that for example does not penetrate the CNS thus improving pharmacokinetics and reducing the risk of adverse effects.

Although this study evaluated transport of Ile-thiazolidide and related molecules in order to evaluate the PEPT assays rather than to investigate the mechanism of, or to optimize this therapeutic candidate, the study does open to the way to further development of the peptidomimetic thiazolidides.

As mentioned above DPP-IV inhibitors have been implicated in the progression of cancer. Lung cancer is one type of cancer that is particularly difficult to treat and improving drug delivery technology is viewed as a major step towards improved options in the treatment of cancers including lung cancers. It therefore remains an intriguing possibility that by modifying the peptidyl thiazolidides so that they are more effectively transported across the airway epithelium on the PEPT2 transporter it may be possible to develop new generation anti-cancer drugs. It is envisioned that such drugs could be administered by inhalation and which effectively reach their desired site of action.

This study thus demonstrates how, by modifying the ability of peptidyl thiazolodides to be transported by PEPT1 or 2 proteins the pharmacokinetic properties and targeting of these therapeutics can be modified. The study evaluated only a few molecules and either by modeling the thiazolodide moiety or the amino acid residues it is clear that a large library of candidate therapeutics could be generated. The assays described here would facilitate the screening of such a library and could therefore lead to improvement to Ile-thiazolidide as an anti-diabetic treatment or for other indications. The utility of the currently technology however extends to other therapeutics including poorly absorbed drugs which can be turned into rapidly and efficiently transported compounds by rendering them into PEPT-substrates. This has been exemplified by the antiviral nucleoside acyclovir which can be esterified with L-valine to improve its pharmacokinetics. Hence this technology stands to open up important new avenues in the field of drug delivery.


Entry date Thursday, June 10, 2004

Adapted from Foltz et al, J Pharmacol Exp Ther. 2004 Mar 29 [Epub ahead of print].
 

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