Factors limiting the oral bioavailability of N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) and enhancement of absorption in rats by delivery in a water-in-oil microemulsion

doi:10.1016/S0378-5173(00)00349-5

International Journal of Pharmaceutics

Volume 199, Issue 1, 10 April 2000, Pages 17-28

https://doi.org/10.1016/S0378-5173(00)00349-5 Get rights and content

Abstract

The bioavailability (BA) of radio-labelled N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) was low when administered by oral gavage as an aqueous solution to conscious male Sprague–Dawley rats (8.3±4.4% (mean±S.D., n=3)). To assess the likely factors contributing to the poor BA of GMDP, the stability of GMDP in the lumen of the gastrointestinal (GI) tract was examined in vitro, using ex vivo GI contents. GMDP was degraded by the contents of the small intestine, caecum and large intestine but was more stable in stomach contents. The permeability coefficient (p_app) of GMDP in isolated sections of rabbit ileum was 1.67×10⁻⁶ cm/s in the mucosal to serosal direction and was not significantly different in the serosal to mucosal direction, indicating that GMDP is poorly permeable and passively transported across the intestinal wall. First pass metabolism was considered to be unlikely to be the primary limitation to the oral bioavailability of GMDP and therefore, that the oral bioavailability of GMDP was likely limited by instability in the lumen of the gastrointestinal tract and low intestinal permeability. A water-in-oil (w/o) microemulsion formulation subsequently developed to address these problems was trialed in a preliminary bioavailability study in rats and enhanced the bioavailability of GMDP ten-fold when administered intraduodenally, indicating that w/o microemulsions may represent a viable mechanism for enhancing the bioavailability of poorly GI-stable and poorly permeable peptide-based molecules.

Introduction

N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) is an N-acetylglucosamine analogue of muramyl dipeptide (MDP). MDP is the smallest fragment of the bacterial cell wall which initiates an immune response in man (Ellouz et al., 1974). MDP and MDP analogues have been identified as having potential clinical utility as vaccine adjuvants (Ellouz et al., 1974), immunostimulators (Tsubura et al., 1988), anti-cancer agents (Utsugi et al., 1991) and antiinflammatory agents (Leclerc et al., 1979) however, MDP is highly pyrogenic in man (Dinarello et al., 1978). GMDP retains the favourable activity of MDP but is less pyrogenic.

Oral administration is desirable for most therapeutic agents, including peptides and proteins, as it avoids the pain and risk of infection associated with parenteral administration and thereby leads to greater patient compliance. The development of peptides and proteins as oral formulations has however, been limited by their low bioavailability when administered via this route. In this regard, initial studies examining the oral bioavailability of (¹⁴C)GMDP in beagle dogs have indicated that the absolute bioavailability of GMDP is low (6.7%, unpublished data), however, limited information is available regarding the factors contributing to the poor oral bioavailability.

The low oral bioavailability of GMDP was confirmed in the current study in the rat. The stability of GMDP in the lumen of the gastrointestinal tract and intestinal permeability were subsequently examined and the data reported here lead to the conclusion that the oral bioavailability of GMDP is limited primarily by instability in the lumen of the gastrointestinal tract and low intestinal permeability. The dissolution of GMDP in an aqueous environment is unlikely to contribute to the low oral bioavailability as the peptide is highly water-soluble (1 g of peptide is dissolved in 0.9 ml of water). Consequently formulation strategies which protect the compound from breakdown in the gut and increase the intestinal permeability will likely enhance the oral bioavailability of GMDP.

In the light of these findings, a water-in-oil microemulsion system similar to that previously reported to enhance the oral bioavailability of peptides in rats (Constantinides et al., 1994, Constantinides, 1995, Constantinides et al., 1996) was selected as a prototype delivery system with which to conduct a preliminary bioavailability study. The data described indicate that water-in-oil microemulsion systems may improve the delivery characteristics of peptides when enzymatic/chemical degradation in the lumen of the gut and limited intestinal permeability are the cause of reduced oral bioavailability.

Section snippets

Materials

Three batches of [¹⁴C]GMDP (specific activity of 175 μCi/μmole, 194 μCi/μmole and 182 μCi/μmole and one batch of (³H)GMDP (specific activity 1.27 Ci/mmole) were custom synthesised by Auspep Pty. (Parkville, Vic., Australia). Peptech (North Ryde, NSW, Australia) supplied the GMDP. (¹⁴C)mannitol (Cat No. NEC 314), (³H)mannitol (Cat No. NET 101) and (¹⁴C]diazepam (Cat No. NET 564) were obtained from NEN (Boston, MA). Captex 355 and Capmul MCM were obtained from Abitec (Janesville, WI). Tween 80

Pharmacokinetics of GMDP following intravenous administration

The plasma concentration-time profile for parent (¹⁴C)GMDP after i.v. administration (mean±S.D., n=3) is shown in Fig. 1. Total ¹⁴C activity is also shown and expressed as ng GMDP equivalents. Total ¹⁴C activity may also represent activity associated with species other than [¹⁴C]GMDP and therefore, total ¹⁴C activity in ng GMDP equivalents will only accurately express GMDP concentration if the only labelled species present is GMDR Limited deviation of the total ¹⁴C activity trace from the

Discussion

The low absolute oral bioavailability of (¹⁴C)GMDP (8.3±4.4%) found in the current study confirms previous unpublished studies for GMDP in beagle dogs (oral bioavailability 6.7%, unpublished data, Peptech, Sydney) and is consistent with the low oral bioavailability of many peptide-like drugs. The oral bioavailability of peptide-like drugs may be limited by poor dissolution, breakdown in the gastrointestinal (GI) tract prior to absorption, significant first pass metabolism or limited

Acknowledgements

The authors wish to thank Peptech Limited (Australia) for funding, Dr Fiona Dunagan for assessment of this manuscript and Richard Gleisner for his technical assistance.

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Factors limiting the oral bioavailability of N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) and enhancement of absorption in rats by delivery in a water-in-oil microemulsion

Abstract

Introduction

Section snippets

Materials

Pharmacokinetics of GMDP following intravenous administration

Discussion

Acknowledgements

J. Pharm. Sci.

Biochem. Biophys. Res. Commun.

Peptides

Cell. Immunol.

Peptides

Lipid microemulsions for improving drug dissolution and oral absorption: physical and biopharmaceutical aspects

Pharm. Res.

Formulation and intestinal absorption enhancement evaluation of water-in-oil microemulsions incorporating medium-chain glycerides

Pharm. Res.

Water-in-oil microemulsions containing medium-chain fatty acids/salts: formulation and intestinal absorption enhancement evaluation

Pharm. Res.

The pyrogenicity of the synthetic adjuvant muramyl dipeptide and two structural analogues

J. Infect. Dis.

The effect of beta-turn structure on the passive diffusion of peptides across Caco-2 cell monolayers

Pharm. Res.