In vitro behavior of a phosphate ester prodrug of amprenavir in human intestinal fluids and in the Caco-2 system: Illustration of intraluminal supersaturation

doi:10.1016/j.ijpharm.2006.12.011

International Journal of Pharmaceutics

Volume 336, Issue 2, 24 May 2007, Pages 302-309

https://doi.org/10.1016/j.ijpharm.2006.12.011 Get rights and content

Abstract

As a result of their improved aqueous solubility, the development of phosphate ester prodrugs is an interesting approach to increase intestinal absorption of poorly water-soluble drugs. Absorption of a drug from its phosphate ester prodrug is based on intestinal dephosphorylation of the prodrug which may result in intraluminal supersaturation of the parent drug, followed by an increased absorptive flux across the intestinal mucosa. In this study, we evaluated the behavior of fosamprenavir, a phosphate ester prodrug of amprenavir, in the Caco-2 system and in aspirated human intestinal fluids (HIF), both showing phosphatase activity. Starting from a solution of fosamprenavir in HIF, a supersaturated solution of amprenavir was generated and maintained during a time period sufficient for absorption. Moreover, supersaturation of amprenavir resulted in an enhanced flux across Caco-2 monolayers. To our knowledge, this is the first illustration of supersaturation in real intestinal media. Next, we showed an inhibitory effect of inorganic phosphate on the dephosphorylation of fosamprenavir, both in the Caco-2 model and in HIF. As a consequence, phosphate-buffered media, including fasted state simulated intestinal fluid (FaSSIF), are incompatible with the study of phosphate ester prodrugs and should be replaced with media containing a biorelevant phosphate concentration (0.4–1 mM) and another buffering compound such as 2-morpholinoethanesulfonic acid (MES).

Introduction

As an alternative to formulation-related approaches, synthesis of water-soluble prodrugs is one of the strategies to increase the intestinal absorption of poorly water-soluble drugs (Fleisher et al., 1996). As a result of their relatively straightforward synthesis, inherent chemical stability and potentially improved dissolution/solubility characteristics, phosphate esters may be interesting oral prodrugs of poorly soluble drugs, especially for moderate to high permeability compounds (Heimbach et al., 2003a). Commercialized examples include estramustine phosphate, prednisolone phosphate, fludarabine phosphate, and fosamprenavir. Fosamprenavir is a phosphate ester prodrug of the poorly water-soluble HIV protease inhibitor amprenavir (0.036 mg/ml at pH 7 (Yu et al., 1999)). The calcium salt of fosamprenavir has been commercialized as Telzir^® (Lexiva^®), which should replace the standard amprenavir formulation (Agenerase^®). With the Agenerase formulation, the intraluminal concentration of amprenavir is increased by the solubilizing excipient d-α-tocopheryl polyethyleneglycol 1000 succinate (TPGS) (Yu et al., 1999, Brouwers et al., 2006). Due to high amounts of TPGS required, this formulation suffers from a high pill burden (8 capsules of 150 mg as a standard dose). On the contrary, because of the improved solubility of fosamprenavir (pH-dependent: from 0.3 mg/ml at pH 7 to 54 mg/ml at pH 3.3) (Furfine et al., 2004), only two tablets (equivalent to 2 × 700 mg amprenavir) of the prodrug formulation suffice to obtain similar plasma concentrations of amprenavir (Falcoz et al., 2002, Chapman et al., 2004, Wire et al., 2006).

As transepithelial transport of phosphate ester prodrugs is in general very limited, formation of the parent drug before or during intestinal uptake is a prerequisite for absorption. Dephosphorylation of an orally administered phosphate ester prodrug to its parent drug is mediated by intestinal alkaline phosphatase (EC 3.1.3.1), present in the intestinal lumen and in the apical membrane of enterocytes. The activity of alkaline phosphatase can be affected by various components (e.g. amino acids, metal ions and inorganic phosphate) (Fernley and Walker, 1967, Komoda et al., 1982, Tardivel et al., 1992). Alkaline phosphatase activity in the intestine will therefore depend on intraluminal conditions, especially considering the complex and variable (intra- and inter-subject) composition of intestinal fluids. This dependency potentially affects the intestinal absorption of drugs administered as a phosphate ester prodrug. For instance, inorganic phosphate, a reaction product of dephosphorylation, has been recognized as a competitive inhibitor of alkaline phosphatase (Fernley and Walker, 1967). The physiological impact of this inhibition on phosphatase activity in serum has been investigated (Coburn et al., 1998). However, the potential impact of the modulatory effect of inorganic phosphate on intestinal phosphatase activity, and thus on drug absorption, has, to the best of our knowledge, not been explored yet.

If fast intraluminal dephosphorylation occurs, the higher solubility of a phosphate ester prodrug may result in parent drug concentrations at the intestinal mucosa that exceed the saturation solubility (supersaturation), possibly creating an enhanced absorptive flux. Whether or not a phosphate ester prodrug really increases the absorption of its parent drug will partly depend on the time window during which this intraluminal supersaturation is maintained in relation to the permeability of the parent drug (Heimbach et al., 2003b). Despite its importance, studies on supersaturation in relevant intraluminal conditions are rather limited. Kostewicz et al. (2004) investigated supersaturation and precipitation of weak bases upon entry into the small intestine in simulated intestinal fluids, but to our knowledge, supersaturation has not been studied yet in real intestinal media.

In the present study, we characterized the behavior of the phosphate ester prodrug fosamprenavir applied in the Caco-2 model system and in aspirated human intestinal fluids (HIF). Human Caco-2 monolayers express intestinal alkaline phosphatase at their apical membrane and have previously been used to study the absorption of phosphate ester prodrugs (Heimbach et al., 2003a, Heimbach et al., 2003b). The use of intestinal fluids allowed to explore the influence of real intraluminal conditions on the stability of fosamprenavir. For reasons mentioned above, particular attention was paid to (1) the potential for maintained supersaturation in intraluminal conditions and the resulting effects on transepithelial transport, (2) the impact of modulation of phosphatase activity by physiological inorganic phosphate concentrations and (3) the use of biorelevant conditions in intestinal absorption studies.

Section snippets

Materials

Amprenavir and fosamprenavir calcium were kindly provided by GlaxoSmithKline (Middlesex, UK). Stock solutions were prepared in DMSO and diluted at least 100× before use in experiments. Acetonitrile and methanol were purchased from Fisher Scientific (Leicestershire, UK). BDH Laboratory Supplies (Poole, UK) provided NaH₂PO₄, KH₂PO₄, NaOH and NaCl. Ammonium molybdate tetrahydrate, sodium taurocholate, glucose and 2-morpholinoethanesulfonic acid (MES) were obtained from Sigma–Aldrich (St. Louis,

Fosamprenavir in the Caco-2 system

In a first set of experiments, the behavior of fosamprenavir was evaluated in the Caco-2 system. The stability of fosamprenavir in transport medium at 37 °C was confirmed in advance (data not shown). Upon incubation in the apical compartment of the Caco-2 system (60 min in transport medium pH 6.5), fosamprenavir was converted to amprenavir in a concentration-dependent way: 42 ± 4% at 10 μM, 33 ± 3% at 100 μM and 7 ± 1% at 1000 μM. The conversion increased with increasing apical pH, as illustrated in Fig. 1

Conclusion

As a phosphate ester prodrug as such cannot cross the intestinal epithelium, dephosphorylation to its parent drug by intestinal alkaline phosphatase is a necessary step in the absorption of the parent drug. Despite this importance, certain aspects of the dephosphorylation step are relatively unexplored, especially considering relevant intraluminal conditions. Therefore, we evaluated in this study the behavior of a phosphate ester prodrug of the poorly water-soluble amprenavir in the Caco-2

Acknowledgements

This study was supported by grants from the ‘Fonds voor Wetenschappelijk Onderzoek’ (FWO), Flanders and from the ‘Onderzoeksfonds’ of the K.U. Leuven, Belgium. We also wish to thank Rita Vos (Gastroenterology, University Hospitals Leuven, Belgium) for her assistance during the in vivo studies.

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In vitro behavior of a phosphate ester prodrug of amprenavir in human intestinal fluids and in the Caco-2 system: Illustration of intraluminal supersaturation

Abstract

Introduction

Section snippets

Materials

Fosamprenavir in the Caco-2 system

Conclusion

Acknowledgements

J. Pharm. Sci.

Adv. Drug Deliv. Rev.

Int. J. Pharm.

Int. J. Pharm.

Effect of P-glycoprotein modulators on alkaline phosphatase activity in cultured rat hepatocytes

Cell Physiol. Biochem.

Fosamprenavir: a review of its use in the management of antiretroviral therapy-naive patients with HIV infection

Drugs

Alkaline phosphatase (EC 3.1.3. 1) in serum is inhibited by physiological concentrations of inorganic phosphate

J. Clin. Endocrinol. Metab.

Pharmacokinetics of GW433908, a prodrug of amprenavir, in healthy male volunteers

J. Clin. Pharmacol.

Studies on alkaline phosphatase. Inhibition by phosphate derivatives and the substrate specificity

Biochem. J.