The effect of amiloride on the in vivo effective permeability of amoxicillin in human jejunum: experience from a regional perfusion technique

Abstract

The purpose of this human intestinal perfusion study (in vivo) was twofold. Firstly, we aimed to determine the effective in vivo jejunal permeability (P_eff) of amoxicillin and to classify it according to the Biopharmaceutics Classification System (BCS). Secondly, we investigated the acute effect of amiloride on the jejunal P_eff of amoxicillin. Amoxicillin, a β-lactam antibiotic, has been reported to be absorbed across the intestinal mucosa by both passive diffusion and active transport. A regional single-pass perfusion of the jejunum was performed using a Loc-I-Gut^® perfusion tube in 14 healthy volunteers. Each perfusion lasted for 200 min and was divided into two periods of 100 min each. The concentration of amoxicillin entering the jejunal segment was 300 mg/l in both periods, and amiloride, an inhibitor of the Na⁺/H⁺ exchanger, was added at 25 mg/l in period 2. The concentrations of amoxicillin and amiloride in the outlet jejunal perfusate were measured with two different HPLC-methods. Antipyrine and [¹⁴C]PEG 4000 were added as internal standards to the perfusion solution. Amiloride had no significant effect on the jejunal P_eff of amoxicillin. The human in vivo jejunal P_eff for amoxicillin was 0.34±0.11×10⁻⁴ and 0.46±0.12×10⁻⁴ cm/s in periods 1 and 2, respectively. The high jejunal P_eff for amiloride was 1.63±0.51×10⁻⁴ cm/s which predicts an intestinal absorption of more than 90%. Following the BCS amoxicillin was classified as a low P_eff drug, and amiloride had no acute effect on the in vivo jejunal P_eff of amoxicillin.

Introduction

Amoxicillin, a β-lactam antibiotic, has been reported to be absorbed across the intestinal mucosa by both passive diffusion and active transport in humans and rats (Paintaud et al., 1992, Chulavatnatol and Charles, 1994, Oh et al., 1992, Tsuji, 1995). The active transport component is most likely mediated through the oligopeptide carrier localised at the apical enterocyte membrane (Paintaud et al., 1992, Chulavatnatol and Charles, 1994, Oh et al., 1992, Tsuji, 1995, Sinko and Balimane, 1998). The oligopeptide carrier is a symport carrier transporting a substrate with a proton across the apical enterocyte membrane. The access of protons for the oligopeptide carrier is a result of a sodium carrier, the Na⁺/H⁺ exchanger, located in the brush-border membrane of the enterocytes (Adibi, 1997). In humans the intestinal absorption of amoxicillin decreased on average from 72±9 to 45±11% when the oral dose increased from 500 to 3000 mg (Paintaud et al., 1992). The dose-dependent intestinal absorption was further supported in patients with ileostomy (inflammatory bowel disease), where the recovery of the dose from the ileostomy increased from 8 to 77% at the lowest and highest dose (375 and 6000 mg), respectively (Sjöwall et al., 1992). An intestinal intubation study in humans showed that amoxicillin was absorbed from various regions in the small intestine, but not from the colon, which agrees with the localisation of the oligopeptide carrier (Barr et al., 1994, Kramer et al., 1995). In rats similar results were obtained in the small intestine where the permeability was saturable and could be competitively inhibited. In addition, the colonic permeability was low and only involved passive diffusion (Tsuji, 1995).

Various factors affecting the intestinal transport through the oligopeptide carrier (PepT1) across the intestinal epithelium are of pharmaceutical and clinical interest, as there exists an increasing number of drugs that are substrates for this carrier, such as β-lactam antibiotics, cephalosporins, antiviral drugs, anticancer compounds, angiotensin-converting enzyme inhibitors and renin inhibitors (Yang et al., 1999). For instance, diet may be one of the most important factors that regulate the activity of intestinal transport of di- and tripeptides. A recent report demonstrated that the up-regulation of di- and tripeptide intestinal transport activity by dietary proteins is due to a transcriptional activation of the PEPT1 gene by selective amino acids and dipeptides in the diet (Shiraga et al., 1999). Another plausible important factor for the activity of the oligopeptide carrier is the modulation of the complex neural network which is a very rapid response and therefore excludes transcription (Berlioz et al., 1999, Berlioz et al., 2000). For instance, Berlioz et al. (1999) proposed that sympathetic noradrenergic fibers, intestinal sensory neurons, and nicotinic synapses are involved in the increased carrier-mediated transport of cephalexin through the oligopeptide carrier caused by the calcium channel blocker nifedipine. In a pharmacokinetic study in humans it has been reported that amiloride, a well-known inhibitor of the Na⁺/H⁺ exchanger, may affect the efficacy of the intestinal peptide transporter. This inhibition was suggested to be mediated by a decreased proton gradient across the intestinal mucosa that usually drives the peptide transporter. This mechanism may have decreased bioavailability of amoxicillin by 27% after oral co-administration of amiloride (Westphal et al., 1995).

Bioavailability of drugs following oral administration is influenced by several factors such as dissolution, transit time, gastrointestinal stability, intestinal permeability and first-pass metabolism in the gut and/or liver. Among these, the intestinal permeability is a major determinant of the fraction of drug absorbed and quantitatively represents the principle membrane transport coefficient of the intestinal mucosa of a drug, which is possible to use regardless of the transport mechanism across mucosa (Csáky, 1984, Amidon et al., 1995). In the present human jejunal perfusion study (in vivo) we have determined the jejunal P_eff of three drugs. This intestinal perfusion technique has been widely applied to investigate drug absorption, presystemic metabolism and drug dissolution (Lennernäs et al., 1992, Lennernäs, 1998, Lennernäs, 1997, Lindahl et al., 1996, Bönlökke et al., 1997). Recently, it has also been used to establish a human permeability data base for the proposed Biopharmaceutical Classification System (BCS) for oral immediate release products (Amidon et al., 1995). These human in vivo P_eff-value correlations, obtained during appropriate physiological conditions, provide a basis for establishing in vitro–in vivo correlation that can be used for predictions of oral drug absorption as well as setting bioequivalence standards for drug approval. BCS takes three major factors into account: solubility and permeability properties of the drugs and as well the dissolution of the immediate release drug product (Amidon et al., 1995). The reference P_eff-values for BCS have been determined in the human proximal jejunum, the intestinal region where the major drug absorption occurs from immediate release dosage form. In subsequent publications we will report in vivo values of jejunal P_eff for drugs representing different BCS, pharmacological and chemical structural classes.

In the present human study one objective was to determine the in vivo jejunal P_eff for amoxicillin and to classify the drug according to the Biopharmaceutics Classification System (BCS). The second objective was to investigate the acute effect of amiloride on the in vivo jejunal P_eff of amoxicillin. Finally, we also determined the in vivo P_eff for amiloride and antipyrine.