Interaction between pivaloylcarnitine and l-carnitine transport into L6 cells overexpressing hOCTN2

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Abstract

Patients ingesting pivalic acid containing prodrugs develop hypocarnitinemia. Pivalic acid is cleaved from such drugs and excreted renally as pivaloylcarnitine. Plasma concentrations (reflecting the concentration in the glomerular filtrate entering the proxinmal tubule) in patients treated with cefditoren pivoxil are approximately 5 μM for pivaloylcarnitine and 10 μM for carnitine. Kinetic studies were performed using L6 cells overexpressing the human kidney carnitine transporter (hOCTN2) to assess the mechanisms leading to hypocarnitinemia in such patients. l-carnitine transport showed saturation kinetics (Km 6.3 μM) and could be inhibited competitively by pivaloylcarnitine (Ki 70 μM). Pivaloylcarnitine was also transported by OCTN2 (Km 212 μM) and its transport could be inhibited competitively by l-carnitine (Ki 7.8 μM). Haldane and Eadie-Hofstee plots were linear for both carnitine and pivaloylcarnitine. Our data indicate that both carnitine and pivaloylcarnitine bind to OCTN2 at a single, identical site. Considering the low plasma and tubular pivaloylcarnitine concentration, the high Km of pivaloylcarnitine regarding OCTN2 and the inhibition of pivaloylcarnitine transport by carnitine, pivaloylcarnitine is unlikely to be reabsorbed under these conditions. On the other hand, our data indicate that the renal reabsorption of carnitine is not impaired in patients treated with pivalic acid containing prodrugs. Hypocarnitinemia in such patients therefore develops due to massive renal losses of pivaloylcarnitine and not due to inhibition of carnitine reabsorption by pivaloylcarnitine.

Introduction

To improve the bioavailability of drugs, hydroxyl groups can be esterified with carboxylic acids [1]. After having been absorbed, such prodrugs are rapidly hydrolyzed by carboxylesterases in enterocytes, liver or blood [2], [3]. Pivalic acid (trimethyl acetic acid, Fig. 1) is one of the carboxylic acids used for this purpose, and cefditoren pivoxil, as well as cefetamet pivoxil, are examples for such prodrugs. After hydrolysis of the ester bond, the pivalic acid formed can undergo glucuronidation or activation to pivaloyl-CoA, which can be converted to the respective carnitine ester [4], [5], [6], [7]. In contrast to the CoA esters, acylcarnitines such as pivaloylcarnitine can be exported from organelles (e.g. mitochondria) and from cells, enter the plasma and be excreted by the kidneys [8].

Soon after the introduction of pivalic acid containing prodrugs on the market, patients with very low carnitine plasma concentrations have been described [5], [8]. Detailed investigations in healthy volunteers demonstrated that the decrease in the plasma carnitine concentration appears within 1 week after starting and is completely reversible within days after stopping cefditoren pivoxil [4]. Since carnitine is filtered by the kidneys [9] and very efficiently reabsorbed in the proximal tubule by the sodium-dependent carnitine carrier OCTN2 [10], [11], an interaction between pivaloylcarnitine and carnitine could be suspected. Since children with systemic carnitine deficiency, a disease associated with mutations in the OCTN2 gene and a loss of function of the corresponding protein [10], [11], can have a complete loss of renal reabsorption of carnitine [12], OCTN2 appears to be the only significant renal carnitine transporter in humans. In vitro, it has clearly been demonstrated that acylcarnitines can impair the transport of carnitine by OCTN2 [13], [14], [15]. Regarding the structural similarity between acylcarnitines and carnitine (Fig. 1), this interaction may be competitive. In support of this assumption, a competitive antagonism between the carnitine structural analogue trimethylhydrazoniumpropionate and carnitine transport into rat kidney brush border membrane vesicles has been described [16]. More recently, Okamura et al. [17] reported that pivaloylcarnitine can compete competitively with carnitine transport into LLC-PK1 cells, but that pivaloylcarnitine is not transported by OCTN2 [17]. In a subsequent study by the same research group, the competitive nature of the antagonism between carnitine and pivaloylcarnitine was confirmed and the authors demonstrated that pivaloylcarnitine is indeed transported by OCTN2 [18]. They could not fully explain the development of carnitine deficiency in patients treated with pivalic acid containing prodrugs, however, because they studied only the inhibition of carnitine transport by pivaloylcarnitine, but not the effect of carnitine on the transport of pivaloylcarnitine [18].

We therefore decided to study the kinetics of carnitine and pivaloylcarnitine in L6 cells with stable overexpression of hOCTN2, which we have characterized previously in detail [14]. In contrast to Ohnishi et al. [18], we not only performed a full kinetic characterization of the transport of carnitine, but also of the transport of pivaloylcarnitine by hOCTN2. Our study confirms the results obtained by Ohnishi et al. [18] and at the same time provides the mechanisms responsible for the lack of renal reabsorption of pivaloylcarnitine in patients treated with pivalic acid containing prodrugs.

Section snippets

Reagents

l-[Methyl-3H]-carnitine hydrochloride (3.00 TBq/mmol) was purchased from General Electrics/Amersham-Pharmacia Biotech (Little Chalfont, Buckinghamshire, England), l-carnitine was purchased from Fluka (Buchs, Switzerland), trifluoroacetic acid was from Riedel-de Haen (Buchs, Switzerland). Pivaloylchloride was purchased from ACROS Organics (Chemie Brunschwig, Basel, Switzerland). All other chemicals were from Sigma (Buchs, Switzerland) or Merck (Dietikon, Switzerland).

Synthesis of pivaloylcarnitine and [3H] pivaloylcarnitine

The synthesis and

Synthesis of pivaloylcarnitine

The synthesized products (pivaloylcarnitine and 3H-pivaloylcarnitine) yielded one spot (Rf 0.26) with thin layer chromatography. The purity was >96% for both products synthesized as was approved by NMR analysis and <1% carnitine was detectable in the products.

l-Carnitine kinetics

hOCTN2 is strongly expressed in the kidney [10], [11], and is responsible for the reabsorption of filtered l-carnitine. Accordingly, brush border membrane vesicles isolated from renal cortex have been shown to possess Na+-dependent, high

Discussion

After ingestion of pivalic acid containing prodrugs, pivalic acid is cleaved rapidly and converted to a high extent to pivaloylcarnitine, which enters the plasma and is excreted by the kidney. As shown in our study, pivaloylcarnitine and carnitine are competitive antagonists for binding to and transport with hOCTN2.

As shown in Fig. 3, pivaloylcarnitine inhibits the transport of carnitine by hOCTN2 and could therefore possibly be associated with increased renal excretion of carnitine. In the

Conflict of interest

None declared.

Acknowledgement

The study was supported by a grant of the Swiss National Science Foundation to SK (310000-112483).

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