Long-Circulating Vasoactive 1,18-Octadecanedioic Acid-Terlipressin Conjugate

Abstract

Hepatorenal syndrome (HRS) is a life-threatening complication of end-stage liver disease first reported over a century ago, but its management still poses an unmet challenge. A therapeutic agent found to stabilize the condition is a short cyclic peptide, vasopressin analogue, terlipressin (TP). While TP is commonly prescribed for HRS patients in most parts of the world, it was only recently approved for use in the United States. TP exhibits short circulation half-lives and adverse side effects associated with the dose required. Herein, we present a 1,18-octadecanedioic acid (ODDA) conjugate of the cyclic peptide (ODDA-TP), which enables noncovalent binding to serum albumin via native fatty acid binding modes. ODDA-TP is demonstrated to outperform TP alone in studies including in vitro cellular receptor activation, stability in plasma, pharmacokinetics, and performance in vivo in rats. Specifically, ODDA-TP had an elimination half-life 20 times that of TP alone while exhibiting a superior safety profile.

Figure 1

Design of ODDA–TP and albumin binding. (A) Schematic representation of ODDA–TP bound to a hydrophobic channel in HSA with favorable electrostatic contacts shown at the base of the channel as for native fatty acids. (B) The binding isotherm for ODDA–TP and HSA fits a multiple-site model. (C) High and low binding affinities were identified.

Figure 2

Comparison of TP and ODDA–TP in cell-based assays. V1 receptor activation as indicated by the calcium flux assay in SMCs stimulated by either TP (A) or ODDA–TP (B). The results indicate a dose-dependent effect for both the peptide and the conjugate. (C) The ability of the peptides to bind to the V1a receptor expressed in HEK-293T cells was measured using a flow cytometry-based binding competition assay employing Alexa647-PhAcALVP. (D) Peptide activity was tested using a cell-based pCRE β-gal reporter gene assay that measures Gq activation downstream of GPCR activation.

Figure 3

Plasma stability of TP and ODDA–TP. Remaining amount of test agent following incubation in plasma from (A) rat, (B) minipig, or (C) human was quantified by LC–MS. TP was rapidly degraded in rat and minipig plasma. ODDA–TP remained stable in all three plasma samples. F = female animals, M = male animals. No gender-related differences were observed.

Figure 4

(A) PK profile of TP and ODDA–TP administrated at 150 nmol kg^–1 to male Sprague-Dawley rats by IV injection. TP plasma concentrations were quantifiable up to 0.5 h. ODDA–TP plasma concentrations were quantifiable up to the terminal time point in the study, 24 h. A value of 16.5 h was obtained as the elimination half-life time of ODDA–TP. Metabolite identification data demonstrate the improved serum stability of the conjugate as TP starts degrading immediately (B), while ODDA-TP remains intact for 24 h (C) following injection to the animals.

Figure 5

Radiotelemetry recordings of (A) systolic blood pressure, (B) diastolic blood pressure, (C) mean arterial pressure, and (D) heart rate following IV injection of either TP (2 mg kg^–1) or ODDA–TP (2.48 mg kg^–1) to Sprague-Dawley rats.