In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V₁ and V₂

Introduction

Terlipressin (Nα-tryglycl-8-lysine-vasopressin), a pro-drug for the endogenous/natural porcine hormone [Lys₈]-vasopressin (LVP),^1,2 is a synthetic vasopressin analog derived from the natural/endogenous human hormone [Arg₈]-vasopressin (AVP). Terlipressin differs from AVP in the substitution of lysine for arginine at the eighth position of endogenous AVP and the addition of three glycyl residues at the amino terminus (Figure 1). Large randomized clinical studies in patients with hepatorenal syndrome type 1 have shown that intravenous administration of terlipressin improves renal function in this setting.^3,4 Terlipressin exerts its effects via its partial vasopressin-1 (V₁) agonist activity and the full V₁ agonist activity of its metabolite, LVP.⁵ Findings from experimental studies conducted in rat and human tissues provide convincing evidence of the rapid, acute vasopressor function of terlipressin following intravenous bolus administration of clinically relevant doses, as well as the longer term vasoconstrictive effects achieved through terlipressin conversion to LVP.⁶

Figure 1 Molecular formulas for terlipressin, lysine-vasopressin, and arginine-vasopressin. Abbreviations: AVP, [Arg8]-vasopressin; LVP, [Lys8]-vasopressin.

V₁ receptors (previously known as V_1a receptors) are located on vascular smooth muscle in the kidney, myometrium, bladder, liver, spleen, platelets, and fat tissue; V₁ receptors mediate vasoconstriction, hepatic glycogenolysis, and platelet aggregation.^7,8 Vasopressin-2 (V₂) receptors are located on the basolateral membrane of the distal tubule and collecting ducts in the kidney; V₂ receptors mediate the antidiuretic effects of vasopressin.⁷ Cleavage of the N-triglycyl residue on terlipressin occurs in vivo, resulting in a prolonged duration of action for terlipressin compared with LVP.⁷ The purpose of this study was to investigate the binding activities and receptor-mediated cellular activities of terlipressin, LVP, and AVP at vasopressin receptor subtypes V₁ and V₂ in vitro. Notably, vasopressin-3 receptors (previously known as V_1b receptors) were not investigated in this study because they are not relevant to the hepatorenal syndrome type 1 disease state.

Materials and methods

Cell lines

Chinese hamster ovary (CHO) cells expressing human V₁ receptors (line CHO-S; study numbers 20404, 21104, and 100009187; Cerep, Celle l’Evescault, France) and human V₂ receptors (line CHO-K1; study numbers 20404, 21104, and 100009187; Cerep)⁹ were used for the competitive binding and functional assays (Table 1).^9,10 The expression of vasopressin receptors was approximately fivefold higher in the V₂ cell line; thus, different amounts of cell membrane proteins or cells were used in the assays (Table 2).

Table 1 Cells and reference compounds Abbreviations: AVP, [Arg8]-vasopressin; Bmax, maximum number of receptor-binding sites; CHO, Chinese hamster ovary; fmol, fentomole; V1, vasopressin-1; V2, vasopressin-2.

Table 2 Experimental conditions Abbreviations: AVP, [Arg8]-vasopressin; cAMP, cyclic adenosine monophosphate; HTRF, homogeneous time-resolved fluorescence; V1, vasopressin-1; V2, vasopressin-2.

Competitive binding assays

Cell membrane homogenates were prepared from cells of the CHO-S and CHO-K1 cell lines (study numbers 20404, 21104, and 100009187; Cerep). [³H]AVP at a concentration of 0.3 nM was added to each cell membrane homogenate (V₁ assay: 40-µg protein; V₂ assay: 16-µg protein), which was then incubated (V₁ assay: 60 minutes; V₂ assay: 120 minutes) at 22°C with various concentrations of terlipressin, LVP, and AVP in assay buffer containing Tris–HCl 50 mM (pH 7.4), MgCl₂ 5 mM, and 0.1% bovine serum albumin (Table 2). The following terlipressin concentrations were tested (molar): 1.0 × 10⁻³, 3.0 × 10⁻⁴, 1.0 × 10⁻⁴, 3.0 × 10⁻⁵, 1.0 × 10⁻⁵, 3.0 × 10⁻⁶, 1.0 × 10⁻⁶, 3.0 × 10⁻⁷, 1.0 × 10⁻⁷, and 1.0 × 10⁻⁸ M. The following LVP and AVP concentrations were tested (molar): 1.0 × 10⁻⁶, 1.0 × 10⁻⁷, 3.0 × 10⁻⁸, 1.0 × 10⁻⁸, 3.0 × 10⁻⁹, 1.0 × 10⁻⁹, 3.0 × 10⁻¹⁰, 1.0 × 10⁻¹⁰, 3.0 × 10⁻¹¹, 1.0 × 10⁻¹¹, and 1.0 × 10⁻¹² M. Nonspecific binding was determined in the presence of AVP 1 µM. All assays were performed in triplicate.

After incubation, samples were filtered rapidly under vacuum through glass fiber filters (GF/B; Packard Bioscience Company, Meriden, CT, USA [now PerkinElmer, Inc., Waltham, MA, USA]) presoaked with 0.3% polyethylenimine and rinsed several times with ice-cold Tris–HCl 50 mM buffer in a 96-sample cell harvester (UniFilter; PerkinElmer, Inc.). The filters were air dried, and the radioactivity retained in the filters was counted in a scintillation counter (TopCount; PerkinElmer, Inc.) in a scintillation cocktail (MicroScint 0; PerkinElmer, Inc.). The standard reference agonist for V₁ was [d(CH₂)₅¹,Tyr(Me)₂]-AVP, and the standard reference agonist for V₂ was AVP. These reference compounds were tested at several concentrations to obtain competition curves from which the concentration achieving half-maximal inhibition concentration (IC₅₀) was calculated.

Receptor-mediated cellular activity assays

Cell cultures were established from the same CHO cell lines used in the binding assays described earlier (Table 1).^9,10 Cells expressing human V₁ receptors were suspended in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Waltham, MA, USA), complemented with 0.1% fetal calf serum, and then distributed in microplates at a density of 45,000 cells per well (Table 2). The fluorescent probe (Fluo4 NW; Thermo Fisher Scientific) was mixed with probenecid in Hank’s balanced salt solution buffer (Thermo Fisher Scientific) complemented with N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) 20 mM buffer (pH 7.4; Thermo Fisher Scientific) and added into each well and equilibrated with the cells for 60 minutes at 37°C, and then for 15 minutes at 22°C. Assay plates were then positioned in a microplate reader (CellLux; PerkinElmer, Inc.) for the addition of various concentrations of terlipressin, LVP, and AVP and for subsequent measurements of activity. The following terlipressin concentrations were tested (molar): 1.0 × 10⁻³, 1.0 × 10⁻⁴, 1.0 × 10⁻⁵, 3.0 × 10⁻⁶, 1.0 × 10⁻⁶, 3.0 × 10⁻⁷, 1.0 × 10⁻⁷, 3.0 × 10⁻⁸, 1.0 × 10⁻⁸, 3.0 × 10⁻⁹, 1.0 × 10⁻⁹, 1.0 × 10⁻¹⁰, and 1.0 × 10⁻¹¹ M. The following LVP and AVP concentrations were tested (molar): 1.0 × 10⁻⁴, 1.0 × 10⁻⁵, 1.0 × 10⁻⁶, 1.0 × 10⁻⁷, 1.0 × 10⁻⁸, 3.0 × 10⁻⁹, 1.0 × 10⁻⁹, 3.0 × 10⁻¹⁰, 1.0 × 10⁻¹⁰, 3.0 × 10⁻¹¹, 1.0 × 10⁻¹¹, 3.0 × 10⁻¹², 1.0 × 10⁻¹², 1.0 × 10⁻¹³, and 1.0 × 10⁻¹⁴ M. All assays were performed in triplicate. Changes in fluorescence intensity reflected changes in the concentration of free cytosolic calcium. The standard reference agonist was AVP.

The activity at V₂ receptors was measured based on cyclic adenosine monophosphate (cAMP) production using the homogeneous time-resolved fluorescence method. Cells expressing human V₂ receptors were suspended in Hank’s balanced salt solution buffer complemented with HEPES 20 mM buffer (pH 7.4), 0.01% bovine serum albumin, and isobutyl-methylxanthine 0.5 mM, and then distributed in microplates at a density of 3000 cells per well and preincubated for 5 minutes at room temperature in the absence (control) or in the presence of the various concentrations of terlipressin, LVP, AVP, and the reference antagonist ([adamantaneacetyl¹, O-Et-d-Tyr², Val⁴, aminobutyryl⁶]-AVP). Next, the reference agonist, AVP, was added to a final concentration of 0.03 nM. After 30 minutes of incubation at room temperature, the cells were lysed, and the fluorescence acceptor (D2-labeled cAMP) and the fluorescence donor (anti-cAMP antibody labeled with europium cryptate) were added. After 60 minutes of incubation at room temperature, the plates were positioned in a microplate reader (RUBYstar; BMG Labtech, Cary, NC, USA), and fluorescence transfer was measured at λex = 337 nM and λem = 620 and 665 nM. The following terlipressin concentrations were tested (molar): 1.0 × 10⁻³, 1.0 × 10⁻⁴, 1.0 × 10⁻⁵, 3.0 × 10⁻⁶, 1.0 × 10⁻⁶, 3.0 × 10⁻⁷, 1.0 × 10⁻⁷, 3.0 × 10⁻⁸, 1.0 × 10⁻⁸, 3.0 × 10⁻⁹, 1.0 × 10⁻⁹, 1.0 × 10⁻¹⁰, and 1.0 × 10⁻¹¹ M. The LVP and AVP concentrations were tested (molar): 1.0 × 10⁻⁴, 1.0 × 10⁻⁵, 1.0 × 10⁻⁶, 1.0 × 10⁻⁷, 1.0 × 10⁻⁸, 3.0 × 10⁻⁹, 1.0 × 10⁻⁹, 3.0 × 10⁻¹⁰, 1.0 × 10⁻¹⁰, 3.0 × 10⁻¹¹, 1.0 × 10⁻¹¹, 3.0 × 10⁻¹², 1.0 × 10⁻¹², 1.0 × 10⁻¹³, and 1.0 × 10⁻¹⁴ M. All assays were performed in triplicate.

The reference compounds for V₁ and V₂ were tested at several concentrations to obtain concentration–response curves from which the concentration producing half-maximal effective concentration (EC₅₀) or IC₅₀ was calculated.

Data analysis

For the binding assays, specific ligand binding to receptors was defined as the difference between the total binding and the nonspecific binding determined in the presence of an excess of unlabeled ligand. Results were expressed as a percentage of control-specific binding (measured specific binding/control-specific binding × 100) and as percent inhibition of control radioligand-specific binding (100 – [measured specific binding/control-specific binding] × 100) obtained in the presence of terlipressin, LVP, or AVP. Selectivity of terlipressin and LVP for V₁ and V₂ receptors was calculated as compared with AVP.

Values for the IC₅₀ and the Hill coefficient (nH) were determined by nonlinear regression analysis of the competition curves generated with mean replicate values using Hill equation curve fitting (Y = D + [A – D]/[1 + (C/C₅₀)^nH], where Y = specific binding, D = minimum specific binding, A = maximum specific binding, C = test article concentration, C₅₀ = IC₅₀, and nH = slope factor). Inhibition constants (K_i) were calculated from the Cheng Prusoff equation (K_i = IC₅₀/1+[L/KD], where L = concentration of radioligand in the assay and KD = affinity of the radioligand for the receptor).

For the functional assays, results were expressed as a percentage of control agonist response (measured response/control response × 100) in the presence of the test article. The EC₅₀ values were determined by nonlinear regression analysis of the functional assay curves generated with mean replicate values using the Hill equation curve fitting (Y = D + [A – D]/[1 + (C/C₅₀)^nH], where Y = response, D = right asymptote of the curve, A = left asymptote of the curve, C = test article concentration, C₅₀ = EC₅₀, and nH = slope factor). The results for V₁ were expressed as a percentage of the control response to AVP 1 µM, and the results for V₂ were expressed as percent inhibition of the control response to AVP 0.03 nM.

Results

Binding affinity

Results of the binding affinity assays are summarized in Table 3 and illustrated in Figure 2. The potency of binding affinity of the tested compounds to both V₁ and V₂ receptors was AVP>LVP>>terlipressin. For V₁ receptors, the binding affinity of LVP was about twofold less than that of AVP, and the binding affinity of terlipressin was about 600-fold less than that of LVP. For V₂ receptors, the binding affinity of LVP was about 12-fold less than that of AVP, and the binding affinity of terlipressin was about 700-fold less than that of LVP.

Figure 2 Binding affinity as a function of concentration for the tested compounds. Abbreviations: AVP, [Arg8]-vasopressin; LVP, [Lys8]-vasopressin; V1, vasopressin-1; V2, vasopressin-2.

Table 3 Binding affinity assay results Abbreviations: AVP, [Arg8]-vasopressin; IC50, half-maximal inhibition concentration; Ki, inhibition constant; LVP, [Lys8]-vasopressin; V1, vasopressin-1; V2, vasopressin-2.

AVP bound V₁ and V₂ receptors with similar high affinities (8.0 × 10⁻¹⁰ and 8.5 × 10⁻¹⁰, respectively). LVP also bound V₁ and V₂ receptors with high affinity, but affinity for V₁ was about sixfold higher than that for V₂ (1.8 × 10⁻⁹ and 1.0 × 10⁻⁸, respectively). Terlipressin bound V₁ and V₂ receptors with low affinity, but affinity for V₁ was about sixfold higher than that for V₂ (1.1 × 10⁻⁶ and 6.9 × 10⁻⁶, respectively).

Cellular activity

Results of the vasopressin-mediated functional activity assays are summarized in Table 4 and illustrated in Figure 3. The order of potency for cell activation was the same as for the binding assay: AVP>LVP>>terlipressin. LVP was a full agonist at both V₁ and V₂ receptors, producing maximal cell activation about equal to that of AVP. Terlipressin produced maximal cell activation about equal to that of AVP at V₂ receptors, and thus was a full agonist at V₂ receptors, but it produced only 41% of the maximal effect observed with AVP at V₁ receptors, indicating that it is a partial agonist at V₁ receptors.

Figure 3 Vasopressin-mediated cell activation as a function of concentration for the tested compounds. Abbreviations: AVP, [Arg8]-vasopressin; LVP, [Lys8]-vasopressin; V1, vasopressin-1; V2, vasopressin-2.

Table 4 Functional activity assay results Abbreviations: AVP, [Arg8]-vasopressin; EC50, half-maximal effective concentration; Ki, inhibition constant; LVP, [Lys8]-vasopressin; V1, vasopressin-1; V2, vasopressin-2.

Discussion

Terlipressin is a pro-drug for the endogenous/natural porcine hormone LVP.^1,2 In the current study, the results of radioligand competitive binding assays in cell membrane homogenates of CHO cells expressing human V₁ and V₂ receptors demonstrated that terlipressin binds to V₁ and V₂ receptors with much lower affinities than LVP or AVP. Terlipressin was selective for V₁ receptors at a V₁:V₂ ratio of 6:1. Results of the functional assays indicated that terlipressin behaves as a partial agonist at V₁ receptors and a full agonist at V₂ receptors.

Our results support findings from previously reported studies and provide additional evidence regarding the pharmacologic properties of terlipressin.^2,11,12 Colson et al,¹¹ using vascular smooth muscle cells isolated from rat aorta and human uterine artery, found that the relative binding affinity (K_i [nM]) of LVP, AVP, and terlipressin for human and rat V₁ receptors was AVP>LVP>>terlipressin. The mean binding affinity of terlipressin for human V₁ receptors (1229 nM) was about 120-fold less than the binding affinity of LVP (10 nM); about a 700-fold difference in terlipressin (852 nM) versus LVP binding (1.2 nM) was observed at rat V₁ receptors.¹¹ Mean maximal cell activation with terlipressin, as measured by intracellular calcium mobilization, was 48% at human V₁ receptors and 66% at rat V₁ receptors versus 100% with LVP.¹¹ The selectivity of terlipressin for V₁ receptors was 1.9-fold higher than that for V₂ receptors compared with the 6.3-fold higher selectivity observed in our study. This difference may be a reflection of the different cell lines utilized for competitive binding assays (rat and human vascular smooth muscle cells versus CHO cells, respectively) in the two studies. Bernadich et al¹² found that the mean potency of terlipressin for V₁ activation in vasopressor tests in a rat model of portal hypertension (9 IU×µmol⁻¹) was about 66-fold less than the mean potency of vasopressin (614 IU×µmol⁻¹); they reported that terlipressin was selective for V₁ receptors, with a V₁:V₂ ratio of 2.2:1.0 compared with 1:1 for vasopressin. Wisniewski et al² investigated the potency of terlipressin in vitro for V₁ activation in a recombinant system with a functional assay and found that the EC₅₀ (148 nM) was about 150-fold higher than that for LVP (0.93 nM). In an in vivo model in rats, the investigators showed that LVP and AVP were similar in time to effect and magnitude of effect in raising arterial blood pressure when given at identical doses; in contrast, a 100-fold higher dose of terlipressin was required to achieve a similar effect with a slower onset of action.²

Because terlipressin is metabolized to LVP, which is a potent, full V₁ receptor agonist in vivo, our data support the theory that the principal pharmacologic response to terlipressin administration is most likely attributable to the LVP metabolite.¹

Conclusion

These data suggest that the in vivo pharmacologic response to the administration of terlipressin primarily reflects LVP-mediated activation of V₁ receptors. However, terlipressin demonstrated agonist activity at both V₁ and V₂ receptors. Results from the current study provide additional, supportive evidence for previous findings and further establish the binding affinity, potency, and selectivity of terlipressin at vasopressin receptors.

Acknowledgments

This study was sponsored by Mallinckrodt Pharmaceuticals. Michael Morren, RPh, MBA, of Peloton Advantage, LLC, provided administrative and publishing support, funded by Mallinckrodt Pharmaceuticals.

Disclosure

Khurram Jamil and Krishna R Devarakonda are employees of Mallinckrodt Pharmaceuticals. Stephen Chris Pappas is an employee of Orphan Therapeutics, LLC. The authors report no other conflicts of interest in this work.

References

1.		Forsling ML, Aziz LA, Miller M, Davies R, Donovan B. Conversion of triglycylvasopressin to lysine-vasopressin in man. J Endocrinol. 1980;85(2):237–244.
2.		Wisniewski K, Alagarsamy S, Taki H, et al. Synthesis and biological activity of terlipressin and its putative metabolites. In: Blondelle SE, editor. Understanding Biology Using Peptides: Proceedings of the Nineteenth American Peptide Symposium. San Diego, CA: Springer; 2006:489–490.
3.		Sanyal AJ, Boyer T, Garcia-Tsao G, et al; Terlipressin Study Group. A randomized, prospective, double-blind, placebo-controlled trial of terlipressin for type 1 hepatorenal syndrome. Gastroenterology. 2008;134(5):1360–1368.
4.		Boyer TD, Sanyal AJ, Wong F, et al; REVERSE Study Investigators. Terlipressin plus albumin is more effective than albumin alone in improving renal function in patients with cirrhosis and hepatorenal syndrome type 1. Gastroenterology. 2016;150(7):1579–1589.
5.		Saner FH, Canbay A, Gerken G, Broelsch CE. Pharmacology, clinical efficacy and safety of terlipressin in esophageal varices bleeding, septic shock and hepatorenal syndrome. Expert Rev Gastroenterol Hepatol. 2007;1(2):207–217.
6.		Ryckwaert F, Virsolvy A, Fort A, et al. Terlipressin, a provasopressin drug exhibits direct vasoconstrictor properties: consequences on heart perfusion and performance. Crit Care Med. 2009;37(3):876–881.
7.		Kam PC, Williams S, Yoong FF. Vasopressin and terlipressin: pharmacology and its clinical relevance. Anaesthesia. 2004;59(10):993–1001.
8.		Petersen MB. The effect of vasopressin and related compounds at V1a and V2 receptors in animal models relevant to human disease. Basic Clin Pharmacol Toxicol. 2006;99(2):96–103.
9.		Tahara A, Saito M, Sugimoto T, et al. Pharmacological characterization of the human vasopressin receptor subtypes stably expressed in Chinese hamster ovary cells. Br J Pharmacol. 1998;125(7):1463–1470.
10.		Cotte N, Balestre MN, Phalipou S, et al. Identification of residues responsible for the selective binding of peptide antagonists and agonists in the V2 vasopressin receptor. J Biol Chem. 1998;273(45):29462–29468.
11.		Colson PH, Virsolvy A, Gaudard P, et al. Terlipressin, a vasoactive prodrug recommended in hepatorenal syndrome, is an agonist of human V1, V2 and V1B receptors: implications for its safety profile. Pharmacol Res. 2016;113(Pt A):257–264.
12.		Bernadich C, Bandi JC, Melin P, Bosch J. Effects of F-180, a new selective vasoconstrictor peptide, compared with terlipressin and vasopressin on systemic and splanchnic hemodynamics in a rat model of portal hypertension. Hepatology. 1998;27(2):351–356.