Kazimierz Wisniewski

Novel d-amino acid tetrapeptides produce potent antinociception by selectively acting at peripheral κ-opioid receptors,

Kappa-(kappa) opioid receptors are widely distributed in the periphery and activation results in antinociception; however supraspinal acting kappa-agonists result in unwanted side effects. Two novel, all d-amino acid, tetrapeptide kappa-opioid receptor agonists, FE 200665 and FE 200666, were identified and compared to brain penetrating (enadoline) and peripherally selective (asimadoline) kappa-agonists as potential analgesics lacking unwanted central nervous system (CNS) side effects. In vitro characterization was performed using radioligand binding and GTP gamma S binding. Antinociception was evaluated in both mice and rats. Rotarod tests were performed to determine motor impairment effects of the kappa-agonists. FE 200665 and FE 200666 showed high affinity for human kappa-opioid receptor 1 (Ki of 0.24 nM and 0.08 nM, respectively) and selectivity for human kappa-opioid receptor 1 (human kappa-opioid receptor 1/human mu-opioid receptor/human delta-opioid receptor selectivity ratios of 1/16,900/84,600 and 1/88,600/>1,250,000, respectively). Both compounds demonstrated agonist activity in the human kappa-opioid receptor 1 [35S]GTP gamma S binding assay (EC50 of 0.08 nM and 0.03 nM) and resulted in dose-related antinociception in the mouse writhing test (A50: 0.007 and 0.013 mg/kg, i.v., respectively). Markedly higher doses of FE 200665 and FE 200666 were required to induce centrally-mediated effects in the rotarod assay (548- and 182-fold higher doses, respectively), and antinociception determined in the mouse tail-flick assay (>1429- and 430-fold fold higher doses, respectively) after peripheral administration supporting a peripheral site of action. The potency ratios between central and peripheral activity suggest a therapeutic window significantly higher than previous kappa-agonists. Furthermore, FE 200665 has entered into clinical trials with great promise as a novel analgesic lacking unwanted side effects seen with current therapeutics.

Pharmacological Characterization of FE 202158, a Novel, Potent, Selective, and Short-Acting Peptidic Vasopressin V1a Receptor Full Agonist for the Treatment of Vasodilatory Hypotension

FE 202158, ([Phe2,Ile3,Hgn4,Orn(iPr)8]vasopressin, where Hgn is homoglutamine and iPr is isopropyl), a peptidic analog of the vasoconstrictor hormone [Arg8]vasopressin (AVP), was designed to be a potent, selective, and short-acting vasopressin type 1a receptor (V1aR) agonist. In functional reporter gene assays, FE 202158 was a potent and selective human V1aR agonist [EC50 = 2.4 nM; selectivity ratio of 1:142:1107:440 versus human vasopressin type 1b receptor, vasopressin type 2 receptor (V2R), and oxytocin receptor, respectively] contrasting with AVPs lack of selectivity, especially versus the V2R (selectivity ratio of 1:18:0.2:92; human V1aR EC50 = 0.24 nM). This activity and selectivity profile was confirmed in radioligand binding assays. FE 202158 was a potent vasoconstrictor in the isolated rat common iliac artery ex vivo (EC50 = 3.6 nM versus 0.8 nM for AVP) and reduced rat ear skin blood flow after intravenous infusion in vivo (ED50 = 4.0 versus 3.4 pmol/kg/min for AVP). The duration of its vasopressor effect by intravenous bolus in rats was as short as AVP at submaximally effective doses. FE 202158 had no V2R-mediated antidiuretic activity in rats by intravenous infusion at its ED50 for reduction of ear skin blood flow, in contrast with the pronounced antidiuretic effect of AVP. Thus, FE 202158 seems suitable for treatment of conditions where V1aR activity is desirable but V2R activity is potentially deleterious, such as vasodilatory hypotension in septic shock. In addition to the desirable selectivity profile, its short-acting nature should allow dose titration with rapid onset and offset of action to optimize vasoconstriction efficacy and safety.

Unlike arginine vasopressin, the selective V1a receptor agonist FE 202158 does not cause procoagulant effects by releasing von Willebrand factor.

Objective:To compare the effects on von Willebrand factor release of the mixed vasopressin type 1a and type 2 receptor agonist arginine vasopressin and the selective vasopressin type 1a receptor agonist FE 202158, [Phe2,Ile3,Hgn4,Orn(iPr)8]vasopressin, at doses required for the treatment of septic shock. Design:Prospective, randomized, controlled laboratory experiment. Setting:University animal research facility. Subjects:Twenty-four chronically instrumented sheep. Interventions:After a 5-day recovery from instrumentation, sheep were randomly assigned to receive a single intravenous bolus of the selective vasopressin type 2 receptor agonist desmopressin (1 nmol·kg−1) or continuous intravenous infusions of arginine vasopressin (3 pmol·kg−1·min−1), the selective vasopressin type 1a receptor agonist FE 202158 (10 pmol·kg−1·min−1), or vehicle (0.9% NaCl) (n = 6 each). Measurements and Main Results:The von Willebrand factor antigen activity relative to hemoglobin concentration (vWF:Ag/Hb ratio) was measured at different time points during the 120-min study period. Maximal vWF:Ag/Hb ratio expressed as percentage of baseline level was significantly increased compared to vehicle-infused animals (3 ± 2%) in the desmopressin (40 ± 6%, p < .001) and arginine vasopressin groups (25 ± 4%, p < .001). The ratio for the FE 202158 group was not statistically different from the sham group (9 ± 2%, p = .208). Notably, maximal vWF:Ag/Hb ratio was lower in the FE 202158 than the arginine vasopressin group (p < .005). Conclusions:Unlike the mixed vasopressin type 1a receptor/vasopressin type 2 receptor agonist arginine vasopressin, the selective vasopressin type 1a receptor agonist FE 202158 does not release von Willebrand factor. Because von Willebrand factor is involved in coagulatory and inflammatory pathways during septic shock, future studies should clarify the role of the vasopressin type 2 receptor–mediated von Willebrand factor increase by arginine vasopressin and the potential benefit of selective vasopressin type 1a receptor–agonists like FE 202158.

Pharmacological characterization of FE 201874, the first selective high affinity rat V1A vasopressin receptor agonist

Distinct vasopressin receptors are involved in different physiological and behavioural functions. Presently, no selective agonist is available to specifically elucidate the functional roles of the V1A receptor in the rat, one of the most widely used animal models. FE 201874 is a new derivative of the human selective V1A receptor agonist F180. In this study, we performed a multi‐approach pharmacological and functional characterization of FE 201874 to determine whether it is selective for V1A receptors.

Synthesis and Biological Activity of Terlipressin and its Putative Metabolites

Introduction Terlipressin (H-Gly3-LVP, GLYPRESSIN ® [1]), 1, is a peptide drug approved in some European and Asian countries for the treatment of bleeding esophageal varices and in France for hepatorenal syndrome. It presumably acts on the vasopressin 1a receptor (V1a-R), whose endogenous ligand is arginine vasopressin (AVP), to increase peripheral vascular resistance leading to increase in arterial blood pressure (ABP). Although 1 and AVP have both been used clinically to correct syndromes of inappropriate vasodilatation, 1 has much longer onset and duration of action [2,3]. It is hypothesized that 1 acts as a pro-drug and that its onset and duration of action are due to successive cleavage of the glycine N-terminal moieties to ultimately produce lysine vasopressin (LVP, 4). Each of the putative metabolites 2-4 would be biologically active. To determine the biological activity of the metabolites and further investigate the mechanism of action of 1, the peptides 1-4 were synthesized and tested both in vivo and in vitro.

Selective and non-selective OT receptor agonists induce different locomotor behaviors in male rats via central OT receptors and peripheral V1a receptors

Oxytocin (OT) continues to inspire much research due to its diverse physiological effects. While the best-understood actions of OT are uterine contraction and milk ejection, OT is also implicated in maternal and bonding behaviors, and potentially in CNS disorders such as autism, schizophrenia, and pain. The dissection of the mechanism of action of OT is complicated by the fact that this peptide activates not only its cognate receptor but also vasopressin type 1a (V1a) receptors. In this study, we evaluated OT and a selective OT receptor (OTR) agonist, FE 204409, in an automated assay that measures rat locomotor activity. The results showed: 1) Subcutaneous (sc) administration of OT decreased locomotor behavior (distance traveled, stereotypy, and rearing). This effect was reversed by a V1a receptor (V1aR) antagonist ([Pmp1,Tyr(ME)2]AVP, sc), suggesting that OT acts through peripheral V1aR to inhibit locomotor activity. 2) A selective OTR agonist (FE 204409, sc) increased stereotypy. This effect was reversed by an OTR antagonist dosed icv, suggesting a central OTR site of action. Our findings identify distinct behavioral effects for OT and the selective agonist FE 204409, adding to the growing body of evidence that the V1aR mediates many effects attributed to OT and that peptides administered systemically at supra-physiological doses may activate receptors in the brain. Our studies further emphasize the importance of utilizing selective agonists and antagonists to assess therapeutic indications.

In Vitro Evaluation of New, Potent, and Selective V2 Receptor Agonists

The vasopressin analogue desmopressin (dDAVP, 1) is a potent V2 receptor agonist that also activates the related V1b receptor [1]. dDAVP is approved in many countries for the treatment of diabetes insipidus, primary nocturnal enuresis, nocturia, and coagulation disorders including hemophilia A and von Willebrand’s disease. In search of novel, potent, selective and pharmacologically useful peptidic V2R agonists, we synthesized a series of C-terminally truncated [Val4]dDAVP (2) [2] analogs modified in positions 2, 3, 7 and/or at the disulfide bridge. The peptides were evaluated for in vitro potency at the humanV2 receptor (hV2R) and selectivity versus related receptors (hV1aR, hV1bR, hOTR). Here we present comprehensive in vitro data for the new compounds and describe synthetic methods used to prepare the analogues.