On the mechanism of degradation of oxytocin and its analogues in aqueous solution.

Oxytocin (OT) is a cyclic nonapeptide containing one internal disulfide bond between its Cys(1) and Cys(6) residues. Although OT is one of the most commonly used peptidic drugs, the mechanism of its degradation in aqueous solution and the identity of its degradants have not been fully elucidated. To investigate the pathways and products of OT degradation in slightly acidic to neutral solutions, we prepared the peptides: OT, [D-Cys(1)]OT, a series of N-alkylated OT analogues, [[(13)C3,(15) N]Cys(1)]OT, and OT where each sulfur atom was systematically replaced by either methylene, (34)S, or Se. The peptides were incubated at 40°C and the degradation products studied by HPLC, LCMS, and (13)C-NMR. Our findings suggest that the degradation begins with ß-elimination of the disulfide linkage to form a putative intermediate linear peptide containing an S-thiocysteine (a persulfide) in position 6 and a dehydroalanine in position 1. This intermediate persulfide appears to donate a sulfur atom to an intact OT molecule to form OT trisulfide and higher monomeric polysulfides, while the dehydroalanine residue is hydrolyzed with loss of the N-terminal amino group to yield a linear N-pyruvoylated octapeptide containing a reduced Cys(6). Based on the MS and (13)C-NMR data of the products from degradation of [[(13)C3,(15)N]Cys(1)]OT, we postulate that the ultimate degradation products of OT are dimers composed of two pyruvoylated octapeptides held together by one disulfide bridge between the two Cys(6) residues and by one more, non-reducible, linkage resulting from an aldol-type condensation between the two N-terminal pyruvoyl groups.

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, ([Phe(2),Ile(3),Hgn(4),Orn(iPr)(8)]vasopressin, where Hgn is homoglutamine and iPr is isopropyl), a peptidic analog of the vasoconstrictor hormone [Arg(8)]vasopressin (AVP), was designed to be a potent, selective, and short-acting vasopressin type 1a receptor (V(1a)R) agonist. In functional reporter gene assays, FE 202158 was a potent and selective human V(1a)R agonist [EC(50) = 2.4 nM; selectivity ratio of 1:142:1107:440 versus human vasopressin type 1b receptor, vasopressin type 2 receptor (V(2)R), and oxytocin receptor, respectively] contrasting with AVP's lack of selectivity, especially versus the V(2)R (selectivity ratio of 1:18:0.2:92; human V(1a)R EC(50) = 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 (EC(50) = 3.6 nM versus 0.8 nM for AVP) and reduced rat ear skin blood flow after intravenous infusion in vivo (ED(50) = 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 V(2)R-mediated antidiuretic activity in rats by intravenous infusion at its ED(50) 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 V(1a)R activity is desirable but V(2)R 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.

Novel D-amino acid tetrapeptides produce potent antinociception by selectively acting at peripheral kappa-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.

Synthesis and Pharmacological Characterization of Novel Glucagon-like Peptide-2 (GLP-2) Analogues with Low Systemic Clearance.

Glucagon-like peptide-2 receptor agonists have therapeutic potential for the treatment of intestinal diseases. The native hGLP-2, a 33 amino acid gastrointestinal peptide, is not a suitable clinical candidate, due to its very short half-life in humans. In search of GLP-2 receptor agonists with better pharmacokinetic characteristics, a series of GLP-2 analogues containing Gly substitution at position 2, norleucine in position 10, and hydrophobic substitutions in positions 11 and/or 16 was designed and synthesized. In vitro receptor potency at the human GLP-2, selectivity vs the human GLP-1 and GCG receptors, and PK profile in rats were determined for the new analogues. A number of compounds more potent at the hGLP-2R than the native hormone, showing excellent receptor selectivity and very low systemic clearance (CL) were discovered. Analogues 69 ([Gly(2),Nle(10),D-Thi(11),Phe(16)]hGLP-2-(1-30)-NH2), 72 ([Gly(2),Nle(10),D-Phe(11),Leu(16)]hGLP-2-(1-33)-OH), 73 ([Gly(2),Nle(10),D-Phe(11),Leu(16)]hGLP-2-(1-33)-NH2), 81 ([Gly(2),Nle(10),D-Phe(11),Leu(16)]hGLP-2-(1-33)-NHEt), and 85 ([Gly(2),Nle(10),D-Phe(11),Leu(16)]hGLP-2-(1-33)-NH-((CH2)2O)4-(CH2)2-CONH2) displayed the desired profiles (EC50 (hGLP-2R) < 100 pM, CL in rat <0.3 mL/min/kg, selective vs hGLP-1R and hGCGR). Compound 73 (FE 203799) was selected as a candidate for clinical development.

Relaxation processes in a system with logarithmic growth.

We discuss relaxation and aging processes in the one- and two-dimensional ABC models. In these driven diffusive systems of three particle types, biased exchanges in one direction yield a coarsening process characterized in the long-time limit by a logarithmic growth of ordered domains that take the form of stripes. From the time-dependent length, derived from the equal-time spatial correlator, and from the mean displacement of individual particles different regimes in the formation and growth of these domains can be identified. Analysis of two-times correlation and response functions reveals dynamical scaling in the asymptotic logarithmic growth regime as well as complicated finite-time and finite-size effects in the early and intermediate time regimes.

New, potent, and selective peptidic oxytocin receptor agonists.

Mothers of preterm babies frequently have difficulty establishing or maintaining lactation, thought to be due to interference with the milk ejection reflex. Administration of exogenous oxytocin can produce alveolar contraction and adequate breast emptying resulting in establishment of successful lactation. The natural hormone oxytocin is not receptor-selective and may cause hyponatremia via V2 receptor mediated antidiuresis. We have designed a series of potent oxytocin analogues containing N-alkylglycines in position 7 with excellent selectivity versus the related V1a, V1b, and V2 vasopressin receptors and short half-life: agonists 31 ([2-ThiMeGly(7)]dOT), 47 (carba-6-[Phe(2),BuGly(7)]dOT), 55 (carba-6-[3-MeBzlGly(7)]dOT), and 57 (carba-1-[4-FBzlGly(7)]dOT) have EC50 values at hOTR < 0.1 nM, selectivity ratios versus related human vasopressin receptors of >2000, IC50 at hV1aR > 500 nM, and total clearance in rats in the range of 60-80 mL min(-1) kg(-1). Compound 57 (FE 202767) is currently in clinical development for the treatment of preterm mothers requiring lactation support.

New, potent, selective, and short-acting peptidic V1a receptor agonists.

[Arg(8)]vasopressin (AVP) produces vasoconstriction via V(1a) receptor (V(1a)R)-mediated vascular smooth muscle cell contraction and is being used to increase blood pressure in septic shock, a form of vasodilatory hypotension. However, AVP also induces V(2) receptor (V(2)R)-mediated antidiuresis, vasodilation, and coagulation factor release, all deleterious in septic shock. The V(1a)R agonist terlipressin (H-Gly(3)[Lys(8)]VP) also lacks selectivity vs the V(2)R and has sizably longer duration of action than AVP, preventing rapid titration of its vasopressor effect in the clinic. We designed and synthesized new short acting V(1a)R selective analogues of general structure [Xaa(2),Ile(3),Yaa(4),Zaa(8)]VP. The most potent and selective compounds in in vitro functional assays (e.g., [Phe(2),Ile(3),Asn(Me(2))(4),Orn(8)]VP (31), [Phe(2),Ile(3),Asn((CH(2))(3)OH)(4),Orn(8)]VP (34), [Phe(2),Ile(3),Hgn(4),Orn(iPr)(8)]VP (45), [Phe(2),Ile(3),Asn(Et)(4),Dab(8)]VP (49), [Thi(2),Ile(3),Orn(iPr)(8)]VP (59), [Cha(2),Ile(3),Asn(4),Orn(iPr)(8)]VP (68)) were tested by intravenous bolus in rats for duration of vasopressive action. Analogues 31, 34, 45, and 49 were as short-acting as AVP. Compound 45, FE 202158, is currently undergoing clinical trials in septic shock.