Cx43 (connexin 43) – no IC50, Ki, EC50, or DC50 values are reported in these two papers. The cellular target molecule for Rotigaptide is unknown; however, it prevents dephosphorylation of Ser297 and Ser368 on Cx43 during ischaemia. [2][3]

In cultured neonatal ventricular myocytes, rogaptide (100 nM; 24 hours) positively affects the reduction of Cx43 protein levels mediated by cycloheximide [1]. Rotigaptide (100 nM; 24 h) has no effect on the apoptosis of INS-1 cells in and of itself. When the concentration of IL-1b was greater than 15 pg/ml, it significantly decreased apoptosis in cytokine-exposed cells by approximately 10% in insulin-producing rat INS-1 cells [2].

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Rotigaptide (1-1000 nM) produced a dose-dependent increase in Cx43 protein expression in cultured neonatal rat ventricular cardiomyocytes after 24 h, reaching a maximum 3-fold increase at 100 nM (determined by quantitative Western blot analysis with C8093 antibody, normalized to p70 internal control). Treatment with 20 mM LiCl served as a positive control. [2]
Immunocytochemical staining with C6219 antibody showed that 100 nM Rotigaptide markedly increased Cx43 immunoreactivity (9-fold by semiquantitative fluorescence analysis) and Cx43-positive gap junction plaques at cell-cell borders. [2]
In the presence of cycloheximide (10 μg/ml), Cx43 protein levels were reduced to 39% of vehicle; cotreatment with 100 nM Rotigaptide reduced Cx43 levels to 56% of vehicle, suggesting that rotigaptide's effect on Cx43 expression is partly due to increased biosynthesis. [2]
Rotigaptide (50 nM, 5 h) increased GJIC by 40% in HeLa cells expressing Cx43-GFP, with similar findings in neonatal rat cardiomyocytes and HL-1 atrial cells. No effect was observed in HeLa cells expressing Cx32 or Cx26. [3]
Rotigaptide stimulates PKC activity in HeLa-Cx43 cells, an effect blocked by the selective PKC-α inhibitor CGP54345. [3]
In isolated perfused rat hearts, Rotigaptide significantly increased the time to ischaemia-induced asystole and suppressed dephosphorylation of Ser297 and Ser368 at 30 min of ischaemia, as determined by mass spectrometry (MALDI and LC-ESI-MS/MS) phosphorylation analysis on Cx43. [3]
In double-cell patch clamp studies on isolated guinea pig cardiomyocytes, Rotigaptide increased gap junction conductance without affecting membrane conductance. [3]
In isolated rat atrial strips under metabolic stress (hypoxia + glucose deprivation), Rotigaptide concentration-dependently prevented and reverted stress-induced atrial conduction slowing, with a bell-shaped dose-response (maximal efficacy at 10-100 nM). [3]
In isolated rabbit hearts subjected to acidosis (pH 6.0), Rotigaptide significantly reduced increased dispersion of activation-recovery intervals and inhibited conduction slowing in both longitudinal and transverse directions (effect most pronounced transversely, ~60% reduction). [3]

The administration of rotigaptide (IV; 300 mg/kg; single dose) to mice and rats did not result in any toxic effects on either species. In studies involving dose escalation, rats (100 mg/kg) and dogs (10 mg/kg) responded well to continuous intravenous infusion of rotigaptide over a period of 5–14 days. Furthermore, no compound-related side effects or toxicological or histological outcomes were observed [3].
In open-chest dogs following LAD ligation, Rotigaptide (plasma concentrations ~1, 8, 70 nM) prevented induction of re-entrant ventricular tachycardia (VT) with high reproducibility (~90% in saline-treated dogs; all doses of rotigaptide convincingly prevented re-entrant VT). Prevention correlated with reversal of functional unidirectional conduction block. Rotigaptide failed to prevent focal VT/VF (11 out of 13 had focal VT/VF following rotigaptide vs 13 out of 14 after saline), suggesting GJIC plays a minor role in focal arrhythmias. [3]
In open-chest dogs subjected to 1 h LCX ligation followed by 4 h reperfusion, Rotigaptide (administered 10 min prior to reperfusion) significantly suppressed premature ventricular complexes and VT with a maximal reduction of ~95% at the highest dose (P<0.05). [3]
Rotigaptide (30 mg/kg/day for 14 days in rats) significantly reduced infarct size following myocardial infarction in canine ischaemia/reperfusion and rat chronic ischaemia models. [3]
In dog models of AF, Rotigaptide (100 nM plasma level) showed significant anti-arrhythmic effect in chronic mitral regurgitation-induced atrial dilatation (atrial CV increased by 42%) and completely prevented ischaemia-induced increase in AF duration in a selective atrial ischaemia model. In atrial and ventricular tachypacing models, rotigaptide increased atrial conduction (5-10%) but had no anti-arrhythmic effect. [3]

Western Blot Analysis[1]
Cell Types: Neonatal cardiomyocytes
Tested Concentrations: 100 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: Partially prevents the loss of Cx43 protein expression in neonatal cardiomyocytes.

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Apoptosis analysis[2]
Cell Types: Cx43-deficient INS-1 cells
Tested Concentrations: 100 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: Improved cytokine-induced apoptosis and improved mitochondrial function.

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Neonatal rat ventricular cardiomyocyte culture: Hearts from Sprague-Dawley newborn rats were dissociated with trypsin and collagenase, seeded on collagen-coated plates at densities of 65,000-125,000 cells/cm², and cultured in MEM with Earle's salts, glutamine, penicillin/streptomycin, vitamin B12, 10% horse serum, 5% fetal calf serum. On day 5, cells received Rotigaptide (1-1000 nM) or 20 mM LiCl for 24 h. For protein synthesis inhibition, 10 μg/ml cycloheximide dissolved in 17 mM ethanol was used with or without 100 nM Rotigaptide. Western blot: cells lysed in RIPA buffer, protein content determined by DC protein assay, samples denatured and run on 10% NuPAGE gels, transferred to nitrocellulose, blocked with casein buffer, incubated with anti-Cx43 antibody C8093 (1:40,000), biotinylated secondary antibodies, and streptavidin-HRP; chemiluminescence detected with SuperSignal Femto; Cx43 bands normalized to p70 internal control. Immunofluorescence: cells fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton, blocked with 10% normal goat serum, incubated with rabbit anti-Cx43 C6219 (1:400), Alexa568-conjugated secondary antibodies, imaged under fluorescence microscope; fluorescence intensities analyzed semiquantitatively. [2]
Double-cell patch clamp: Isolated guinea pig cardiomyocytes were used to measure gap junction conductance and membrane conductance before and after Rotigaptide treatment. [3]
Dye transfer assay: Rat neonatal cardiac myocytes, HL-1 cells, and HeLa cells expressing Cx43-GFP, Cx32-GFP, or Cx26 were microinjected with fluorescent dyes; Rotigaptide (50 nM, 5 h) increased dye transfer in Cx43-expressing cells only. [3]
Mass spectrometry phosphorylation analysis: Cx43 was purified from isolated perfused rat hearts subjected to global ischaemia with or without Rotigaptide; phosphorylation sites were identified using MALDI and LC-ESI-MS/MS; 13 serine phosphorylation sites were identified; Ser297 and Ser368 dephosphorylation during ischaemia was suppressed by rotigaptide. [3]
PKC activity assay: HeLa-Cx43 cells were treated with Rotigaptide or AAP10; PKC activity was measured, and blockade by PKC-α inhibitor CGP54345 was assessed. [3]
Isolated rat atrial strip: Atria were subjected to metabolic stress (hypoxia + glucose deprivation) with or without Rotigaptide (concentration range 1 nM to 1 μM); conduction velocity was measured; both prevention and reversion of conduction slowing were assessed. [3]

Rotigaptide was administered via intravenous bolus or continuous intravenous infusion in animal models. Doses and routes varied by species and model: in dog VT models, doses achieving plasma concentrations of ~1, 8, and 70 nM were used; in reperfusion studies, rotigaptide was given 10 min prior to reperfusion; in infarct size studies, doses ranged from 1 ng/kg bolus + 10 ng/kg/hr infusion to 1000 ng/kg bolus + 10 ng/kg/hr infusion IV. [3]

Intravenous pharmacokinetics in animals: In mice, rats and dogs, Rotigaptide is characterized by moderate total body clearance (CLT) of 0.226 to 0.981 L/hr/kg, low steady-state volume of distribution (Vdss) of 0.175 to 0.306 L/kg, and short apparent half-life (t1/2) of 0.29 to 0.9 hr. In rats: CLT ~121 ml/min/kg, Vd ~90 ml/kg, t1/2 ~16 min. In dogs: CLT ~42 ml/min/kg, Vd ~37 ml/kg, t1/2 ~4.3 min. Exposure increased with increasing dosages in a dose-proportional manner. ~93% of the hexapeptide is excreted in the urine as whole peptide; no metabolites observed in liver microsomes or hepatocytes; no inhibition of cytochrome P450 isozymes. [3]
Human pharmacokinetics (Phase I): Intravenous bolus in healthy volunteers: CLT of 133 ml/min, Vdss of 21.4 L, t1/2 of 2.7 hr. Continuous intravenous infusion: AUCss was consistent with predicted levels and increased in a dose-proportional manner. 61-84% of injected Rotigaptide was excreted unchanged in the urine; no metabolites apparent in plasma. [3]

Preclinical toxicology: Single intravenous bolus: Rotigaptide was well tolerated at 100 mg/kg in rats and 10 mg/kg in dogs; no toxicity in rats and mice at doses up to 300 mg/kg. Repeated-dose (5-14 days continuous IV infusion): well tolerated at up to 100 mg/kg/day for 14 days in rats and 10 mg/kg/day for 14 days in dogs; no compound-related effects. Non-genotoxic in bacterial reverse mutation, chromosome aberration, and mouse micronucleus assays. [3]
Cardiovascular safety: Rotigaptide (bolus IV, 10 mg/kg) had no effect on arterial blood pressure, heart rate, or ECG (PR, QRS, QT) in telemetry-instrumented dogs; no pro-arrhythmic effects. Plasma concentrations at this dose were ~47,674 ng/ml (~77,267 times minimum efficacious concentration in dog VT model). No effect on APD or ERP in rabbit Langendorff hearts; no effect on resting membrane potential or action potential amplitude in guinea pig hearts; no hERG channel blockade in patch clamp studies. [3]
Clinical safety (Phase I): No drug-related adverse effects or drug-drug interactions observed in ~200 healthy volunteers in Phase I trials. Single ascending dose (0.03-30 mg IV infusion; 2-3 mg IV bolus), continuous infusion (1-20 mg/day for 6 days), and drug interaction studies with digoxin and atenolol showed no safety concerns. [3]

[1]. The connexin 43 regulator Rotigaptide reduces cytokine-induced cell death in human islets.

[2]. The antiarrhythmic peptide rotigaptide (ZP123) increases connexin 43 protein expression in neonatal rat ventricular cardiomyocytes.Cell Commun Adhes. 2006 Jan-Apr;13(1-2):21-7.

[3]. Pharmacological modulation of gap junction function with the novel compound rotigaptide: a promising new principle for prevention of arrhythmias.Basic Clin Pharmacol Toxicol. 2007 Oct;101(4):215-30.

Rotigaptide is a peptide. Rotigaptide is currently in the basic scientific research stage related to heart and vascular diseases.

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Rotigaptide (Ac-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly-NH₂) is a stable hexapeptide antiarrhythmic peptide developed from the AAP10 analogue via rotation-inversion incorporating unnatural D-amino acids for improved proteolytic stability (in vitro half-life in human plasma: 14 days). The compound is highly water-soluble. [3]
Mechanism of action: Increases gap junction intercellular communication (GJIC) via connexin43 (Cx43) without affecting membrane currents. It prevents ischaemia-induced dephosphorylation of Cx43 at Ser297 and Ser368, which are considered important for gating of Cx43 gap junction channels during ischaemia. The cellular target molecule is unknown; signalling may involve G-protein-dependent membrane receptor and PKC-α activation, as AAP10-mediated effects were blocked by GDP-βS, PKC inhibitors, and PKC-α-specific inhibitors. [3]
Efficacy profile: Prevents re-entrant ventricular tachycardia during acute myocardial ischaemia in dogs; prevents reperfusion arrhythmias; reduces infarct size; prevents ischaemia-induced atrial fibrillation; increases atrial conduction in chronic volume overload models. No effect on focal arrhythmias. Bell-shaped dose-response relationship in some models. [3]
Clinical development: Phase I trials completed in ~200 healthy volunteers with no drug-related adverse effects. Phase II trials in patients with acute coronary syndrome initiated in June 2005. The compound is being developed for prevention of life-threatening re-entry VT/VF in MI patients. [3]

C28H39N7O9

617.650766611099

617.28

355151-12-1

9938933

White to light yellow solid powder

1.4±0.1 g/cm3

1187.3±65.0 °C at 760 mmHg

671.8±34.3 °C

0.0±0.3 mmHg at 25°C

1.607

-3.53

7

9

12

44

1110

5

O[C@@H]1CN(C([C@H]2CCCN2C([C@@H](CC2C=CC(=CC=2)O)NC(C)=O)=O)=O)[C@@H](C(NCC(N[C@@H](C(NCC(N)=O)=O)C)=O)=O)C1

GFJRASPBQLDRRY-TWTQBQJDSA-N

InChI=1S/C28H39N7O9/c1-15(25(41)30-12-23(29)39)32-24(40)13-31-26(42)22-11-19(38)14-35(22)28(44)21-4-3-9-34(21)27(43)20(33-16(2)36)10-17-5-7-18(37)8-6-17/h5-8,15,19-22,37-38H,3-4,9-14H2,1-2H3,(H2,29,39)(H,30,41)(H,31,42)(H,32,40)(H,33,36)/t15-,19+,20-,21-,22-/m1/s1

(2R,4S)-1-[(2R)-1-[(2R)-2-acetamido-3-(4-hydroxyphenyl)propanoyl]pyrrolidine-2-carbonyl]-N-[2-[[(2R)-1-[(2-amino-2-oxoethyl)amino]-1-oxopropan-2-yl]amino]-2-oxoethyl]-4-hydroxypyrrolidine-2-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~200 mg/mL (~323.81 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (8.10 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (8.10 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 5 mg/mL (8.10 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)