Rotigaptide

Cat No.:V8703 Purity: ≥98%

COA Product Data Instructions for use SDS

Rotigaptide (ZP123) is a specific modulator of connexin 43 (Cx43).

Rotigaptide Chemical Structure CAS No.: 355151-12-1
Product category: New1

This product is for research use only, not for human use. We do not sell to patients.

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Product Description
Bioactivity & Protocols
Physicochemical Properties
Solubility Data
Calculators
Clinical Trial Information
Biological Data

Product Description

Rotigaptide (ZP123) is a specific modulator of connexin 43 (Cx43). Rotigaptide prevents Cx43-mediated uncoupling of gap junction communication and normalizes intercellular communication during acute metabolic stress. Rotigaptide is an antiarrhythmic peptide (AAP) with potential for research into cardiac arrhythmias, particularly atrial fibrillation.

Biological Activity I Assay Protocols (From Reference)

ln Vitro	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].
ln Vivo	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].
Cell Assay	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. 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.
References	[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.
Additional Infomation	Rotigaptide is a peptide. Rotigaptide is under investigation for the basic science of Heart Disease and Vascular Disease.

These protocols are for reference only. InvivoChem does not independently validate these methods.

Physicochemical Properties

Molecular Formula	C28H39N7O9
Molecular Weight	617.650766611099
Exact Mass	617.28
CAS #	355151-12-1
PubChem CID	9938933
Appearance	White to light yellow solid powder
Density	1.4±0.1 g/cm3
Boiling Point	1187.3±65.0 °C at 760 mmHg
Flash Point	671.8±34.3 °C
Vapour Pressure	0.0±0.3 mmHg at 25°C
Index of Refraction	1.607
LogP	-3.53
Hydrogen Bond Donor Count	7
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	12
Heavy Atom Count	44
Complexity	1110
Defined Atom Stereocenter Count	5
SMILES	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
InChi Key	GFJRASPBQLDRRY-TWTQBQJDSA-N
InChi Code	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
Chemical Name	(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
HS Tariff Code	2934.99.9001
Storage	Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility Data

Solubility (In Vitro)

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

Solubility (In Vivo)

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.

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.

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.6190 mL	8.0952 mL	16.1904 mL
	5 mM	0.3238 mL	1.6190 mL	3.2381 mL
	10 mM	0.1619 mL	0.8095 mL	1.6190 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

Calculator

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Molarity Calculator allows you to calculate the mass, volume, and/or concentration required for a solution, as detailed below:

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See Example

An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?

Enter 350.26 in the Molecular Weight (MW) box
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Enter 5 in the Volume box and choose the correct unit (mL)
Click the “Calculate” button
The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

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Dilution Calculator allows you to calculate how to dilute a stock solution of known concentrations. For example, you may Enter C1, C2 & V2 to calculate V1, as detailed below:

What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:

Enter 10 into the Concentration (Start) box and choose the correct unit (mM)
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The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box

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Molecular Weight Calculator allows you to calculate the molar mass and elemental composition of a compound, as detailed below:

Note: Chemical formula is case sensitive: C12H18N3O4 c12h18n3o4
Instructions to calculate molar mass (molecular weight) of a chemical compound:

To calculate molar mass of a chemical compound, please enter the chemical/molecular formula and click the “Calculate’ button.

Definitions of molecular mass, molecular weight, molar mass and molar weight:

Molecular mass (or molecular weight) is the mass of one molecule of a substance and is expressed in the unified atomic mass units (u). (1 u is equal to 1/12 the mass of one atom of carbon-12)
Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.

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Reconstitution Calculator allows you to calculate the volume of solvent required to reconstitute your vial.

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The answer appears in the Volume (to add to vial) box

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)

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Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)

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Calculation results

Working concentration： mg/mL；

Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O，mix and clarify.

Note： (1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.

Clinical Trial Information

NCT Number	Recruitment	interventions	Conditions	Sponsor/Collaborators	Start Date	Phases
NCT00901563	COMPLETED	Drug: Rotigaptide Other: Forearm vascular study	Heart Disease Vascular Disease	University of Edinburgh	2009-03	Not Applicable
NCT00137332	TERMINATED	Drug: GAP-486 (ZP-123) Drug: 0.9% Sodium Chloride, USP	Arrhythmia	Wyeth is now a wholly owned subsidiary of Pfizer	2005-11	Phase 2
NCT00137293	TERMINATED	Drug: GAP-486 (ZP-123) Drug: 0.9% Sodium Chloride, USP	Arrhythmia	Wyeth is now a wholly owned subsidiary of Pfizer	2005-11	Phase 2

Biological Data

Rotigaptide reduces cytokine-induced apoptosis in Cx43-expressing human islets. Fifty pancreatic islets per condition were pre-incubated with or without 500 nM rotigaptide/ZP123 (RG) or control peptide ZP119 (CP) for one hour in the presence or absence of cytokine mixture (300 pg/mL IL-1β + 10 ng/mL IFNɣ + 10 ng/mL TNFα; Cyt) for 4 days. (A) The Cx43 expression level was determined in untreated human and rat islets, and in INS-1 cells exposed to the cytokine combination (150 pg/mL IL-1β +0.1 ng/mL IFNɣ) using specific primers with qPCR. The expression of the genes normalized to HPRT1 was calculated by −∆Ct. (B) Apoptosis was measured by Roche cell death assay according to the manufacturer’s protocol. Data are means ± SEM of n = 5 donor human islets. * ≤ 0.05, ττ ≤ 0.01. The symbols τ and * indicate the Bonferroni-corrected paired t-test values of cytokine (Cyt) exposure versus control (CTL) and Cyt exposure versus exposure to Cyt and RG, respectively. NS: not significant.[1]. Seyed Mojtaba Ghiasi, et al. The connexin 43 regulator Rotigaptide reduces cytokine-induced cell death in human islets.

Rotigaptide ameliorates inflammation-induced apoptosis associated with improved mitochondrial function in Cx43-deficient INS-1 cells. INS-1 cells were pre-incubated with or without 100 nM rotigaptide (RG) or control peptide ZP119 (CP) for one hour in the presence or absence of cytokine combination (IL-1β in the concentrations indicated + 0.1 ng/mL IFNγ; Cyt) or glucolipotoxicity (0.5 µM palmitate + 25 mM glucose; GLT) for 24 h. (A) Apoptosis was measured by Roche cell death assay according to the manufacturer’s protocol. (B) Mitochondrial function was determined using MTT assay. Results are means ± SEM of n = 6 independent experiments. * or τ ≤ 0.05, ** or ττ ≤ 0.01, *** or τττ ≤ 0.001, ττττ ≤ 0.0001. The symbols τ and * star indicate the Bonferroni-corrected paired t-test values of cytokine (Cyt) exposure versus control (CTL) and Cyt exposure versus exposure to Cyt and RGs, respectively. NS: not significant.[1]. Seyed Mojtaba Ghiasi, et al. The connexin 43 regulator Rotigaptide reduces cytokine-induced cell death in human islets.

Rotigaptide does not reduce inflammatory or glucolipotoxicity-induced intracellular ROS in INS-1 cells. INS-1 cells were pre-incubated with or without 100 nM rotigaptide (RG) or control peptide ZP119 (CP) for one hour in the presence or absence of cytokine mixture (IL-1β in the concentrations indicated + 0.1 ng/mL IFNɣ; Cyt) or glucolipotoxicity (0.5 µM Palmitate + 25 mM glucose; GLT) for 18 h. (A) Cellular ROS level was determined using DCF assay and presented as MFI. (B) The mRNA level of NADH-dehydrogenase subunit 2 (ND2) and cytochrome C oxidase II (Sco2) genes was determined using specific primers with qPCR. The expression of the genes normalized to HPRT1 was calculated by -∆Ct. Data are the means ± SEM of n = 6 (for A)/n = 4 (for B) independent experiments. τ ≤ 0.05, ττ ≤ 0.01, τττ ≤ 0.001, ττττ or **** ≤ 0.0001. The symbols τ and * indicate the Bonferroni-corrected paired t-test values of cytokine (Cyt) exposure versus control (CTL) and Cyt exposure versus exposure to Cyt and RG, respectively. NS: not significant. ROS: reactive oxygen species, MFI: mean fluorescent intensity, DCF: dichlorodihydrofluorescein.[1]. Seyed Mojtaba Ghiasi, et al. The connexin 43 regulator Rotigaptide reduces cytokine-induced cell death in human islets.