Endogenous Metabolite
Phosphocreatine targets the creatine kinase system, which catalyzes the reversible transfer of a phosphoryl group between phosphocreatine and ADP to regenerate ATP. The compound serves as a substrate for creatine kinase, which is present in high concentrations in skeletal muscle, cardiac muscle, and brain. By providing a reservoir of high-energy phosphate, phosphocreatine helps maintain ATP levels during periods of high energy demand. The creatine/phosphocreatine system is essential for maintaining energy homeostasis in tissues with high and fluctuating energy demands.

Research indicates that Phosphocreatine possesses neuroprotective and renoprotective potential. In terms of neuroprotection, Phosphocreatine (5-20 mM, 24 h) attenuates MGO (Methylglyoxal)-induced PC12 cell damage, inhibits apoptosis [3], and prevents the loss of mitochondrial membrane permeability [3]. Its mechanism of action involves normalizing mitochondrial function and reducing oxidative stress through the Akt-mediated Nrf2/HO-1 pathway [3]. Regarding renoprotection, Phosphocreatine (5-40 mM, 24 h) protects NRK-52E cells from MGO-induced kidney injury in a concentration-dependent manner [4], and its application at 10-40 mM for 4 h inhibits the production of renal oxidative stress metabolites [4].

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In vitro studies have demonstrated that Phosphocreatine is an effective substrate for creatine kinase and can rapidly regenerate ATP from ADP. The compound's ability to buffer ATP levels has been characterized in various in vitro systems, including isolated mitochondria, muscle homogenates, and purified enzyme preparations. Phosphocreatine has been shown to protect cells from energy depletion and maintain cellular function under conditions of metabolic stress. The compound's in vitro activity is well-characterized and forms the basis for its clinical use as a cardioprotective agent.

In the context of DOX-induced cardiotoxicity, Phosphocreatine treatment (200 mg/kg, i.p., every other day for 7 weeks) attenuated oxidative stress and apoptosis, and rescued myocardial tissue from necroptosis [2]. Additionally, in a diabetic nephropathy model, Phosphocreatine (20-40 mg/kg, i.v., daily for 6 weeks) demonstrated renoprotective effects by preserving renal function in Sprague-Dawley rats [4].

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In vivo studies have demonstrated that Phosphocreatine is effective in protecting the heart from ischemia-reperfusion injury and improving cardiac function. The compound is used clinically as a cardioprotective agent in the treatment of heart failure, myocardial ischemia, and other cardiac conditions. Phosphocreatine has also been studied for its effects on muscle function, exercise performance, and neurological disorders. The compound is administered intravenously or orally and has been shown to improve energy metabolism and tissue function in various clinical settings.

The in vitro enzyme assays for Phosphocreatine typically involve measuring the activity of creatine kinase in the presence of the compound. In these assays, creatine kinase is incubated with phosphocreatine and ADP, and the formation of ATP and creatine is measured. The reaction is monitored using coupled enzyme assays or HPLC. The compound's ability to serve as a substrate for creatine kinase is confirmed by measuring the rate of ATP production. These assays are standard for characterizing the activity of the creatine/phosphocreatine system.

Cellular assays for Phosphocreatine are conducted using various cell types, including cardiomyocytes, muscle cells, and neurons. Cells are treated with phosphocreatine or incubated in media containing the compound, and cellular ATP levels, mitochondrial function, and cell viability are assessed. The compound's ability to protect cells from energy depletion and metabolic stress is evaluated by exposing cells to hypoxia, ischemia, or other stress conditions. These cell-based assays confirm the compound's role in energy metabolism and its cytoprotective effects.

In vivo animal studies for Phosphocreatine are conducted in rat, mouse, and dog models of cardiac ischemia, heart failure, and other conditions. The compound is administered intravenously or orally, and its effects on cardiac function, energy metabolism, and tissue protection are assessed. Hemodynamic parameters, ATP levels, and markers of tissue damage are measured. These studies confirm the compound's in vivo efficacy and support its clinical use as a cardioprotective agent.

Absorption, Distribution and Excretion
Creatine phosphate is excreted via the kidneys. The final product of creatine degradation is creatinine, which enters the bloodstream from its storage sites in muscles. Once creatinine enters the renal parenchyma, it is filtered by the glomeruli and ultimately excreted in the urine.

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Phosphocreatine has a molecular weight of 211.11 and a molecular formula of C4H10N3O5P. The compound is soluble in water and is typically administered intravenously for clinical use. When administered intravenously, phosphocreatine is rapidly taken up by tissues with high energy demands, including the heart and skeletal muscle. The compound is metabolized to creatine and phosphate, which are excreted primarily in urine. The half-life of phosphocreatine in plasma is short, and it is rapidly cleared from the circulation.

Phosphocreatine is generally well-tolerated at therapeutic doses. The most common adverse effects are mild and include gastrointestinal disturbances when taken orally. No significant toxicity has been reported with the use of phosphocreatine at recommended doses. The compound has been used clinically as a cardioprotective agent with a favorable safety profile. However, as with any medication, phosphocreatine should be used with caution in patients with specific medical conditions and under the supervision of a healthcare professional.

[1]. Creatine: a dietary supplement and ergogenic aid. Nutr Rev. 1999 Feb;57(2):45-50.

[2]. Phosphocreatine attenuates doxorubicin-induced cardiotoxicity by inhibiting oxidative stress and activating TAK1 to promote myocardial survival in vivo and in vitro. Toxicology. 2021 Aug;460:152881.

[3]. Neuroprotective effect of phosphocreatine on oxidative stress and mitochondrial dysfunction induced apoptosis in vitro and in vivo: Involvement of dual PI3K/Akt and Nrf2/HO-1 pathways. Free Radic Biol Med. 2018 May 20;120:228-238.

[4]. Protection of diabetes-induced kidney injury by phosphocreatine via the regulation of ERK/Nrf2/HO-1 signaling pathway. Life Sci. 2020 Feb 1;242:117248.

N-Creonic acid phosphate (N-COP) is a phosphate amino acid, formed by attaching a phosphate group to the primary nitrogen atom of the guanidine group in the creatine molecule. It is a metabolite in both humans and mice. It is both a phosphate amino acid and a phosphagen. Its function is related to creatine. It is the conjugate acid of N-COP (2-). Creatine phosphate (COP) is the phosphorylated form of creatine. It is primarily found endogenously in the skeletal muscle of vertebrates, playing a crucial role in rapid energy buffering activities such as muscle cell contraction through its ability to regenerate adenosine diphosphate (ADP) into adenosine triphosphate (ATP). It is an endogenous substance mainly found in the skeletal muscle of vertebrates. It has been used to treat heart disease and has been added to cardioplegic solutions. (Reynolds JEF (Editor-in-Chief): Martindale Pharmacopeia (Electronic Edition). Micromedex, Englewood, Colorado, 1996)

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Drug Indications
Creonic phosphate is a naturally occurring substance found primarily in the skeletal muscle of vertebrates. Its main function in the body is to maintain and recycle adenosine triphosphate (ATP) to support muscle activity, such as muscle contraction. Given creatine phosphate's role in ATP recycling, its most likely therapeutic use is to treat conditions caused by insufficient energy or increased energy demand, such as ischemic stroke and other cerebrovascular diseases. However, it is worth noting that although creatine phosphate has been used to treat cardiovascular diseases in some countries, there are currently relatively few clinical studies for this indication, insufficient to provide adequate evidence. Furthermore, because creatine phosphate is not a regulated substance, some professional athletes take it as a supplement to enhance short-duration bursts of muscle strength or energy, thereby improving athletic performance.

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Mechanism of Action
Adenosine triphosphate (ATP) is the primary chemical energy source for muscle contraction in the human body. During muscle contraction, ATP molecules are hydrolyzed and converted into adenosine diphosphate (ADP), which is then consumed. To maintain muscle homeostasis, ATP in muscles must be regenerated periodically. Creatine phosphate, naturally present in the body, can regenerate ATP by transferring its high-energy phosphate group to ADP, thus generating ATP and creatine. This process of ATP regeneration using creatine phosphate typically occurs within seconds after strenuous muscle or nerve activity, providing a rapid reserve of high-energy phosphate groups for the recycling of ATP in muscle tissue. Creatine phosphate recycling to generate ATP is actually the fastest known method of ATP regeneration. Creatine is a naturally occurring chemical in the human body, primarily stored in skeletal muscle in both free and phosphorylated forms. Creatine phosphate is the phosphorylated form of creatine. In addition, creatine phosphate is also present in other organs such as the kidneys, liver, and brain. In fact, most creatine synthesis in the body occurs in the liver, where the amidine group of arginine is transferred to glycine with the help of glycine transaminase to form guanidinoacetic acid. Then, guanidinoacetic acid is methylated with the methyl group of S-adenosylmethionine by guanidinoacetic acid methyltransferase to produce creatine. The synthesized creatine is transported to storage sites in skeletal muscle via the bloodstream. Creatine phosphorylation is reversible, involving both forward and reverse reactions. That is, creatine phosphate can donate a phosphate group to adenosine diphosphate (ADP) to regenerate ATP under anaerobic conditions, and simultaneously, when muscle activity is low, excess ATP can be dephosphorylated, converting creatine to creatine phosphate. This dual activity—using excess ATP to synthesize creatine phosphate at rest and using creatine phosphate to regenerate ATP during high-intensity activity—demonstrates the crucial role of creatine phosphate as an energy buffer in human muscle cells. The rapid ATP regeneration from creatine phosphate is considered a coupled reaction—essentially, the energy released from the transfer of the phosphate group by creatine phosphate is used to regenerate ATP. Therefore, creatine phosphate plays a vital role in tissues with high and fluctuating energy demands, such as muscle and brain tissue.

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Phosphocreatine (creatine phosphate) is a naturally occurring compound that serves as a rapidly mobilizable reserve of high-energy phosphates in skeletal muscle and brain【67-07-2】. It has a molecular formula of C4H10N3O5P and a molecular weight of 211.11. Phosphocreatine plays a critical role in energy metabolism by regenerating ATP from ADP via the creatine kinase system. It is used clinically as a cardioprotective agent for the treatment of heart failure, myocardial ischemia, and other cardiac conditions. The compound is also used in research for studying energy metabolism and muscle function.

C4H10N3O5P

211.1131

211.035

C, 22.76; H, 4.77; N, 19.90; O, 37.89; P, 14.67

67-07-2

922-32-7 (di-hydrochloride salt);18838-38-5;67-07-2;19333-65-4;71519-72-7;922-32-7; 57-00-1 (free) 15366-29-7 (ethyl ester); 67-07-2 (phosphate); 1616693-92-5 (riboside)

9548602

White to off-white solid powder

1.8±0.1 g/cm3

449.1±47.0 °C at 760 mmHg

>300 °C(lit.)

225.4±29.3 °C

0.0±2.3 mmHg at 25°C

1.627

-3.39

4

6

4

13

271

0

CN(CC(=O)O)/C(=N/P(=O)(O)O)/N

DRBBFCLWYRJSJZ-UHFFFAOYSA-N

InChI=1S/C4H10N3O5P/c1-7(2-3(8)9)4(5)6-13(10,11)12/h2H2,1H3,(H,8,9)(H4,5,6,10,11,12)

2-[methyl-[(E)-N'-phosphonocarbamimidoyl]amino]acetic acid

phosphocreatine; Creatine phosphate; phosphorylcreatine; N-phosphocreatine; ...; 67-07-2;

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)

H2O: ~175 mg/mL (829 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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