Creatine analog

Cyclocreatine suppresses creatine metabolism and proliferation in prostate cancer cells [3]
Cyclocreatine is a creatine analogue that has been shown to functionally block the phosphagen system. While cellular uptake of cyclocreatine is reported to be mediated by SLC6A8, it remains to be investigated whether cyclocreatine exerts its effects via inhibition of creatine synthesis and/or uptake. We tested the effect of cyclocreatine (varying concentrations up to 1%) on the in vitro proliferative capacity of murine and human prostate cancer cells with varying SPRY2 expression. Cyclocreatine (1%) impaired in vitro proliferation of both human and murine prostate cancer cells (Fig. 3A and B). A dose dependent response to cyclocreatine treatment was observed with growth inhibition detected at a dose as low as 0.125% (Supplementary Fig. S3A–S3F). Furthermore, cyclocreatine treatment significantly impaired the colony-forming ability of PC3 cells (Fig. 3C), along with reduced cellular creatine and phosphocreatine levels (Fig. 3D and E) while leaving ATP levels unaltered (Fig. 3F). Together these data suggest cyclocreatine may have potential as a therapeutic agent for prostate cancer.

To directly study how cyclocreatine affects creatine uptake and metabolism, we performed stable isotope tracing using 13C-creatine (0.1 mmol/L), in the presence/absence of cyclocreatine (0.1 mmol/L to 50 mmol/L, with 50 mmol/L being 0.7% cyclocreatine). Cyclocreatine was efficiently internalized by PC3 CL1 cells (Fig. 4A). We observed a dose-dependent inhibition of 13C-creatine uptake (Fig. 4B), and production of the related metabolites 13C-creatinine and 13C-phosphocreatine (Fig. 4C and D). Collectively, our data show that, when exogenously available to these cells, the main source of intracellular creatine is via uptake, a process that is impaired by cyclocreatine.

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Cyclocreatine suppresses the phosphagen system [4]
Cyclocreatine is a creatine analogue that blocks the phosphagen system by displacing creatine. We applied cyclocreatine treatment (1%) to investigate functional effects of creatine metabolism. Cyclocreatine suppressed in vitro creatine kinase activity (Figure 3A) and impaired in vitro proliferation of both human and murine prostate cancer cells (Figure 3D-E). Furthermore, cyclocreatine treatment significantly impaired the colony forming ability of PC3 cells (Figure 3F), along with reduced cellular creatine and phosphocreatine levels (Figure 3G-H) while the ATP levels were not altered (Figure 3I). Together these data suggest cyclocreatine potently suppresses activities of the phosphagen system.

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Creatine metabolism regulates cellular S-adenosyl methionine (SAM) levels [4]
Consistent with the observation of enhanced creatine uptake in SPRY2 deficient cells, in creatine proficient serum supplemented culture conditions, cyclocreatine drastically reduced creatine uptake (Figure 4A). In addition, in serum-free culture conditions, we observed enhanced creatine synthesis in SPRY2 deficient PC3 cells (Figure 4A), which was accompanied by significantly higher levels of phosphocreatine (Figure 4B). Treatment with cyclocreatine significantly reduced cellular levels of creatine and phosphocreatine irrespective of SPRY2 status (Figure 4A-B), with both creatine uptake and biosynthesis suppressed. Collectively, cyclocreatine treatment blocked creatine uptake, creatine biosynthesis and phosphocreatine formation.
S-adenosyl methionine (SAM) is required for de novo creatine synthesis. In the presence of enhanced cellular creatine synthesis, cellular SAM steady state level may be reduced. We carried out targeted metabolomics analysis to study metabolic changes associated with SPRY2 deficiency with and without cyclocreatine treatment. In the absence of exogenous creatine, SPRY2 deficient cells (with enhanced creatine biosynthesis) exhibited significantly reduced levels of SAM (Figure 4C left side), consistent with increased SAM utilisation for creatine biosynthesis. We therefore reasoned that blockade of creatine biosynthesis may reciprocally lead to SAM accumulation. Indeed, cyclocreatine treatment significantly increased the cellular level of SAM in PC3 cells regardless of SPRY2 status and culture condition (Figure 4C right side, 4D). Consistent with a previous report (Schmidt et al, 2016), SAM treatment decreased the growth of PC3 cells independent of SPRY2 status (Figure 4E). Hence, in addition to the direct effects of reduced creatine levels, cyclocreatine induced effects may result in part from the accumulation of SAM (due to blockade of creatine biosynthesis).

Cyclocreatine (CCr) and CCrP treatment prior to the onset of ischemia, preserved high levels of ATP in ischemic myocardium, reduced myocardial cell injury, exerted anti-inflammatory and anti-apoptotic activities, and restored contractile function during reperfusion in animal models of acute myocardial infarction (AMI), global cardiac arrest, cardiopulmonary bypass, and heart transplantation. [1]

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Cyclocreatine rescues microgliosis and clustering and moderates neurite dystrophy in vivo [2]
Since cyclocreatine rescued metabolism and viability and suppressed autophagy in Trem2−/− BMDMs in vitro and given previous studies showing that cyclocreatine is passively transported across membranes and can accumulate and function as a phosphagen in the mouse brain in vivo (Kurosawa et al., 2012), we asked whether dietary supplementation with cyclocreatine could rescue microglial function and suppress autophagy in vivo in Trem2−/− 5XFAD mice. The drinking water of 5XFAD and Trem2−/− 5XFAD mice was supplemented with cyclocreatine from 10 weeks of age until 8 months of age. Remarkably, significantly fewer multivesicular/multilamellar structures were seen by TEM in microglia of Trem2−/− 5XFAD mice treated with cyclocreatine than in microglia of untreated mice (Fig. 6A, B). Confocal microscopy corroborated that cyclocreatine treatment ameliorated many autophagy and viability parameters in Trem2−/− 5XFAD mice, such as the number of LC3 puncta/high powered field (HPF) (Fig. 6C, D), the percentage of LC3+ microglia (Fig. S5A), and the percentage of cleaved caspase-3+ microglia (Fig. 6E), although others parameter did not reach statistical significance (Fig. S5B–D). Furthermore, clustering of microglia around plaques (Fig. 6C, F) and the number of microglia/HPF in plaque-bearing regions of the cortex (Fig. S5E) were both significantly increased in Trem2−/− 5XFAD mice treated with cyclocreatine compared to untreated Trem2−/− 5XFAD mice. These findings indicate that dietary supplementation with cyclocreatine is sufficient to partially rescue the defect in microgliosis and microglial clustering around plaques in Trem2−/− 5XFAD mice, while concomitantly mitigating autophagy and death of the microglia.

To assess the impact of cyclocreatine on microglial activation, which is also impaired in Trem2−/− 5XFAD mice, we quantified the percentage of microglia that expressed the activation marker osteopontin (Spp1), a protein that, in the brain, is upregulated in microglia in the context of Aβ deposition (Orre et al., 2014; Wang et al., 2015). Untreated Trem2−/− 5XFAD mice had very few Spp1+ microglia, while cyclocreatine-treated Trem2−/− 5XFAD mice had significantly more Spp1+ microglia, as did TREM2-sufficient 5XFAD mice (Fig. 7A, B). Moreover, biochemical analysis of microglia isolated ex vivo demonstrated that cyclocreatine treatment of Trem2−/− 5XFAD mice also restored microglial mTOR signaling and limited autophagy compared to untreated Trem2−/− 5XFAD mice (Fig. 7C, D).

As a major function of TREM2 in vivo is enabling microglia to form a barrier around plaques that prevents spreading of Aβ fibrils and alleviates dystrophy of plaque-adjacent neurites (Wang et al., 2016; Yuan et al., 2016), we asked whether cyclocreatine treatment of Trem2−/− 5XFAD mice impacted plaque morphology and/or neuronal dystrophy. While plaques in untreated Trem2−/− 5XFAD mice had a lower density than those in 5XFAD mice as measured by methoxy-X04 staining intensity, the density of plaques in cyclocreatine treated Trem2−/− 5XFAD mice resembled that of plaques in 5XFAD mice (Fig. 7E), although plaque shape complexity was not significantly altered (Fig. S5F). Despite reducing plaque density, cyclocreatine did not moderate plaque accumulation or the engulfment of plaque particulates by microglia, at least at this time point (Fig. S5G–I). As APP is known to accumulate as distinct rounded particles in dystrophic neurites, we used APP deposition around plaques to assess neurite dystrophy (Masliah et al., 1996; Wang et al., 2016; Yuan et al., 2016). Cyclocreatine treatment of Trem2−/− 5XFAD mice significantly reduced plaque-associated neurite dystrophy compared to untreated Trem2−/− 5XFAD mice to levels observed in 5XFAD mice (Fig. 7F, G). Taken together, these data indicate that cyclocreatine administration improves microglial metabolism and the protective response to Aβ plaques in TREM2-deficient 5XFAD mice.

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Effects of cyclocreatine on in vivo prostate carcinogenesis and metastatic potential [3]
To investigate the tumor suppressive effects of cyclocreatine, we treated prostate tumor bearing Ptenpc−/− Spry2pc−/−mice with 1% cyclocreatine for one month. Treatment was well tolerated and we did not observe evidence of any cyclocreatine-related pathologies as indicated by normal mouse weights and histological examination of the major organs (namely heart, kidney, liver, and spleen; Supplementary Table S3; Supplementary Fig. S4A). Cyclocreatine treatment significantly decreased tumor cell proliferation as shown by reduced Ki67 staining (Fig. 5A and B), while the trend for reduced tumor weights did not reach statistical significance (Fig. 5C). We next performed metabolic analysis of tumor tissues and whole blood, collected from control or cyclocreatine-treated animals. Cyclocreatine was detected in both blood (Fig. 5D) and tumor tissues (Fig. 5E), confirming that the drug had reached the tissue of interest. Further data analysis showed alterations in creatine metabolism upon cyclocreatine treatment. Whereas no changes were observed in the steady-state level of creatine (Fig. 5F) or phosphocreatine (Fig. 5G), the intratumoral levels of its breakdown product creatinine (Fig. 5H) were significantly decreased. Furthermore, cyclocreatine increased the arginine levels, and decreased guanidinoacetate levels in tumors (Fig. 5I and J). Such metabolic alterations would be in line with inhibition of de novo creatine synthesis. Although we could not demonstrate de novo synthesis of creatine in human prostate cancer cells cultured in vitro (Fig. 2C), these observations may indicate synthesis in one or more cell populations within the tumor microenvironment in vivo. The observed changes in the tumoral levels of arginine or guanidinoacetate were not observed in the blood samples (Supplementary Fig. S4B and S4C), but a systemic effect of cyclocreatine affecting intratumoral metabolites cannot be ruled out.

Regardless of the tumor genotype, the process of cancer metastasis is metabolically demanding in terms of both energy and biomass. We therefore tested the effect of cyclocreatine in a model of liver metastasis, which may affect patients with advanced and treatment refractory prostate cancer. To this purpose, splenic injections of PC3M cells were performed and mice were treated with 1% cyclocreatine for a month. Liver metastatic burden was significantly reduced by cyclocreatine (Fig. 5K and L).

Overall, our data highlight the importance of creatine in prostate cancer, and reveal new molecular insights into the mechanism of cyclocreatine treatment on creatine metabolism, with creatine uptake being the dominant source.

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Creatine Transporter Deficiency (CTD) is an inborn error of metabolism presenting with intellectual disability, behavioral disturbances and epilepsy. There is currently no cure for this disorder. Here, we employed novel biomarkers for monitoring brain function, together with well-established behavioral readouts for CTD mice, to longitudinally study the therapeutic efficacy of cyclocreatine (cCr) at the preclinical level. Our results show that cCr treatment is able to partially correct hemodynamic responses and EEG abnormalities, improve cognitive deficits, revert autistic-like behaviors and protect against seizures. This study provides encouraging data to support the potential therapeutic benefit of cyclocreatine or other chemically modified lipophilic analogs of Cr. [5]

Metabolism assays [2]
For real-time analysis of extracellular acidification rates (ECAR) macrophages were analyzed using an XF96 Extracellular Flux Analyzer. Cells were incubated overnight in complete RPMI in the indicated concentration of LCCM with or without Cyclocreatine (10 mM). Measurements were taken under basal conditions and following the sequential addition of 1 μM oligomycin and 1.5 μM fluoro-carbonyl cyanide phenylhydrazone (FCCP).

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13C1-creatine tracing [3]
PC3 CL1 cells were seeded in 6-well plates (7.5×104 cells/well; 3 wells per experimental condition) in RPMI supplemented with 10% FBS and 2 mmol/L glutamine (day 0). After 48 hours (day 2), medium was changed to RPMI supplemented with 10% dialyzed FBS and 2 mmol/L glutamine. On day 3, medium was changed to 7 mL RPMI (10% dialyzed FBS, 2 mmol/L glutamine) in the presence/absence of 13C-creatine (0.1 mmol/L), and various concentrations of Cyclocreatine (0, 0.1, 1, 10, and 50 mmol/L). After 24 hours, metabolites were extracted as described above.

The generation of Trem2−/− and Trem2−/− 5XFAD mice has been described previously (Oakley et al., 2006; Turnbull et al., 2006; Wang et al., 2015). All mice were on a C57BL/6 background. Age and sex matched mice were used for all experiments; experimental cohorts of mice were cohoused from birth to control for the microbiota.. For in vivo Cyclocreatine treatment 10-week old mice were put on cyclocreatine-containing water, treatment was continued until mice reached 8 months of age. Desired intake of cyclocreatine was approximately 0.28 mg/g of body weight/day, which is approximately the same as the standard creatine dose used in humans of 285 mg/kg of body weight/day (Kurosawa et al., 2012). Cyclocreatine was administered in drinking water at a final concentration of 2.33 mg/ml. [2]

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\n\n\nTo investigate the effects of creatine on in vivo prostate tumors, creatine (40 mg daily) was administered via gavage of 1% creatine solution for 2 months. Similarly, 1% Cyclocreatine (w/v; 2-imino-1-imidazolidineaceticacid) was supplied in drinking water ad libitum for 1 month.\n\nCD-1 nude mice (6–8 weeks old; male) were used to study metastatic tumor cell growth in the liver following splenic injection of PC3M cells (5 million cells per injection into the spleen of each experimental mouse). One percent Cyclocreatine (w/v; 2-imino-1-imidazolidineaceticacid; Sigma #377627) in drinking water was started one week after tumor cell implantation ad libitum for 1 month. The PREPARE guidelines were used to design and execute experiments. Tumor burden in the liver was assessed at necropsy by inspection for visible tumor lesions, and the number of hepatic lobes involved was used to calculate the percentage involvement. The presence or absence of metastasis in each lobe was then confirmed histologically by hematoxylin and eosin staining. [3]

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\n\n\nTo investigate the effects of creatine supplement on GEMM prostate tumour growth, creatine (40 mg daily) was administered via gavage of 1% creatine solution for two months. Similarly, 1% Cyclocreatine (w/v) (2-imino-1-imidazolidineaceticacid) (Sigma #377627) was supplied in drinking water ad libitum for one month.\n\nCD-1 Nudes 6-8 weeks old male mice were used to study the extent of liver metastases following splenic injection of PC3M cells (five million cells per injection into the spleen of each experimental mouse). 1% Cyclocreatine (w/v) (2-imino-1-imidazolidineaceticacid) in drinking water was started one week after tumour cell implantation ad libitum for one month. The ARRIVE guidelines were used to design and execute experiments. Matastasis burden in the liver was obtained histologically as previously described (Patel et al., 2018). [4]

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\n\nSatellite pharmacokinetic and histology study [4]
\nSatellite mice were dosed in the same manner as the efficacy study animals and used for brain pharmacokinetics (PK) and histology evaluation. CrT−/y mice were either not dosed (CrT−/y) or treated daily with 1% low-fat chocolate milk (vehicle, V-CrT−/y) or 140 mg/kg cCr (H-CrT−/y). Of the 21 animals, 7 per group were terminated at each time point (PND44, PND114, or PND184). Twenty-four hours after the last dose, brains were harvested from the mice following whole-body perfusion with cold saline. One hemisphere was immediately snap frozen for PK study and the other hemisphere was fixed in 10% neutral-buffered formalin for histology. For PK, LC–MS/MS assays for cCr (limit of quantification 0.35 nmol/g brain) and phospho-cCr (limit of quantification 0.22 nmol/g brain) were developed and used to measure concentrations of total cCr (summation of cCr and phospho-cCr) against an internal standard (Cyclocreatine-1,4,5-13C3) in the brain hemispheres of all satellite H-CrT−/y animals at PND44, 114, and 184. Additionally, brain PK was measured in 4–5 randomly selected M-CrT−/y and L-CrT−/y animals from the efficacy study at PND195 (after whole body perfusion with saline, see “Drug administration and design of efficacy study” paragraph). For reference, brain creatine and phospho-creatine values in wild-type C57BL/6J mice were obtained in a separate study from the same laboratory that developed and ran the cCr/phospho-cCr assays. The mean total creatine value reported (12,813 nmol/g brain) is very similar to that reported by other groups in wild-type animals10,11,43. For histology, 5 of the 7 animals in each group were randomly selected for evaluation at PND114 and PND184. Hemispheres were trimmed into 8–10 coronal sections, embedded in paraffin, sectioned using a 5-micron block advance, and stained with Hematoxylin and Eosin. Amygdaloid body, basal nuclei/striatum, cerebellum, cerebral cortex, hippocampus, hypothalamus, medulla oblongata, meninges, midbrain, olfactory bulb, pons, thalamus ventricular system, and white matter were evaluated for each animal.\n

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\n\nDrug administration and design of efficacy study [5]
\nCyclocreatine(cCr) was dissolved in 1% low-fat chocolate milk . Dosing solutions were stored at 2–8 °C between use and fresh solutions were made weekly. CCr in 1% chocolate milk was shown to be stable for 3 weeks at room temperature, and solubility was determined to be 15 mg/mL. Animals were left untreated or presented daily with 1% low-fat chocolate milk (vehicle treatment, V) or a 1% low-fat chocolate milk-cCr cocktail (in a volume of 5 ml/kg). cCr was administered once daily at three different dose levels (140, 46 or 14 mg/kg) over the course of 24 weeks starting in mice at postnatal day (PND). Longitudinal IOS imaging was conducted at PND40, PND110 and PND180. After completion of each IOS recording, mice were rested for at least four days and then each animal was subjected to serial neurobehavioral assessments of cognitive and psychomotor functions (Y maze, Morris Water Maze, rotarod and self-grooming). While Y maze, MWM and rotarod were longitudinally administered to the same animals, self-grooming was tested only at PND195 (see Fig. 1a). At the end of the dosing period (PND200), animals were monitored via EEG for 24 h and then challenged with KA. A subset of randomly selected M-CrT−/y (n = 5) and L-CrT−/y mice (n = 4) were not used for EEG and the brain was harvested for cCr pharmacokinetic profiling Otherwise, all animals were subjected to the same tests in the same time intervals with no differences in the experimental timeline was introduced between individuals or groups.[5]

Cyclocreatine (cCr) can cross the blood-brain barrier and enter the brain[5]. To assess the brain permeability of cCr, we monitored the pharmacokinetic characteristics of a group of animals after oral administration. We detected that all mice that received long-term drug treatment were able to enter the brain, with the total cCr level in H-CrT−/y mice being higher than that in M-CrT−/y and L-CrT−/y mice (Fig. 5). In addition, longitudinal quantitative analysis of cCr in mice of three different age groups used for behavioral and functional tests showed that high-dose administration rapidly led to an increase in cCr concentration in the brain, and cCr reached a steady-state level after 23 days of administration and remained stable throughout the treatment period (Fig. 5).

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Effects of cyclocreatine on the occurrence and metastatic potential of prostate cancer in vivo[3]
To investigate the anticancer effect of cyclocreatine, we treated tumor-bearing Ptenpc−/− Spry2pc−/− mice with 1% cyclocreatine for one month. Treatment was well tolerated, with mice maintaining normal body weight and no abnormalities observed in the histological examination of major organs (i.e., heart, kidneys, liver, and spleen) (Supplementary Table S3; Supplementary Figure S4A), indicating no pathological changes related to Cyclocreatine. Cyclocreatine treatment significantly reduced tumor cell proliferation, as evidenced by decreased Ki67 staining (Figures 5A and B), but the trend toward tumor weight reduction was not statistically significant (Figure 5C). Next, we performed metabolic analyses on tumor tissues and whole blood from mice in both the control and Cyclocreatine-treated groups. Cyclocreatine was detected in both blood (Figure 5D) and tumor tissue (Figure 5E), confirming that the drug reached the target tissue. Further data analysis revealed altered creatine metabolism after Cyclocreatine treatment. While the steady-state levels of creatine (Figure 5F) or phosphocreatine (Figure 5G) remained unchanged, the intratumoral level of its breakdown product, creatinine (Figure 5H), was significantly reduced. Furthermore, Cyclocreatine increased arginine levels in tumors and decreased guanidinoacetic acid levels (Figures 5I and J). These metabolic alterations are consistent with the inhibition of de novo creatine synthesis. Although we were unable to confirm de novo creatine synthesis in cultured human prostate cancer cells (Fig. 2C), these observations may indicate creatine synthesis in one or more cell populations within the tumor microenvironment in vivo. No changes in intratumoral arginine or guanidinoacetic acid levels were observed in blood samples (Supplementary Figs. S4B and S4C), but the possibility of systemic effects of Cyclocreatine on intratumoral metabolites cannot be ruled out. To directly investigate how Cyclocreatine affects creatine uptake and metabolism, we performed stable isotope tracing using 13C-creatine (0.1 mmol/L) in the presence/absence of Cyclocreatine (0.1 mmol/L to 50 mmol/L, where 50 mmol/L is 0.7% Cyclocreatine). Cyclocreatine was efficiently internalized by PC3 CL1 cells (Fig. 4A). We observed dose-dependent inhibition of 13C-creatine uptake (Fig. 4B) and the production of related metabolites 13C-creatinine and 13C-phosphocreatine (Fig. 4C and D). Overall, our data suggest that when these cells have access to exogenous creatine, the primary source of intracellular creatine is uptake, and Cyclocreatine inhibits this process.

[1]. Cyclocreatine protects against ischemic injury and enhances cardiac recovery during early reperfusion. Expert Rev Cardiovasc Ther. 2019 Sep;17(9):683-697.

[2]. TREM2 Maintains Microglial Metabolic Fitness in Alzheimer's Disease. Cell. 2017 Aug 10;170(4):649-663.e13.

[3]. Cyclocreatine Suppresses Creatine Metabolism and Impairs Prostate Cancer Progression. Cancer Res. 2022 Jul 18;82(14):2565-2575.

[4]. Cyclocreatine suppresses prostate tumorigenesis through dual effects on SAM and creatine metabolism. bioRxiv, 2021: 2021.02. 26.432994.

[5]. Cyclocreatine treatment ameliorates the cognitive, autistic and epileptic phenotype in a mouse model of Creatine Transporter Deficiency. Sci Rep. 2020 Oct 27;10(1):18361.

Cyclocreatine is an oxygen- and nitrogen-containing organic compound whose function is related to α-amino acids.

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Introduction: One of the key mechanisms leading to irreversible myocardial injury due to hypoxia/ischemia is the depletion of adenosine triphosphate (ATP). Cyclocreatine (CCr) and its water-soluble salt, Cyclocreatine phosphate (CCrP), are potent bioenergetic agents that can maintain high levels of ATP during ischemia. This article covers: Treatment with CCr and CCrP before ischemia can maintain high levels of ATP in ischemic myocardium, reduce cardiomyocyte damage, exert anti-inflammatory and anti-apoptotic effects, and restore contractile function during reperfusion in animal models of acute myocardial infarction (AMI), systemic cardiac arrest, cardiopulmonary bypass, and heart transplantation. Medline and Embase databases (1970–February 2019), WIPO database (as of February 2019); no language restrictions. Expert opinion: This review provides a theoretical basis for the various clinical applications of CCrP and CCr, aiming to minimize ischemic injury and necrosis. One strategy is to use CCrP in patients with acute myocardial infarction (AMI) during or after percutaneous coronary intervention (PCI) in order to protect the myocardium, reduce the infarct size, and thus delay the progression of heart failure. Another clinical application is for predictable myocardial ischemia, where CCrP pretreatment may improve the prognosis and quality of life of patients undergoing cardiopulmonary bypass coronary revascularization, as well as the prognosis and quality of life of end-stage heart failure patients who are scheduled for heart transplantation. [1]

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Increased risk of Alzheimer's disease (AD) is associated with low-activity variants of TREM2. TREM2 is a surface receptor that is essential for microglia to respond to neurodegeneration, including proliferation, survival, aggregation, and phagocytosis. How TREM2 promotes such a diverse range of responses is unclear. This study found a large number of autophagic vesicles in microglia of AD patients carrying the TREM2 risk variant and in microglia of TREM2-deficient mice with AD-like pathology. Similar findings were observed in TREM2-deficient macrophages under growth factor restriction or endoplasmic reticulum (ER) stress conditions. Combining metabolomics and RNA sequencing (RNA-seq), we discovered that this aberrant autophagy is associated with a defect in the mammalian target of rapamycin (mTOR) signaling pathway, which affects ATP levels and biosynthetic pathways. In vitro experiments showed that metabolic disturbances and autophagy can be counteracted by Dectin-1 (a receptor that triggers TREM2-like intracellular signals) and Cyclocreatine (a creatine analog that provides ATP). Dietary Cyclocreatine supplementation inhibited autophagy, restored microglia aggregation around plaques in TREM2-deficient β-amyloid pathological mice, and reduced malnutrition of neurons adjacent to plaques. Therefore, TREM2 promotes microglial responses during Alzheimer's disease by maintaining cellular energy and biosynthetic metabolism. [2] Based on this, our study showed that the mTOR-mediated metabolic activation deficiency in TREM2-deficient cells can be corrected in vitro by creatine kinase pathway or activation of the Dectin-1 pathway (which transmits intracellular signals similar to TREM2). Based on these results, we adopted a Cyclocreatine-based treatment strategy. Cyclocreatine is an analog of creatine that can cross the cell membrane into the brain (Woznicki and Walker, 1979), can be phosphorylated and dephosphorylated by creatine kinase (McLaughlin et al., 1972), and can generate ATP (Kurosawa et al., 2012). Notably, we found that dietary Cyclocreatine supplementation during Aβ accumulation improved the viability, number, and aggregation of microglia around Aβ plaques. As a result, plaque density increased, and more importantly, plaque-associated neuropathy was significantly reduced. Although Cyclocreatine treatment was insufficient to reduce the overall accumulation of Aβ plaques, this may depend on the time point of analysis and/or the dose and duration of Cyclocreatine treatment. While the creatine kinase pathway has previously been shown to play a crucial role in the central nervous system in neurotransmitter release, membrane potential maintenance, Ca2+ homeostasis, and ion gradient restoration (Snow and Murphy, 2001; Wyss and Kaddurah-Daouk, 2000), our results suggest that this system may also be used to maintain microglial metabolism. Notably, Cyclocreatine can inhibit creatine kinase in certain situations, potentially producing systemic effects such as altering pancreatic hormone and glucose metabolism (Ara et al., 1998; Kuiper et al., 2008). Therefore, it must be emphasized that our findings are only a proof-of-concept study and do not recommend Cyclocreatine as a preventative treatment for Alzheimer's disease (AD). Further research is needed to precisely elucidate the mechanisms by which Cyclocreatine affects the microglial response to Aβ. Furthermore, determining whether Cyclocreatine affects the hydrolysis and shedding of TREM2 proteins in microglia is also crucial, as this leads to the release of soluble TREM2, which may have pro-survival functions. In summary, our study suggests that strategies aimed at maintaining microglia metabolism may be promising for therapeutic interventions in Alzheimer's disease (AD) and other neurodegenerative diseases associated with TREM2 deficiency and microglia dysfunction. [2] Prostate cancer is the second leading cause of cancer death in men worldwide. We used a novel genetically engineered mouse model (GEMM) that mimics aggressive prostate cancer driven by deficiencies in the tumor suppressor factors PTEN and Sprouty2 (SPRY2) and found that enhanced creatine metabolism is a core component of disease progression. In vitro experiments showed that creatine treatment enhanced basal cellular respiration, while in vivo experiments showed that creatine treatment promoted tumor cell proliferation. Stable isotope tracing showed that creatine levels in prostate cancer cells were primarily influenced by exogenous creatine supply rather than de novo arginine synthesis. Gene silencing of the creatine transporter SLC6A8 reduced intracellular creatine levels and impaired colony formation in human prostate cancer cells. Correspondingly, in vitro treatment of prostate cancer cells with the creatine analogue Cyclocreatine significantly reduced intracellular levels of creatine and its derivatives phosphocreatine and creatinine, and inhibited cell proliferation. Supplementation with Cyclocreatine inhibited tumor progression in a genetically engineered mouse model of PTEN and SPRY2-deficient prostate cancer (GEMM) and a xenograft liver metastasis model. These results together reveal the metabolic vulnerability of prostate cancer and demonstrate a rational therapeutic strategy to utilize this vulnerability to inhibit tumor progression. [3]

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Prostate cancer has an extremely high incidence rate and is the second leading cause of cancer death in men worldwide. We applied a novel genetically engineered mouse model (GEMM) that mimics invasive prostate cancer driven by defects in the PTEN and SPRY2 (Sprouty 2) tumor suppressor factors and found enhanced creatine metabolism in the phosphagen system during disease progression. The altered creatine metabolism was validated in in vitro and in vivo prostate cancer models as well as in clinical cases. The upregulation of creatine levels was due to increased uptake and de novo synthesis of the SLC6A8 creatine transporter, resulting in enhanced basal cellular respiration. Treatment with Cyclocreatine (a potent and specific creatine analog that blocks the phosphagen system) significantly reduced creatine and phosphocreatine levels. Cyclocreatine inhibits prostate cancer growth in vitro by blocking creatine biosynthesis, leading to the accumulation of the creatine biosynthesis intermediate S-adenosylmethionine (SAM) in cells. In addition, Cyclocreatine treatment inhibited cancer progression in our genetically engineered mouse model (GEMM) and xenograft liver metastasis model. Thus, Cyclocreatine exerts its antitumor effect through a dual mechanism of SAM accumulation and phosphagen system inhibition by targeting the phosphagen system. [4] In this study, we did not observe any adverse reactions to cCr/Cyclocreatine treatment, including no cCr-related death, brain morphological pathological changes, or exacerbation of epilepsy phenotype. In fact, we observed the opposite, with cCr protecting CrT−/y mice from spontaneous and chemically induced seizures. The only possible side effect was that the alpha wave power during sleep may be enhanced after treatment with 140 mg/kg cCr, which may be related to sleep disturbances. This contradicts the results of a recent toxicology study that showed cCr increased seizure rates and brain vacuolation in Sprague-Dawley rats. This difference may be related to the higher dose used in the latter study (vacuolation was observed at 600 mg/kg/day), different species (rats vs. mice), and/or animal genotypes (wild-type vs. animals lacking CrT function). Since cCr does not require CrT for cellular uptake but can be transported via CrT, this may explain the difference in cCr accumulation between wild-type and CrT−/γ animals. Taken together, these data suggest that excessive cCr may have adverse effects on brain circuitry, but further formal toxicology studies in different species are needed to assess the safety profile of this drug. Overall, our findings support the efficacy of cCr in treating connective tissue diseases (CTDs) and lay the foundation for designing interventions targeting this molecule or other chemically modified lipophilic compounds that could be readily translated into clinical applications.

C5H9N3O2

143.15

143.069

35404-50-3

2896

White to yellow solid powder

1.6±0.1 g/cm3

278.4±42.0 °C at 760 mmHg

25-28 °C(lit.)

122.2±27.9 °C

0.0±1.2 mmHg at 25°C

1.660

-1.99

2

3

2

10

178

0

C1CN(C(=N1)N)CC(=O)O

AMHZIUVRYRVYBA-UHFFFAOYSA-N

InChI=1S/C5H9N3O2/c6-5-7-1-2-8(5)3-4(9)10/h1-3H2,(H2,6,7)(H,9,10)

2-(2-amino-4,5-dihydroimidazol-1-yl)acetic acid

cyclocreatine; 35404-50-3; 1-Carboxymethyl-2-iminoimidazolidine; 2-Imino-1-imidazolidineacetic acid; UNII-6732XGX1RK; 6732XGX1RK; AM 285; AM-285;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

1M HCl: 100 mg/mL (698.57 mM)
H2O: 12.5 mg/mL (87.32 mM)
DMSO: < 1 mg/mL

Solubility in Formulation 1: 10 mg/mL (69.86 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with heating and sonication.

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