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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To 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. CD-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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To 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. CD-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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Satellite pharmacokinetic and histology study [4]
Satellite 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.

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Drug administration and design of efficacy study [5]
Cyclocreatine(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]

cCr/Cyclocreatine permeates the blood–brain barrier and gets into the brain [5]
To assess the brain penetration of cCr, we monitored the pharmacokinetic profile in a group of animals following oral administration. We detected brain entry of cCr in all mice chronically treated with the drug, with higher levels of total cCr in H-CrT−/y mice than in M-CrT−/y and L-CrT−/y animals (Fig. 5). Moreover, longitudinal quantification of cCr at the three different ages employed for behavioral and functional testing revealed that high-dose administration promptly leads to a rise in cCr concentration in the brain, with cCr reaching steady state levels following 23 days of dosing and remaining stable through the treatment period (Fig. 5).

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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.

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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.

[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 organooxygen compound and an organonitrogen compound. It is functionally related to an alpha-amino acid.

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Introduction: A critical mechanism of how hypoxia/ischemia causes irreversible myocardial injury is through the exhaustion of adenosine triphosphate (ATP). Cyclocreatine (CCr) and its water-soluble salt Cyclocreatine-Phosphate (CCrP) are potent bioenergetic agents that preserve high levels of ATP during ischemia. Areas covered: 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. Medline and Embase (1970 - Feb 2019), the WIPO databank (up to Feb 2019); no language restriction. Expert opinion: This review provides the basis for a number of clinical applications of CCrP and CCr to minimize ischemic injury and necrosis. One strategy is to administer CCrP to AMI patients in the pre-hospital phase, as well as during, or after Percutaneous Coronary Intervention (PCI) procedure to potentially achieve protection of the myocardium, reduce infarcted-size, and, thus, limit the progression to heart failure. Another clinical applications are in predictable myocardial ischemia where pretreatment with CCrP would likely improve outcome and quality of life of patients who will undergo cardiopulmonary bypass for coronary revascularization and end-stage heart failure patients scheduled for heart transplantation. [1]

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Elevated risk of developing Alzheimer's disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering, and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth-factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA sequencing (RNA-seq) linked this anomalous autophagy to defective mammalian target of rapamycin (mTOR) signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and Cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-β pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism.[2]

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Along these lines, our study shows that the defect in mTOR-mediated metabolic activation in TREM2-deficient cells can be corrected in vitro through the creatine kinase pathway or by triggering the Dectin-1 pathway, which transmits intracellular signals similar to those of TREM2. Based on these results, we adopted a therapeutic strategy based on the use of Cyclocreatine, an analog of creatine that crosses membranes, enters the brain (Woznicki and Walker, 1979), can be phosphorylated and dephosphorylated by creatine kinases (McLaughlin et al., 1972), and can generate a supply of ATP (Kurosawa et al., 2012). Remarkably, we found that administration of dietary cyclocreatine throughout the progression of Aβ accumulation improves microglia viability, numbers and clustering around Aβ plaques. As a result, plaques are denser and, most importantly, plaque-associated neurite dystrophy is greatly reduced. Although cyclocreatine treatment was not sufficient to reduce the overall Aβ plaque accumulation, this may depend on time point chosen for analysis and/or cyclocreatine dosage and duration of treatment. While the creatine kinase pathway has been previously recognized to play an important role in the CNS in neurotransmitter release, membrane potential maintenance, Ca2+ homeostasis, and ion gradient restoration (Snow and Murphy, 2001; Wyss and Kaddurah-Daouk, 2000), our results indicate that this system may also be exploited for sustaining microglial metabolism. It should be noted that in certain settings cyclocreatine can inhibit creatine kinase and can also have systemic effects such as alteration in pancreatic hormones and glucose metabolism (Ara et al., 1998; Kuiper et al., 2008). Thus, it must be emphasized that our findings provide proof of principal studies and the use of cyclocreatine as a preventative treatment for AD is not advisable. Future studies will be required to precisely define the mechanisms through which cyclocreatine impacts microglial responses to Aβ. Additionally, it will be important to determine whether Cyclocreatine has any impact on proteolytic shedding of TREM2 from microglia, which results in the release of soluble TREM2 with potential pro-survival functions. Altogether, our study provides indicates that strategies aimed at sustaining microglial metabolism may be promising for therapeutic intervention in AD and other neurodegenerative diseases linked to TREM2 deficiency and microglial dysfunction in general.[2]

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Prostate cancer is the second most common cause of cancer mortality in men worldwide. Applying a novel genetically engineered mouse model (GEMM) of aggressive prostate cancer driven by deficiency of the tumor suppressors PTEN and Sprouty2 (SPRY2), we identified enhanced creatine metabolism as a central component of progressive disease. Creatine treatment was associated with enhanced cellular basal respiration in vitro and increased tumor cell proliferation in vivo. Stable isotope tracing revealed that intracellular levels of creatine in prostate cancer cells are predominantly dictated by exogenous availability rather than by de novo synthesis from arginine. Genetic silencing of creatine transporter SLC6A8 depleted intracellular creatine levels and reduced the colony-forming capacity of human prostate cancer cells. Accordingly, in vitro treatment of prostate cancer cells with Cyclocreatine, a creatine analog, dramatically reduced intracellular levels of creatine and its derivatives phosphocreatine and creatinine and suppressed proliferation. Supplementation with cyclocreatine impaired cancer progression in the PTEN- and SPRY2-deficient prostate cancer GEMMs and in a xenograft liver metastasis model. Collectively, these results identify a metabolic vulnerability in prostate cancer and demonstrate a rational therapeutic strategy to exploit this vulnerability to impede tumor progression.[3]

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Prostate cancer is highly prevalent, being the second most common cause of cancer mortality in men worldwide. Applying a novel genetically engineered mouse model (GEMM) of aggressive prostate cancer driven by deficiency of PTEN and SPRY2 (Sprouty 2) tumour suppressors, we identified enhanced creatine metabolism within the phosphagen system in progressive disease. Altered creatine metabolism was validated in in vitro and in vivo prostate cancer models and in clinical cases. Upregulated creatine levels were due to increased uptake through the SLC6A8 creatine transporter and de novo synthesis, resulting in enhanced cellular basal respiration. Treatment with Cyclocreatine (a creatine analogue that potently and specifically blocks the phosphagen system) dramatically reduces creatine and phosphocreatine levels. Blockade of creatine biosynthesis by cyclocreatine leads to cellular accumulation of S-adenosyl methionine (SAM), an intermediary of creatine biosynthesis, and suppresses prostate cancer growth in vitro. Furthermore, cyclocreatine treatment impairs cancer progression in our GEMM and in a xenograft liver metastasis model. Hence, by targeting the phosphagen system, cyclocreatine results in anti-tumourigenic effects from both SAM accumulation and suppressed phosphagen system.[4]

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We did not report any detrimental effects of cCr/Cyclocreatine treatment in this study, including no cCr-related deaths, pathological alterations in brain morphology, or exacerbation of the epileptic phenotype. In fact, we observed just the opposite with cCr protecting against spontaneous and chemically-induced convulsions in CrT−/y mice. The only possible side effect could be the gain of alpha power during the sleep phase observed following the treatment with the 140 mg/kg cCr dose, potentially related to sleep disturbances. This is in contrast to a recent toxicology study where cCr has been described to increase seizure incidence and brain vacuolation in Sprague–Dawley rats. This difference could be related to the higher doses used in the latter study (vacuolation was observed at 600 mg/kg/day), the different species (rats vs. mice) and/or the genotype of animals (wild type vs. animals lacking CrT function). Since cCr does not require CrT for cellular uptake, but can be transported by CrT, this could potentially lead to differential build-up of cCr in wild-type compared to CrT−/y animals. Taken together, these data raise the possibility that cCr could have adverse effects on brain circuitry when present in excess, but additional formal toxicology studies in different species would be needed to assess the safety window of this drug. Overall, our findings support the therapeutic efficacy of cCr for treating CTD, laying the foundation for the design of intervention protocols for this molecule or other chemically modified, lipophilic compounds that could be readily translated to the bedside.

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.)