Endogenous Metabolite

In a dose-dependent manner, L-Sepiapterin (Sepiapterin) (0.1-10 μM; 24 hpurs) induces cell proliferation [1]. The phosphorylation of p70S6K induced by VEGF-A (50 ng/ml) is significantly inhibited by L-sepiapterin (1-50 μM; 20 minutes) [1]. Via a NO-dependent mechanism, L-sepiapterin prevents VEGF-A-induced cell migration and proliferation [1].

---

Tetrahydrobiopterin (BH4) is known to be an essential cofactor for the aromatic amino acid hydroxylases, which are involved in the production of neurotransmitters, and for nitric oxide (NO) synthase. In the present study, researchers report that sepiapterin, the more stable form of the BH4 precursor, modulates ovarian cancer cell proliferation and migration by NO-dependent and -independent mechanisms. Sepiapterin induction of cell proliferation and migration in SKOV-3 cells is accompanied by ERK, Akt and p70S6K activation. These stimulatory effects of sepiapterin are reversed by pretreatment with NO synthase inhibitor. Researchers also show that sepiapterin significantly inhibits vascular endothelial growth factor-A (VEGF-A)-stimulated cell proliferation and migration. Pretreatment with NO synthase inhibitor does not alter the ability of sepiapterin to inhibit VEGF-A-induced cell proliferation and migration, indicating that the suppressive effects of sepiapterin on VEGF-A-induced responses are mediated by a NO-independent mechanism. Finally, researchers demonstrate that sepiapterin markedly suppresses VEGF-A-induced p70S6K phosphorylation and VEGFR-2 expression, resulting in inhibition of VEGF-A-induced cell proliferation and migration. Collectively, these findings represent a biphasic effect of sepiapterin on cellular fates, depending on the presence of growth factors, and support further development and evaluation of sepiapterin for the treatment of cancers overexpressing VEGFR-2 [2].

Sepiapterin (10 mg/kg; po (powder feed); once daily for about 8 weeks) significantly increases Ach relaxation in the mesenteric arterioles (SMA) of db/db mice [2].

---

Researchers previously reported that acute incubation with tetrahydrobiopterin (BH4) or sepiapterin, a cofactor for endothelial nitric oxide synthase and a stable precursor of BH4, respectively, enhanced the acetylcholine (Ach)-induced relaxation of isolated small mesenteric arteries (SMA) from diabetic (db/db) mice. In this study, Researchers investigated the effect of chronic oral supplementation of sepiapterin (10 mg kg−1 day−1) to db/db mice on endothelium function, biopterin levels and lipid peroxidation in SMA. Oral dietary supplementation with sepiapterin had no effect on glucose, triglyceride, cholesterol levels and body weight. SMA from db/db mice showed enhanced vascular reactivity to phenylephrine, which was corrected with sepiapterin supplementation. Furthermore, Ach, but not sodium nitroprusside-induced relaxation, was improved with sepiapterin supplementation in db/db mice. BH4 levels and guanosine triphosphate cyclohydrolase I activity in SMA were similar in db/+ and db/db mice. Sepiapterin treatment had no effects on BH4 or guanosine triphosphate cyclohydrolase I activity. However, the level of dihydrobiopterin+biopterin was higher in SMA from db/db mice, which was corrected following sepiapterin treatment. Thiobarbituric acid reactive substance, malondialdehyde, a marker of lipid peroxidation, was higher in SMA from db/db mice, and was normalized by sepiapterin treatment. These results indicate that sepiapterin improves endothelial dysfunction in SMA from db/db mice by reducing oxidative stress. Furthermore, these results suggest that decreased biosynthesis of BH4 may not be the basis for endothelial dysfunction in SMA from db/db mice.

Cell Proliferation Assay[1]
Cell Types: SKOV-3 Cell
Tested Concentrations: 0.1, 1, 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Cell proliferation was induced in a dose-dependent manner.

Animal/Disease Models: Male C57BL/KsJ diabetic mice (db/db)[2]
Doses: 10 mg/kg
Route of Administration: Po (powder feed); one time/day for 8 weeks
Experimental Results: Significant improvement in db/db mice Relaxation of Ach in SMA.

---

Male C57BL/KsJ diabetic mice (db/db) and nondiabetic controls (db/+), 6-week-old, were used. At 8 weeks of age, animals were divided into four groups – group 1: db/+ mice receiving powder chow for 8 weeks, group 2: db/+ mice receiving sepiapterin (10 mg kg−1 day−1) in powder chow for 8 weeks, group 3: db/db mice receiving powder chow for 8 weeks and group 4: db/db mice receiving sepiapterin (10 mg kg−1 day−1) in powder chow for 8 weeks. In accordance with a protocol approved by the University of Calgary animal care committee, mice were killed by cervical dislocation. Heparinized blood samples were collected for blood biochemistry. The mesenteric arcade was removed and first-order branches of the mesenteric artery (measuring approximately 150–200 μm in diameter) were dissected in cold Kreb's solution of the following composition (in mM): NaCl 120, NaHCO3 25, KCl 4.8, NaH2PO4 1.2, MgSO4 1.2, dextrose 11.0, CaCl2 1.8, aerated with 95% O2 and 5% CO2. The rest of the arterial bed was immediately stored at −70°C for biochemical measurements.

[1]. Chronic oral supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in small mesenteric arteries from diabetic (db/db) mice. Br J Pharmacol. 2003;140(4):701‐706.

[2]. Sepiapterin inhibits cell proliferation and migration of ovarian cancer cells via down-regulation of p70S6K-dependent VEGFR-2 expression. Oncol Rep. 2011;26(4):861‐867.

Increased oxidization of BH4 in SMA from diabetic mice may result from an increased reactive oxygen species (ROS) and oxidative stress (Giugliano et al., 1996). ROS may impair endothelium-dependent relaxation by rapidly inactivating NO resulting in the formation of peroxynitrite. BH4 is a powerful reducing agent and it is possible that excess peroxynitrite formation may lead to oxidation and depletion of BH4 (Milstien & Katusic, 1999). In the present study, malondialdehyde levels were elevated in plasma and SMA from db/db mice and were restored to within the control range by sepiapterin treatment, suggesting that the restoration of the endothelial function by sepiapterin could in part be explained by an antioxidant action of sepiapterin. Direct antioxidant properties of BH4 have also been documented by Kojima et al. in xanthine/xanthine oxidase free-radical generating system and phorbol myristate acetate stimulated rat macrophage generated free radicals (Kojima et al., 1995). Oral supplementation with BH4 normalized vascular superoxide production, membrane lipid peroxidation and prevented the activation of nuclear factor kappa B, activating protein-1 in insulin-resistant rats (Shinozaki et al., 2000). Patel et al. (2002) reported that the scavenging efficiency of BH4 was comparable to ascorbate and could be a biologically viable antioxidant (Patel et al., 2002). This will also explain the effect of sepiapterin treatment on enhanced vascular reactivity to PE in SMA from db/db mice. Free radicals are known to impair endothelium integrity, enhance alpha-adrenergic receptor-mediated turnover of phosphoinositol and augment contractility through calcium channels (Chang et al., 1993). Paradoxical negative reports, however, have been reported on the effects of sepiapterin on endothelial dysfunction. Sepiapterin attenuated Ach- and A23187-induced relaxation of isolated aorta from cholesterol-fed rabbits although increased the levels of BH4 (Vasquez-Vivar et al., 2002a). It is possible that prolonged exposure to high concentration of sepiapterin has a direct effect to uncoupled eNOS, resulting in the production of superoxide instead of NO.
In conclusion, increased oxidative stress through oxidation of BH4 uncouples eNOS leading to endothelial dysfunction in SMA from db/db mice, and oral treatment with sepiapterin improves endothelial dysfunction by reducing oxidative stress. [1]

C9H11N5O3

237.219

237.086

C, 45.57; H, 4.67; N, 29.52; O, 20.23

17094-01-8

135398579

White to yellow solid powder

1.9±0.1 g/cm3

448.1ºC at 760 mmHg

> 275 °C (lit.)

224.8ºC

6.54E-10mmHg at 25°C

1.822

-3.93

4

6

2

17

491

1

C[C@@H](C(=O)C1=NC2=C(NC1)N=C(NC2=O)N)O

VPVOXUSPXFPWBN-VKHMYHEASA-N

InChI=1S/C9H11N5O3/c1-3(15)6(16)4-2-11-7-5(12-4)8(17)14-9(10)13-7/h3,15H,2H2,1H3,(H4,10,11,13,14,17)/t3-/m0/s1

2-amino-6-[(2S)-2-hydroxypropanoyl]-7,8-dihydro-3H-pteridin-4-one

LSepiapterin; Sepiapterin; L-Sepiapterin; sepiapterin; L-Sepiapterin; 17094-01-8; Sepiapterine; Sepiapterin [USAN]; CNSA-001; PTC923; Lopac-S-154; Sepiapterinum

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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