Human Endogenous Metabolite; EP

In the presence of VEGF (20 ng/mL), prostaglandin E1 (1 nM-10 μM; 48 hours) concentration-dependently reduces HUVEC proliferation (up to 100% inhibition) with an IC50 of 400 nM [2]. Prostaglandin E1 (1-5 μM; 12-18 hours) inhibits VEGF-induced HUVEC migration in a concentration-suspended manner with an IC of 50 500 nM [2]. Prostaglandin E1 (1-5 μM; 12-18 hours) is produced by suspension cells [2] Prostaglandin E1 (0.01-10 μM; 20 minutes) increases intracellular cAMP levels in HUVECs [2].

Scaffoldin E1 (20 ng/animal/day; subcutaneous injection for 4 days) significantly inhibited FGF-induced angiogenesis in mice [2]. Animal model: C57/bl6 female mice (6-8 weeks) were injected with Matrigel and heparin supplemented with aFGF [2] Dosage: 20 ng/day/animal Administration method: Micropump was placed subcutaneously for 4 days Results: Significantly reduced new blood vessels Formation process.

Stable expression of the prostanoid receptors and ligand binding assay [1]
The CHO cell lines stably expressing the DP, EP1, EP2, EP3, EP4 and IP receptor have been described previously (Sugimoto et al., 1992; Watabe et al., 1993; Hirata et al., 1994; Namba et al., 1994; Nishigaki et al., 1995; Katsuyama et al., 1995). The establishment of the cell lines expressing the TP and FP receptors was performed as previously described (Sugimoto et al., 1992). Brie¯y, a 2.4 kb EcoRI fragment of the FP receptor cDNA (Sugimoto et al., 1994) or an EcoRI fragment of the TP receptor cDNA ML36 (Namba et al., 1992) were subcloned into pdKCR-dhfr, an eukaryotic expression vector containing a mouse dihydrofolate reductase gene as the selection marker. The plasmids were then transfected into CHO-dhfr7 cells de- ®cient in dihydrofolate reductase activity by the lipofection method. Cell populations expressing the FP or TP receptor together with dihydrofolate reductase were selected in amodi®cation of Eagle's medium (a-MEM) lacking ribonucleotides and deoxyribonucleotides. Clonal cell lines expressing each receptor were then isolated by single-cell cloning. Each line of CHO cells was cultured to near con¯uency in aMEM containing 10% foetal calf serum. After the cells were washed with Dulbecco's phosphate bu€ered saline without divalent cations (PBS(7)), they were harvested with PBS(7) containing 5 mM EDTA. The cells were pelleted by centrifugation and homogenized in 0.25 M sucrose containing 25 mM Tris.HCl, pH 7.5, 10 mM MgCl2 1 mM EDTA and 0.1 mM phenylmethylsulphonyl ¯uoride. The membranes were prepared as previously described (Namba et al., 1994). The TP, IP, DP, FP receptors and the four subtypes of the EP receptors were assayed as [3 H]-S-145, [3 H]-iloprost, [3 H]-PGD2, [3 H]- PGF2a and [3 H]-PGE2 binding activities, respectively. Scatchard analyses were performed in an assay mixture containing 25 mM Tris.HCl, pH 7.0, 10 mM MgCl2,1mM EDTA, 0.1 mM phenylmethylsulphonyl ¯uoride, 100 mg protein of each CHO cell membrane and various concentrations of the respective radioligands in a total volume of 200 ml. Nonspeci®c binding was determined as the binding in the presence of over 500 fold excess of non-labelled ligand over the respective radioligand. Incubation was carried out at 308C for 60 min except for the experiments with membranes expressing the DP receptor; these experiments were performed at 48C for 120 min, because incubation at 308C caused high nonspeci®c binding of [3 H]- PGD2 to this membrane (Hirata et al., 1994). Incubation was terminated by the addition of ice-cold 5 mM Tris HCl, pH 7.0. The mixture was ®ltered in vacuo through a Whatman GF/C ®lter. The ®lter was washed with the above bu€er ®ve times, except for the assay of the EP1 receptor binding, which was washed twice. The radioactivity on the ®lter was then determined in Triton-toluene scintillator (Ushikubi et al., 1989). In the displacement experiments, various concentrations of compounds were included in the assay mixture in the presence of each radioligand, which was used at concentrations two fold over the Kd value obtained from the Scatchard analysis.

Proliferation assays[2]
HUVECs, plated at a density of 2×104 cells well−1 in 96-well plates, were pre-treated for 30 min with PGE1/α-cyclodextrin, and then stimulated for 48 h with 20 ng ml−1 VEGF or 20 ng ml−1 bFGF in the presence of the drug. [3H]-Thymidine (1 μCi well−1; specific activity 2 Ci mmol−1) was added during the last 6 h of incubation. The radioactivity associated to the TCA-insoluble fraction was measured after 10% TCA extraction and NaOH solubilization.

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In vitro angiogenesis assays[2]
The formation of vascular-like structures was assessed on a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma (Matrigel), frequently used for the evaluation of in vitro angiogenesis (for reviews see Baatout, 1997; Benelli & Albini, 1999). Twenty-four well plates were coated with Matrigel and the cells were seeded on the polymerized matrix at a density of 5×104 cells well−1. VEGF (10 ng ml−1) and bFGF (10 ng ml−1) were used as angiogenic stimuli. PGE1/α-cyclodextrin was present in the medium during the incubation. After 12–18 h at 37°C in 5% CO2, cells were fixed in 4% paraformaldehyde, and images were acquired using an Axiovert microscope with a PCO SuperVGA SensiCam. The degree of cord formation was quantified by measuring the area occupied by the tubes in five random fields from each well using the National Institute of Health (NIH) Image Program.

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Determination of intracellular cAMP[2]
HUVECs, plated at a density of 1–1.5×105 cells well−1 in 24-well plates, were preincubated for 10 min with 1 mm isobutylmethylxanthine (IBMX) in 199 medium before stimulation for 20 min at 37°C with PGE1/α-cyclodextrin. The reaction was terminated by aspiration of the medium followed by the addition of 0.5 ml of cold absolute ethanol. After overnight freezing at −20°C, the ethanol supernatants were dried, and the intracellular cAMP levels were evaluated with a commercial kit.

C57/bl6 female mice (6-8 weeks) were injected with Matrigel supplemented with aFGF and heparin
20 ng/day/animal
Minipump placed subcutaneously for 4 days

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PGE1/α-cyclodextrin (20 ng day−1) was systemically administered by means of osmotic pumps (Alzet, Charles River) implanted subcutaneously in the back of the animals, posterior to the scapulae. The pumps continuously delivered the drug at controlled rates, with a pumping rate of 0.5 μl h−1. The control animals were implanted with the same pumps, filled with saline. After 4 days, mice were killed, the Matrigel pellets were collected and their haemoglobin content was evaluated using a Drabkin reagent kit. Animal care was in accordance with the Italian State regulation governing the care and the treatment of laboratory animals (permission n° 14/2001).[2]

[1]. Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells. Br J Pharmacol. 1997 Sep;122(2):217-24

[2]. Alprostadil suppresses angiogenesis in vitro and in vivo in the murine Matrigel plug assay. Br J Pharmacol. 2003 Jan;138(2):377-85.

[3]. Prostaglandin E1 long-term self-injection programme for treatment of erectile dysfunction--a follow-up of at least 5 years. Andrologia. 1999;31 Suppl 1:99-103.

Prostaglandin E1 is a prostaglandins E. It has a role as a platelet aggregation inhibitor, a vasodilator agent, an anticoagulant and a human metabolite. It is a conjugate acid of a prostaglandin E1(1-). ChEBI
Alprostadil is a chemically-identical synthetic form of prostaglandin E1 (PGE1), a potent vasodilator produced endogenously. In 1996, the FDA approved the use of alprostadil, administered either with an intracavernosal injection or an intraurethral suppository, for the treatment of erectile dysfunction, and it is used in men for whom oral treatment is either contraindicated or ineffective. After administration, alprostadil promotes smooth muscle relaxation of the corpus cavernosal. Alprostadil is also used in neonatal patients with congenital heart defects that depend on a patent ductus for survival until corrective or palliative surgery can be performed. This drug causes vasodilation by directly affecting vascular and ductus arteriosus (DA) smooth muscle, preventing or reversing the functional closure of the DA that occurs shortly after birth. This results in increased pulmonary or systemic blood flow in infants. DrugBank

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Alprostadil is a Prostaglandin Analog and Prostaglandin E1 Agonist. The mechanism of action of alprostadil is as a Prostaglandin Receptor Agonist. The physiologic effect of alprostadil is by means of Genitourinary Arterial Vasodilation and Venous Vasodilation. FDA Pharm Classes
Alprostadil is a natural product found in Populus balsamifera, Populus candicans, and other organisms with data available. LOTUS - the natural products occurrence database
Alprostadil is the naturally occurring prostaglandin E1 (PGE1) which displays a variety of pharmacologic actions. Alprostadil is a potent vasodilator agent that increases peripheral blood flow, inhibits platelet aggregation, and induces bronchodilation. Used in the treatment of erectile dysfunction, this agent produces corporal smooth muscle relaxation by binding to PGE receptors, resulting in the activation of adenylate cyclase and the subsequent accumulation of 3'5'-cAMP. NCI Thesaurus (NCIt)
A potent vasodilator agent that increases peripheral blood flow.

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Prostaglandin E1 (PGE1) is currently the vasoactive drug of choice for intracavernous self-injection therapy in the treatment of erectile dysfunction. PGE1 is often said to have a low incidence of side-effects. However, real long-term follow-up reports are rare. Here, a report is presented on 32 patients who joined a long-term self-injection programme in which they used PGE1 for a minimum of 5 years under standardized protocol conditions. All these patients had an organic aetiology of erectile dysfunction, and their mean age was 58.7 +/- 8.6 years. The period of observation was on average 75.4 +/- 16.9 months, and the PGE1 dosage 13.5 +/- 5.9 micrograms. A total of 6799 injections were registered. The average number of injections was 213 +/- 127 per patient, which is 2.8 injections per month and patient. As regards side-effects, haematomas were registered in 1.9% of the patients and five cases of prolonged erection (0.07%) caused by unauthorized redosing were noted. Three patients developed reversible penile nodules. In 10 patients, the initial dosage had to be increased. Five patients dropped out after 5 years, none of them due to treatment complications. It is concluded that PGE1 self-injection therapy is a simple and reliable method for long-term use with hardly any side-effects. The patients do not stop treatment because of complications.[3]

C20H34O5

354.48

354.24

C, 67.77; H, 9.67; O, 22.57

745-65-3

Prostaglandin E1-d4;211105-33-8;Prostaglandin E1-d9;2342573-59-3; 745-65-3 (free acid); 27930-45-6 (sodium); 217182-28-0 (isopropyl ester); 35900-16-4 (ethyl ester)

5280723

White to off-white solid powder

1.1±0.1 g/cm3

529.3±50.0 °C at 760 mmHg

115-116 °C

288.0±26.6 °C

0.0±3.2 mmHg at 25°C

1.546

2.24

94.83

CCCCC[C@@H](/C=C/[C@H]1[C@@H](CC(=O)[C@@H]1CCCCCCC(=O)O)O)O

GMVPRGQOIOIIMI-DWKJAMRDSA-N

InChI=1S/C20H34O5/c1-2-3-6-9-15(21)12-13-17-16(18(22)14-19(17)23)10-7-4-5-8-11-20(24)25/h12-13,15-17,19,21,23H,2-11,14H2,1H3,(H,24,25)/b13-12+/t15-,16+,17+,19+/m0/s1

7-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]heptanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.05 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (7.05 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: ≥ 2.5 mg/mL (7.05 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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