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.

---

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.

---

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

---

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]

Absorption, Distribution and Excretion
In patients with erectile dysfunction given 20 μg of alprostadil intracavernously, the systemic plasma concentrations of prostaglandin E1 increased from a baseline of 0.8 pg/mL to a Cmax of 16.8 pg/mL (corrected for baseline). The tmax and AUC for this group of patients were 4.8 min and 173 pg⋅min/mL, respectively. In patients given 20 μg of alprostadil intravenously, AUC was similar to the one detected in patients that received alprostadil intracavernously (174 pg⋅min/mL); however, they had a higher tmax (25.5 min) and a lower Cmax (7.09 pg/mL). Compared to the same dose given by a short-term intravenous infusion, the absolute bioavailability of alprostadil estimated from systemic exposure was about 98%.
Following the degradation of alprostadil by beta- and omega-oxidation, metabolites are excreted primarily by the kidney, and excretion is essentially complete within 24 hours after administration (92%). Approximately 88% and 12% of alprostadil metabolites are excreted through urine and feces, respectively, over 72 hours. Alprostadil and its metabolites are not retained in tissues, and unchanged alprostadil has not been detected in urine.
The volume of distribution of alprostadil has yet to be determined.
In patients with erectile dysfunction given an intravenous infusion of alprostadil (20 μg), the total body clearance was 115 L/min.

---

Metabolism / Metabolites
Alprostadil is rapidly metabolized in the human body. Following intracavernous administration, alprostadil is metabolized in the corpus cavernosum, and a smaller portion is absorbed from the penis into systemic circulation. After intravenous or arterial administration, alprostadil is metabolized and distributed throughout the entire body except for the central nervous system. As much as 60-90% of the circulating alprostadil may be metabolized in the lungs through first-pass pulmonary elimination, in a process known as beta- and omega-oxidation. The enzymatic oxidation of the C15-hydroxy group of alprostadil leads to the formation of 15-keto-PGE₁, while the reduction of the C13, 14-double bond produces 15-keto-PGE₀, and 13,14-dihydro-PGE₁ (PGE₀). The 15-keto metabolites are inactive, but the PGE₀ metabolite has a similar potency to alprostadil in isolated animal organs. The major metabolite of alprostadil is 15-keto-PGE₀.

---

Biological Half-Life
In healthy adults and neonates given a single intravenous dose of alprostadil, half-life goes from 5 to 10 minutes.

Protein Binding
Alprostadil is bound in plasma primarily to albumin (81% bound) and, to a lesser extent, alpha-globulin IV-4 fraction (55% bound).

[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-).
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.
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.
Alprostadil has been reported in Populus balsamifera, Populus candicans, and other organisms with data available.
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.
A potent vasodilator agent that increases peripheral blood flow.

---

Drug Indication
Alprostadil is indicated for palliative, not definitive, therapy to temporarily maintain the patency of the ductus arteriosus until corrective or palliative surgery can be performed in neonates who have congenital heart defects and who depend upon the patent ductus for survival. It is also indicated for the treatment of erectile dysfunction due to neurogenic, vasculogenic, psychogenic, or mixed etiology, and as an adjunct to other diagnostic tests in the diagnosis of erectile dysfunction.

---

Mechanism of Action
Alprostadil is a smooth muscle relaxant that promotes vasodilation and platelet aggregation inhibition. In neonatal patients with ductus arteriosus patency, alprostadil relaxes the 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. Alprostadil appears to be most effective within 96 hours after birth since the DA rapidly loses its responsiveness to alprostadil. When administered by intracavernosal injection or as an intraurethral suppository, alprostadil acts locally to relax the trabecular smooth muscle of the corpora cavernosa and the cavernosal arteries. Swelling, elongation, and rigidity of the penis result when arterial blood rapidly flows into the corpus cavernosum to expand the lacunar spaces. The entrapped blood reduces the venous blood outflow as sinusoids compress against the tunica albuginea leading to penile rigidity. This is referred to as the corporal veno-occlusive mechanism.

---

Pharmacodynamics
Prostaglandin E1 is produced endogenously to relax vascular smooth muscle and cause vasodilation. As a synthetic form of prostaglandin E1, alprostadil has the same pharmacodynamic effects. Alprostadil inhibits platelet aggregation, has anti-inflammatory effects, interferes with immune responses, and stimulates factor X, a blood coagulation enzyme. In adult males, the use of alprostadil may lead to prolonged erection and priapism, penile fibrosis, hypotension, and injection site bleeding. In patients treated up to 24 months with alprostadil, the incidence of prolonged erections (>4 hours long) was 4% of all, and the incidence of priapism (erections greater than 6 hours in duration) was <1%. Patients with preexisting cardiovascular disease treated with alprostadil may also have higher cardiac risk. Neonates with congenital heart defects treated with alprostadil may experience apnea. Apnea is experienced by 10-12% of neonates and is more common in those weighing less than 2 kg at birth. The administration of alprostadil to neonates may also result in gastric outlet obstruction secondary to antral hyperplasia.

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

3

5

13

25

432

4

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

---

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.

---

View More

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.

---

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.

---

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.

---

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