| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
|
||
| 10mg |
|
||
| 25mg |
|
||
| 50mg |
|
||
| 100mg |
|
||
| 250mg | |||
| 500mg | |||
| Other Sizes |
Purity: ≥98%
PF-04447943 (PF04447943) is a novel and potent inhibitor of human recombinant PDE9A with the potential for the treatment of cognitive disorders. It inhibits PDE9A with IC50 of 12 nM and exhibits>78-fold selectivity over other PDE family members (IC50>1000 nM). PF-04447943 was found to have high affinity (Ki of 2.8, 4.5 and 18 nM) for human, rhesus and rat recombinant PDE9 respectively and high selectivity for PDE9 versus PDEs1-8 and 10-11. PF-04447943 significantly increased neurite outgrowth and synapse formation (as indicated by increased synapsin 1 expression) in cultured hippocampal neurons at low (30-100 nM) but not high (300-1000 nM) concentrations. PF-04447943 significantly facilitated hippocampal slice LTP evoked by a weak tetanic stimulus at a concentration of 100 nM but failed to affect response to the weak tetanus at either 30 or 300 nM, or the LTP produced by a theta burst stimulus. Systemic administration of PF-04447943 (1-30 mg/kg p.o.) dose-dependently increased cGMP in the cerebrospinal fluid 30 min after administration indicating target engagement in the CNS of rats. PF-04447943 (1-3 mg/kg p.o.) significantly improved cognitive performance in three rodent cognition assays (mouse Y maze spatial recognition memory model of natural forgetting, mouse social recognition memory model of natural forgetting and rat novel object recognition with a scopolamine deficit). When administered at a dose of 3 mg/kg p.o., which improved performance in novel object recognition, PF-04447943 significantly increased phosphorylated but not total GluR1 expression in rat hippocampal membranes. Collectively these data indicate that PF-04447943 is a potent, selective brain penetrant PDE9 inhibitor that increased indicators of hippocampal synaptic plasticity and improved cognitive function in a variety of cognition models in both rats and mice. Results with PF-04447943 are consistent with previously published findings using a structurally diverse PDE9 inhibitor, BAY73-6199, and further support the suggestion that PDE9 inhibition may represent a novel approach to the palliative remediation of cognitive dysfunction.
| Targets |
IC50: 12 nM (PDE9A)[1]
PF-04447943 is a potent and selective inhibitor of phosphodiesterase 9A (PDE9A). Its affinity (Ki) for recombinant human PDE9A2 is 2.8 ± 0.26 nM, for rhesus PDE9A2 is 4.5 ± 0.13 nM, and for rat PDE9A2 is 18.1 ± 1.9 nM. It is highly selective over other PDE families (PDE1-8, 10-11) with Ki values >5,000 nM for most, demonstrating >1000-fold selectivity for PDE9. [2] |
|---|---|
| ln Vitro |
In a cell-free experiment employing recombinant human, rhesus, and rat PDE9A2, Edelinontrine's Ki values are 2.8±0.26, 4.5±0.13, and 18.1±1.9 nM (n=4, 11 and 9 correspondingly). Edelinontrine exhibits a high degree of selectivity when compared to other PDE enzymes (PDE1, Ki=8600±2121 nM, n = 5; PDE2A3, Ki>99,000 nM; PDE3A, Ki>50,000 nM; PDE4A, Ki>29,000 nM; PDE5A, Ki=14,980±5025 nM, n = 5; PDE6C, Ki=5324±2612 nM, n=4; PDE7A2, Ki>75,000 nM; PDE8A, Ki>50,000 nM; PDE10, Ki>51,250±20,056 nM, n = 4; PDE11, Ki>80,000 nM) and exhibits no other notable activity at ~60 other receptors/enzymes. With an IC50 of 375±36.9 nM (n=16) in HEK whole cells expressing rhesus PDE9A2, Edelinontrine suppresses ANP (0.3 μM) induced cGMP[2].
In primary rat hippocampal neuronal cultures, PF-04447943 at low concentrations (30-100 nM) significantly increased neurite outgrowth per neuron and the number of synapsin 1-positive puncta (an indicator of synapses) per neuron, with maximal effects around 100 nM. Higher concentrations (300-1000 nM) did not show significant effects, indicating an inverted U-shaped concentration-response curve. [2] In mouse hippocampal slices, PF-04447943 at 100 nM significantly facilitated long-term potentiation (LTP) induced by a weak tetanic stimulus (50 Hz, 500 ms), resulting in an approximately 20% enhancement maintained for up to 60 minutes post-tetanus. No significant effect was observed at 30 nM or 300 nM, nor on LTP induced by a robust theta-burst stimulation (TBS). [2] PF-04447943 inhibited atrial natriuretic peptide (ANP, 0.3 µM)-stimulated cGMP accumulation in HEK-293 cells stably expressing rhesus PDE9A2 and hNPR1 with an IC50 of 375 ± 36.9 nM. [2] |
| ln Vivo |
Pharmacokinetic studies using Edelinontrine in rats show a Tmax of 0.3 h, T1/2 of 4.9 h, Cl of 21.7 mL/min/kg, and an oral bioavailability of 47% based on iv and po doses. After rats were given oral medication (1–30 mg/kg), blood, brain, and cerebrospinal fluid (CSF) concentrations of eldinontrine increased in a dose-dependent manner after thirty minutes. Thirty minutes after dosage, the brain:plasma exposure ratios vary from 0.13 at 1 mg/kg to 0.33 at 30 mg/kg. About 50% of brain levels are represented by CSF levels. The plasma and brain concentrations of Edelinontrine in mice are increased dose-dependently by 3, 10, and 30 mg/kg po. The brain to plasma ratio ranges from 0.26 to 0.7, however this is not totally dosage dependent. At the 30 mg/kg dose, CSF cGMP levels rise in a dose-dependent manner from a basal level of 3 pmol/mL to 13.3 pmol/mL (3.5-fold). Additionally, CSF cGMP levels rise in a dose-dependent way; at the 30 mg/kg dose, they increase by 3.5 times, from a basal level of 3 pmol/mL in animals treated with a vehicle to 13.3 pmol/mL. At all studied doses, CSF cGMP levels are raised, reaching a maximum impact of 3.5 times the increase over controls at 30 mg/kg[2].
Oral administration of PF-04447943 (1-30 mg/kg) dose-dependently increased cGMP levels in rat cerebrospinal fluid (CSF) 30 minutes post-dose, indicating central target engagement. Basal CSF cGMP (3 pmol/ml) increased up to 13.3 pmol/ml (3.5-fold) at 30 mg/kg. [2] PF-04447943 (1-3 mg/kg p.o.) significantly improved cognitive performance in rodent models: it enhanced 24-hour social recognition memory in mice at 1 mg/kg, improved spatial recognition memory in the mouse Y-maze at 1 and 3 mg/kg, and reversed scopolamine-induced deficits in novel object recognition in rats at 3 mg/kg. A bell-shaped (inverted U) dose-response was observed, with 10 mg/kg being ineffective in these models. [2] In rats, PF-04447943 (3 mg/kg p.o.), the dose effective in reversing scopolamine-induced cognitive deficit, significantly increased phosphorylation of hippocampal AMPA receptor subunit GluR1 at serine 831, without affecting total GluR1 levels. [2] |
| Enzyme Assay |
The affinity of PF-04447943 for human, rhesus, and rat PDE9A2 was determined using a cell-free fluorescence polarization (FP) assay. Recombinant enzymes (human GST-tagged PDE9A2, or membrane preparations from cells overexpressing rhesus or rat PDE9A2) were incubated with the compound and a FAM-labeled cGMP substrate (final concentration 50 nM) in assay buffer for 60 minutes at room temperature. The enzymatic reaction was stopped by adding a binding solution containing specific reagents. Fluorescence polarization (mP) was measured, and concentration-inhibition curves were fitted to determine the apparent inhibition constant (Ki). Assays were performed in quadruplicate. [2]
Selectivity profiling against other PDE families (PDE1-8, 10-11) was performed using similar FP assays with corresponding recombinant human enzymes and appropriate FAM-labeled cAMP or cGMP substrates at concentrations near their apparent Km values. Assays were performed in duplicate at room temperature. [2] |
| Cell Assay |
The rhesus PDE9A2 construct is subcloned into a pcDNA3.3 TOPO vector and HEK 293 cells, stably transfected to constitutively express rhesus PDE9A2 and hNPR1, are incubated with PF-04447943 (30 μM to 1.5 nM) in assay media at a density of 10,000 cells/well, for 30 min at 37°C. Cyclic GMP formation is stimulated by incubation with 0.3 μM ANP (Atrial Natriuretic Peptide) for another 30 min at 37°C. Following incubation, cells are lysed with Antibody/Lysis buffer and ED Reagent for 1 h at room temperature. After a subsequent incubation with EA Reagent for 30 min at room temperature, followed by incubation with Substrate Reagent for 1 h at room temperature, cGMP concentrations are determined by measuring luminescence on the Envision Microplate Luminometer. The maximal inhibition (100% activity) in the cell based assay is determined using 30 μM PF-04447943 and 0% activity is defined by the DMSO control. PF-04447943 is titrated in quadruplicate, in a 10 point titration. Percentage inhibition is calculated using the maximal inhibition value and IC50 values are calculated from concentration response curves using Prism software[2].
The effect of PF-04447943 on hippocampal neuronal morphology and synaptogenesis was assessed using primary rat embryonic (E18) hippocampal neurons. Neurons were plated at low density (5000 cells/well) and maintained for 6 days. On day 5, cells were treated with PF-04447943 or vehicle for 24 hours. Cells were then fixed, permeabilized, and immunostained for the neuronal marker MAP2 and the synaptic vesicle protein synapsin 1, along with nuclear staining. High-content imaging and analysis were performed to quantify the average neurite length per neuron and the number of synapsin 1-positive puncta per neuron across multiple fields per well. [2] A cell-based assay was used to determine the IC50 for inhibiting cGMP accumulation. HEK-293 cells stably expressing rhesus PDE9A2 and hNPR1 were plated and incubated with PF-04447943 for 30 minutes, followed by stimulation with ANP (0.3 µM) for another 30 minutes. Cells were lysed, and intracellular cGMP levels were quantified using a commercial chemiluminescent immunoassay kit. Luminescence was measured, and IC50 values were calculated from concentration-response curves. [2] |
| Animal Protocol |
For pharmacokinetic (PK) studies in rats, PF-04447943 was administered intravenously (i.v.) and orally (p.o.). Blood and brain samples were collected at various time points. Plasma was separated, and brains were homogenized. Drug concentrations in plasma and brain homogenates were quantified using UPLC coupled with tandem mass spectrometry (UPLC-MS/MS). [2]
For CSF cGMP measurement, rats received PF-04447943 (1-30 mg/kg p.o.) and CSF was collected from the cisterna magna 30 minutes later under CO2 euthanasia. cGMP levels were measured using a commercial enzyme immunoassay (EIA) kit. [2] In cognitive behavioral tests, PF-04447943 was administered orally to rodents. For mouse social and Y-maze tests, it was formulated in 20% hydroxypropyl-β-cyclodextrin and given 30 minutes before the first trial at doses of 1, 3, or 10 mg/kg (10 ml/kg dosing volume). For the rat novel object recognition test, it was formulated in water and administered 60 minutes before the first trial at doses of 1, 3, or 10 mg/kg. Scopolamine (1 mg/kg i.p.) was given 30 minutes before the trial to induce deficits. [2] For the hippocampal GluR1 phosphorylation study, rats received PF-04447943 (3 mg/kg p.o.) or vehicle (water) 1 hour before being euthanized. Hippocampi were rapidly dissected, frozen, and later processed for subcellular fractionation to obtain membrane-enriched fractions for Western blot analysis. [2] |
| ADME/Pharmacokinetics |
In rats, the time to peak concentration (Tmax) of PF-04447943 was 0.3 h, the elimination half-life (T1/2) was 4.9 h, the clearance (Cl) was 21.7 ml/min/kg, and the oral bioavailability was 47%. [2] After oral administration of PF-04447943 (1-30 mg/kg) to rats, the concentrations of PF-04447943 in plasma, brain tissue, and cerebrospinal fluid increased in a dose-proportional manner within 30 minutes after administration. The brain tissue to plasma exposure ratio ranged from 0.13 (1 mg/kg) to 0.33 (30 mg/kg). The drug concentration in cerebrospinal fluid was approximately 50% of the brain tissue concentration. [2] In mice, oral administration (3, 10, 30 mg/kg) also resulted in a dose-dependent increase in plasma and brain tissue concentrations, with brain-plasma ratios ranging from 0.26 to 0.7. [2]
|
| References |
|
| Additional Infomation |
PF-04447943 has been investigated for the treatment of Alzheimer's disease. PF-04447943 is a selective PDE9 inhibitor currently undergoing clinical development for Alzheimer's disease. Its cognitive enhancement effect is thought to be related to the regulation of hippocampal synaptic plasticity, possibly through enhancement of cholinergic and/or glutamate signaling pathways. Improvement in cognitive function is associated with increased levels of hippocampal GluR1 phosphorylation, a marker of AMPA receptor activation and synaptic enhancement. The inverted U-shaped dose-response curves observed in vitro and in vivo suggest a narrow optimal therapeutic window, which may be due to feedback inhibition mechanisms limiting the accumulation of cGMP at high exposures. [2]
|
| Molecular Formula |
C20H25N7O2
|
|---|---|
| Molecular Weight |
395.458203077316
|
| Exact Mass |
395.206
|
| CAS # |
1082744-20-4
|
| PubChem CID |
135564558
|
| Appearance |
Light yellow to yellow solid powder
|
| Density |
1.5±0.1 g/cm3
|
| Index of Refraction |
1.763
|
| LogP |
-0.44
|
| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
7
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
29
|
| Complexity |
632
|
| Defined Atom Stereocenter Count |
2
|
| SMILES |
C[C@@H]1CN(C[C@H]1C2=NC3=C(C=NN3C4CCOCC4)C(=O)N2)CC5=NC=CC=N5
|
| InChi Key |
IWXUVYOOUMLUTQ-CZUORRHYSA-N
|
| InChi Code |
InChI=1S/C20H25N7O2/c1-13-10-26(12-17-21-5-2-6-22-17)11-16(13)18-24-19-15(20(28)25-18)9-23-27(19)14-3-7-29-8-4-14/h2,5-6,9,13-14,16H,3-4,7-8,10-12H2,1H3,(H,24,25,28)/t13-,16-/m1/s1
|
| Chemical Name |
6-((3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl)-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo(3,4-d)pyrimidin-4-one
|
| Synonyms |
PF-04447943; PF-4447943; PF 04447943; PF 4447943; PF04447943; PF4447943.
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO : ≥ 54.6 mg/mL (~138.07 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.32 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 (6.32 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 (6.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5287 mL | 12.6435 mL | 25.2870 mL | |
| 5 mM | 0.5057 mL | 2.5287 mL | 5.0574 mL | |
| 10 mM | 0.2529 mL | 1.2644 mL | 2.5287 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
COA