Monday, July 13, 2015

Drugs in Clinical Pipeline: AZD6482

AZD6482 [(R)-2-((1-(7-methyl-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-9-yl)ethyl)amino)benzoic acid] is a potent, selective and ATP competitive Phosphoinositide 3-Kinase β (PI3Kβ) inhibitor (IC50 = 0.01 uM). PI3Kβ is given an important role in platelet adhesion and aggregation. AZD6482 showed a maximal anti-platelet effect at 1 uM in the in vitro and ex vivo tests both in dog and in man [1].

This novel isoform-selective inhibitor of PI3Kβ was originally developed as a racemic mixture named KN-309 (later AZ12379678) [2]. The racemate (AZ12379678) and the S-enantiomer (AZ12502334) were about three-fold (IC50 = 0.03 uM) and greater than 200-fold (IC50 = 2.3 uM) less potent inhibitor of PI3Kβ as compared with the R-enantiomer AZD6482, respectively. AZD6482 was assessed in functional assays against 88 protein kinases including the closely related PI3K-like protein kinases; DNA-dependent protein kinase (DNA-PK, IC50 = 0.42 uM), ataxia telangiectasia mutated protein (ATM, IC50 greater than 30 uM) and mammalian target of rapamycin (mTOR, IC50 = 8 uM). The selective ratio was high (greater than 200-fold) with the exception of the other PI3K isoforms and one related protein kinase (DNA-PK), 8-08-fold, whereas ATM and mTOR displayed high selectivity in spite of also being PI3K like.

AZD6482 had a short plasma half-life owing to a high clearance and a relatively small distribution volume. With this profile, AZD6482 may be useful as a parenteral antiplatelet agent in situations where a low bleeding risk is desirable such as during acute stroke and cardiothoracic surgery.

The activity of AZD6482 is as follows:

IC50 (PI3Kα enzyme assay) = 0.87 uM
IC50 (PI3Kδ enzyme assay) = 0.08 uM
IC50 (PI3Kβ enzyme assay) = 0.01 uM
IC50 (PI3Kγ enzyme assay) = 1.09 uM

Common Name: AZD6482
Synonyms: AZD6482; AZD-6482; AZD 6482; KN-309; KN309; KN 309
IUPAC Name: (R)-2-((1-(7-methyl-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-9-yl)ethyl)amino)benzoic acid
CAS Number: 1173900-33-8
Mechanism of Action: Kinase Inhibitor; PI3K Inhibitor; PI3Kβ Inhibitor
Indication: Anti-platelet Therapy; Treatment for Strokes
Development Stage: Phase I
Company: AstraZeneca

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the D3 hydroxyl group of membrane phosphoinositides (PIs). They are divided into three distinct classes (Class I, II and III) based on their primary structure, mode of regulation and substrate specificity. Class I PI3Ks are heterodimers with a catalytic (p110α, β, γ and δ) and a regulatory subunit (p85 or p101). They are involved in signal transduction through tyrosine kinase receptors and G protein-coupled receptors and are responsible for agonist-induced production of the second messenger phosphatidylinositol (3,4,5)trisphosphate (PIP3) by phosphorylation of the 3-position in the inositol ring of phosphatidylinositol (4,5)bisphosphate (PIP2) using ATP as a phosphate donor.

PI3Kα and β are ubiquitously expressed whereas PI3Kδ is mainly expressed in leukocytes and PI3Kγ in leukocytes, platelets and cardiomyocytes. PI3Ks have been attributed a central role in cell signaling from a number of receptors involved in proliferation, motility, immune system function etc. Insulin signaling is also modulated through PI3Ks, which promotes glucose uptake and lipogenesis. The literature indicates that the effect on insulin signaling is mediated via PI3Kα but a minor role for PI3Kβ cannot be excluded.

Platelets contain all class I isoforms (α, β, γ and δ) although the levels of PI3Kδ are very low. The role of the individual PI3K isoforms in regulating platelet functional responses has started to be defined during the recent years and PI3Kβ has been attributed the most prominent role of the four. Inhibition of PI3Kβ has been shown to inhibit thrombus formation in different in vivo models without significantly affecting primary hemostasis [1].

AZD6482 potently inhibited Akt(Ser 473) phosphorylation in adenocarcinoma MDA-MB- 468 cells (IC50 = 0.04 uM).

The human anti-platelet potency of AZD6482 was evaluated in a number of in vitro assays using several agonists. The most potent effect was seen on shear induced platelet adhesion and aggregation. In this assay, 5 uM AZD6482 appears not to inhibit primary adhesion but has a strong effect on aggregate formation. The racemate, AZ12379678, had about 25% the potency of AZD6482, close to the theoretical 50%, using ADP as an agonist (impedance aggregometry) whereas the S-enantiomer, AZ12502334, was inactive with no inhibition detected at concentrations less than 10 uM. AZD6482 was also evaluated in vitro in dog and rat blood using ADP-induced impedance aggregometry. AZD6482 was more potent in human blood than in dog and rat blood with IC50 values of 0.27, 1.4 and 1.8 uM in human, dog and rat blood, respectively. Protein binding in man, dog and rat was 8%, 10% and 11% free unbound, respectively. In vivo in dog, AZD6482 produced a complete antithrombotic effect without significantly compromising hemostasis as no increase in bleeding time or blood loss was seen at plasma exposure that achieved a full anti-thrombotic effect, 1 uM. The anti-platelet effect measured ex vivo in dog directly mirrors the antithrombotic effect in vivo and full inhibition of both occurred at approximately 1 uM plasma exposure [1].

1. Nylander, S.; et. al. Human target validation of phosphoinositide 3-kinase (PI3K)β: effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kß inhibitor. J Thromb Haemost 2012, 10(10), 2127-2136.
2. Jackson, S. P.; et. al. Preparation of morpholinyl- and pyridinyl-substituted heterobicyclic ketones as selective inhibitors of phosphoinositide 3- kinase b for use against thrombosis. WO2004016607
3. Study to Investigate Safety and Tolerability of a Single Dose of AZD6482. NCT00688714 (retrieved on 01-07-2015).
4. Bleeding Time Study With AZD6482, Clopidogrel and ASA. NCT00853450 (retrieved on 01-07-2015).