Istradefylline (formerly KW6002; KW 6002; KW-6002; Nourianz), a caffeine derivative, is an orally bioavailable and selective adenosine A2A receptor (A2AR) antagonist with anti-PD (Parkinson's disease) effects. It suppresses the A2A receptor (A2AR) with a Ki of 2.2 nM. It is authorized for use as a supplement to levodopa/carbidopa in Parkinson's disease patients who are having "off" episodes. Istradefylline lessens the "off" periods brought on by continuous administration of the Parkinson's disease medication levodopa.

Physicochemical Properties

Molecular Formula	C20H24N4O4
Molecular Weight	384.43
Exact Mass	384.179
Elemental Analysis	C, 62.49; H, 6.29; N, 14.57; O, 16.65
CAS #	155270-99-8
Related CAS #	Istradefylline-13C,d3; 2749234-46-4
PubChem CID	5311037
Appearance	Light green to green solid powder
Density	1.2±0.1 g/cm3
Boiling Point	601.0±65.0 °C at 760 mmHg
Melting Point	189-193
Flash Point	317.3±34.3 °C
Vapour Pressure	0.0±1.7 mmHg at 25°C
Index of Refraction	1.598
LogP	2.84
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	6
Heavy Atom Count	28
Complexity	613
Defined Atom Stereocenter Count	0
SMILES	O=C1C2=C(N=C(/C=C/C3=CC(OC)=C(OC)C=C3)N2C)N(CC)C(N1CC)=O
InChi Key	IQVRBWUUXZMOPW-PKNBQFBNSA-N
InChi Code	InChI=1S/C20H24N4O4/c1-6-23-18-17(19(25)24(7-2)20(23)26)22(3)16(21-18)11-9-13-8-10-14(27-4)15(12-13)28-5/h8-12H,6-7H2,1-5H3/b11-9+
Chemical Name	8-[(E)-2-(3,4-dimethoxyphenyl)ethenyl]-1,3-diethyl-7-methylpurine-2,6-dione
Synonyms	KW-6002; KW6002; KW 6002
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)

Biological Activity

Targets	Adenosine A2A receptor ( Ki = 2.2 nM )
ln Vitro	In vitro activity: Istradefylline has a 70-fold higher affinity for the A2AR than it does for the A1 receptor.[1] Istradefylline exposure in primary rat striatal astrocytes abolishes the concentration-dependent induction of astrogliosis caused by bFGF in vitro.[5] Istradefylline has binding affinities (Ki) of >287 nM, 9.12 nM, and >681 nM for A1 receptor, A2A receptor, and A3 receptor in humans, respectively; 50.9 nM and 1.57 nM for A1 receptor and A2A receptor in rats, and 105.02 nM and 1.87 nM for A1 receptor and A2A receptor in mice.[6]
ln Vivo	Istradefylline reverses the effects of reserpine- and CGS21680-induced catalepsy with ED50 of 0.05 mg/kg and 0.26 mg/kg, respectively. In these models, istradefylline is more than ten times more potent than other medications that act as dopamine agonists and adenosine antagonists. When administered in conjunction with L-dopa (50 mg/kg), istradefylline has a significant impact on catalepsy brought on by reserpine and haloperidol.[2] When oral istradefylline (10 mg/kg) is given to common marmosets receiving MPTP, the animals' locomotor activity increases to about twice that of control, and their motor disability improves. When administered in conjunction with SKF80723 (1 mg/kg, ip), quinpirole (0.06 mg/kg ip), or L-DOPA (2.5 mg/kg po), istradefylline (10 mg/kg, po) administered 90 minutes prior to SKF80723/quinpirole/L-DOPA results in a significant additive effect on locomotor activity and improves motor disability, but not dyskinesia.[3] Istradefylline considerably reduces striatal dopamine depletion in the MPTP mice model in a number of ways. One week later, partial dopamine and DOPAC depletions in striata are lessened when istradefylline (3.3 mg/kg, i.p.) is administered prior to a single MPTP dose.[1] Oral administration of Istradefylline protects against the loss of nigral dopaminergic neuronal cells induced by 6-hydroxydopamine in rats, and prevents the functional loss of dopaminergic nerve terminals in the striatum and the ensuing gliosis caused by MPTP in mice.[4] In rats depleted of dopamine, chronic istradefylline treatment does not improve the reversal deficits.[7] Co-administration of either tropicamide or istradefylline significantly reduces the tremulous jaw movements induced by pimozide. Behaviorally effective doses of istradefylline decrease pimozide-induced increases in ventrolateral striatal c-Fos expression, while tropicamide actually increases c-Fos expression in rats treated with pimozide.[8]
Cell Assay	The human adenosine A1 or A2A receptor is permanently expressed in a CHO cell line that is cultured in α-MEM supplemented with 10% (v/v) fetal bovine serum, 50 U/mL penicillin, and 50 μg/mL streptomycin. In a 5% CO2 environment, cells are grown at 37°C. These cells are cultivated for twenty-four hours after being seeded at a density of 15,000 cells per well on black 96-well assay plates.
Animal Protocol	The animals are kept in standard housing with a 12-hour light-dark cycle, at a temperature of 24-26°C and a relative humidity of 50–60%. They can be kept in pairs or alone. The diet was composed of fresh fruit, standard food pellets, and marmoset jelly from Mazuri. For five days, the animals receive a daily dosage of 2.0 mg/kg sc of MPTP. The animals are given six to eight weeks to recuperate from the acute effects of MPTP treatment. The animals are hand-fed Mazuri marmoset jelly and fresh fruit puree during MPTP treatment and in the ensuing weeks until they are able to sustain themselves. Prior to behavioral testing, all animals exhibit a significant decrease in basal locomotor activity, slower and less coordinated movements, abnormal postures of some body parts, and a reduction in blinking and checking movements between 6-8 weeks and 8 months after exposure to MPTP. By oral gavage, istradefylline (KW-6002) is given in a final volume of 2.0 mL/kg body weight after being suspended in a solution of 0.3% Tween-80 and 10% sucrose. Researchers evaluated the efficacy and potency of istradefylline (KW-6002) and other reference compounds in the selective adenosine A2A receptor agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21680)-, haloperidol- or reserpine-induced catalepsy models. The effect of KW-6002 on reserpine or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride(MPTP)-induced hypolocomotion was also examined. Results: The ED50s of KW-6002 in the reversal of CGS21680-induced and reserpine-induced catalepsy were 0.05 mg/kg, PO and 0.26 mg/kg, PO, respectively. Compared to the ED50 of other adenosine antagonists and dopamine agonist drugs, KW-6002 is over 10 times as potent in these models. istradefylline (KW-6002) also ameliorated the hypolocomotion (minimum effective dose; 0.16 mg/kg) induced by nigral dopaminergic dysfunction with MPTP or reserpine treatment. Combined administrations of subthreshold doses of KW-6002 and L-dopa (50 mg/kg, PO) exerted prominent effects on haloperidol-induced and reserpine-induced catalepsy, suggesting that there may be a synergism between the adenosine A2A receptor antagonist KW-6002 and dopaminergic agents.[2]
ADME/Pharmacokinetics	Absorption, Distribution and Excretion Istradefylline reaches a Cmax of 181.1ng/mL with a Tmax of 2.0h and an AUC of 11,100ng\*h/mL. M1, the primary active metabolite, reaches a Cmax of 4.34ng/mL with a Tmax of 3.5h. The M8 metabolite reaches a Cmax of 12.6ng/mL with a Tmax of 3.0h and an AUC of 610ng\*h/mL. A 3mg/kg oral dose given to male rats was 17.6% elminated in the urine and 68.3% eliminated in the feces. In urine, 5.31% of the total dose was the M3 metabolite and 1.96% of the total dose was the M1 metabolite. In feces, 30.60% of the total dose was the M3 metabolite, 9.34% of the total dose was the M1 metabolite, 8.33% of the total dose was the M10 metabolite, and 1.62% of the total dose was unchanged istradefylline. The apparent volume of distribution of istradefylline is 448-557L. The apparent clearance of istradefylline is 4.1-6.0L/h. Metabolism / Metabolites The primary metabolite found in urine is the active 4'-O-monodesmethyl istradefylline (M1). Istradefylline is metabolized mainly by CYP1A1, CYP3A4, and CYP3A5. CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18, and CYP2D6 also partly contribute the the metabolism of istradefylline. Other identified metabolites are 1-β-hydroxylated-4’-O-demethyl istradefylline (M2), 3’,4’-O-didemethyl istradefylline (M3), M1 sulfate conjugate (M4), M1 glucuronide (M5), 1-β-hydroxylated istradefylline (M8) and hydrogenated M3 (M10). Biological Half-Life The terminal elimination half life of istradefylline was 64-69 hours.
Toxicity/Toxicokinetics	Hepatotoxicity In prelicensure controlled trials, serum ALT elevations occurred in 4% to 11% of istradefylline-treated subjects compared to 5% to 6% of placebo recipients, most of whom were taking multiple other agents for Parkinson disease. The elevations were usually mild-to-moderate in severity, asymptomatic and self-limited in course. ALT elevations above 3 times the ULN occurred in less than 1% recipients and rarely led to discontinuation. None of the aminotransferase elevations were accompanied by symptoms or jaundice. In preregistration clinical trials and subsequently with its more widespread use, istradefylline has not been linked to instances of clinically apparent liver injury. It has, however, had limited clinical use. Likelihood score: E (unlikely cause of clinically apparent liver injury). Protein Binding Istradefylline is approximately 98% protein bound in plasma, mostly to serum albumin and alpha-1-acid glycoprotein.
References	[1]. J Neurosci . 2001 May 15;21(10):RC143. [2]. Psychopharmacology (Berl) . 1999 Nov;147(1):90-5. [3]. Exp Neurol . 2000 Apr;162(2):321-7. [4]. J Neurochem . 2002 Jan;80(2):262-70. [5]. Glia . 2003 Aug;43(2):190-4. [6]. J Pharmacol Exp Ther . 2007 Nov;323(2):708-19.
Additional Infomation	Pharmacodynamics Istradefylline is a selective adenosine A2A receptor inhibitor. It has a long duration of action as it is given once daily and has a half life of 64-69 hours. Patients taking this medication should be monitored for dyskinesia, hallucinations, and lack of impulse control. Consider dose reductions for these patients.

Solubility Data

Solubility (In Vitro)

DMSO: 6~25.3 mg/mL (15.6~65.9 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.41 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 2: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6013 mL	13.0063 mL	26.0125 mL
	5 mM	0.5203 mL	2.6013 mL	5.2025 mL
	10 mM	0.2601 mL	1.3006 mL	2.6013 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.