Adenosine A2A receptor ( Ki = 2.2 nM )
Adenosine A2A receptor (Ki = 2.2 nM, human; Ki = 1.7 nM, rat) [6]
- Adenosine A1 receptor (Ki = 740 nM, human; Ki = 580 nM, rat) [6]
- Adenosine A2B receptor (Ki > 10000 nM) [6]
- Adenosine A3 receptor (Ki > 10000 nM) [6]

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]

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Istradefylline (KW-6002) is a highly selective antagonist of the adenosine A2A receptor, with >300-fold selectivity over A1 receptors and negligible affinity for A2B/A3 receptors [6]
- In human A2A receptor-expressing CHO cells, Istradefylline dose-dependently inhibited CGS 21680-induced cAMP accumulation, with an IC50 of 4.1 nM; this effect was reversible and competitive [6]
- In primary rat striatal neurons, Istradefylline (1-100 nM) blocked A2A receptor-mediated PKA activation and CREB phosphorylation, reversing the inhibitory effect of adenosine on dopamine D2 receptor signaling [4][6]
- In mouse microglial cells (BV2), Istradefylline (0.1-10 μM) reduced LPS-induced TNF-α and IL-6 production by 35-55% via inhibiting NF-κB activation, indicating anti-inflammatory activity [5]
- It had no significant effect on dopamine D1/D2 receptors, serotonin receptors, or GABA receptors at concentrations up to 10 μM [2][6]

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]

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In MPTP-induced Parkinson’s disease (PD) mice, oral administration of Istradefylline (0.1-1 mg/kg, once daily for 7 days) dose-dependently improved motor deficits, increasing locomotor activity by 40-70% and reducing akinesia [3][6]
- In rats with unilateral 6-OHDA lesions (PD model), Istradefylline (0.3 mg/kg, i.p.) enhanced apomorphine-induced rotational behavior by 2.3 fold, potentiating dopamine-mediated motor responses [1]
- In mice elevated plus maze test, Istradefylline (1-10 mg/kg, p.o.) increased open arm exploration time by 30-50%, showing anxiolytic effects mediated by striatal A2A receptor blockade [2]
- In focal cerebral ischemia rats, Istradefylline (1 mg/kg, i.v., 1 hour post-occlusion) reduced infarct volume by 38% and improved neurological function, associated with reduced microglial activation [5]

Adenosine A2A receptor binding assay: Membrane preparations from human/rat A2A receptor-expressing cells were incubated with [3H]-SCH 58261 (0.5 nM) and Istradefylline (0.01-10000 nM) at 25°C for 90 minutes. Non-specific binding was determined with excess unlabeled SCH 58261. Bound ligands were separated by filtration, and radioactivity was quantified to calculate Ki values [6]
- cAMP inhibition assay: CHO cells expressing human A2A receptors were preincubated with Istradefylline (0.1-100 nM) for 15 minutes, then stimulated with CGS 21680 (100 nM) for 30 minutes. Intracellular cAMP was extracted and quantified by ELISA to determine IC50 values [6]
- NF-κB activity assay: BV2 microglial cells were pretreated with Istradefylline (0.1-10 μM) for 1 hour, then stimulated with LPS (1 μg/ml) for 6 hours. Nuclear extracts were prepared, and NF-κB DNA-binding activity was measured by electrophoretic mobility shift assay (EMSA) [5]

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.

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Striatal neuron signaling assay: Primary rat striatal neurons were cultured for 7-10 days, pretreated with Istradefylline (1-100 nM) for 20 minutes, then exposed to adenosine (1 μM) for 15 minutes. Cells were lysed, and phosphorylated CREB/total CREB were detected by Western blot; band intensity was quantified by densitometry [4][6]
- Microglial inflammation assay: BV2 cells were seeded in 24-well plates and cultured for 24 hours. Istradefylline (0.1-10 μM) was added 1 hour before LPS (1 μg/ml) stimulation. After 24 hours, culture supernatants were collected, and TNF-α/IL-6 levels were measured by ELISA [5]
- Receptor selectivity assay: Cells expressing human dopamine D1/D2, serotonin 5-HT1A/2A, or GABA-A receptors were treated with Istradefylline (0.1-10 μM) for 30 minutes. Receptor activation/inhibition was assessed by respective signaling assays (cAMP, calcium imaging) to confirm selectivity [2][6]

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.

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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]

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MPTP-induced PD mouse model: Male C57BL/6 mice (20-25 g) were injected with MPTP (20 mg/kg, i.p.) once daily for 4 days to induce PD-like symptoms. Istradefylline was dissolved in 0.5% CMC-Na and administered orally at 0.1, 0.3, 1 mg/kg once daily for 7 days starting 1 day post-MPTP. Locomotor activity was measured by open field test, and akinesia was scored by behavioral observation [3][6]
- 6-OHDA-lesioned PD rat model: Male Sprague-Dawley rats (250-300 g) received unilateral stereotaxic injection of 6-OHDA into the substantia nigra. Two weeks post-lesion, Istradefylline (0.3 mg/kg) dissolved in saline was injected intraperitoneally 30 minutes before apomorphine (0.5 mg/kg, s.c.). Rotational behavior was recorded for 60 minutes [1]
- Anxiety mouse model: Female ICR mice (18-22 g) were administered Istradefylline (1, 3, 10 mg/kg) dissolved in 0.5% CMC-Na via oral gavage. One hour later, mice were subjected to the elevated plus maze test, and open arm exploration time was recorded [2]
- Focal cerebral ischemia rat model: Male Wistar rats (200-250 g) were subjected to middle cerebral artery occlusion for 90 minutes. Istradefylline (1 mg/kg) dissolved in saline was injected intravenously 1 hour after occlusion. Infarct volume was measured by TTC staining at 24 hours, and neurological function was scored [5]

Absorption, Distribution and Excretion
The peak plasma concentration (Cmax) of istrafenimine was 181.1 ng/mL, the time to peak concentration (Tmax) was 2.0 h, and the area under the curve (AUC) was 11,100 ng·h/mL. The peak plasma concentration (Cmax) of the major active metabolite M1 was 4.34 ng/mL, and the time to peak concentration (Tmax) was 3.5 h. The peak plasma concentration (Cmax) of metabolite M8 was 12.6 ng/mL, the time to peak concentration (Tmax) was 3.0 h, and the area under the curve (AUC) was 610 ng·h/mL. Following oral administration of a 3 mg/kg dose to male rats, 17.6% of the drug was excreted in the urine and 68.3% in the feces. In urine, M3 metabolite accounted for 5.31% of the total dose, and M1 metabolite accounted for 1.96%. In feces, 30.60% of the total dose was M3 metabolite, 9.34% was M1 metabolite, 8.33% was M10 metabolite, and 1.62% was unmetabolized isoflavone. The apparent volume of distribution of isoflavone is 448-557 liters. The apparent clearance of isoflavone is 4.1-6.0 liters/hour. The major metabolite found in urine is active 4'-O-monodemethylisoflavone (M1). Isofeline is primarily metabolized by CYP1A1, CYP3A4, and CYP3A5. CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18, and CYP2D6 also partially participate in the metabolism of isotretinoin. Other identified metabolites include 1-β-hydroxy-4'-O-demethyl isotretinoin (M2), 3',4'-O-didemethyl isotretinoin (M3), M1 sulfate conjugate (M4), M1 glucuronide (M5), 1-β-hydroxylated isotretinoin (M8), and hydrogenated M3 (M10).
Biological Half-Life
The terminal elimination half-life of isotretinoin is 64–69 hours.

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Oral bioavailability: 70-80% in rats and 65% in humans [6]
-Plasma protein binding: 97-98% in human plasma (concentration range: 0.1-10 μg/mL) [6]
-Metabolism: Mainly metabolized in the liver via cytochrome P450 3A4 (CYP3A4). UDP-glucuronyltransferase (UGT) mediated, inactive metabolites [6]
- Elimination half-life: 8-10 hours in humans; 4-6 hours in rats [6]
- Distribution: Volume of distribution in humans (Vd) = 2.1 L/kg, widely penetrating into brain tissue (brain/plasma ratio = 1.2-1.5) [6]
- Excretion: 60-70% of the dose is excreted in feces as metabolites; 20-25% is excreted in urine; <1% is excreted unchanged [6]

Hepatotoxicity
In premarket controlled trials, 4% to 11% of subjects treated with istrafenine experienced elevated serum ALT levels, compared to 5% to 6% in the placebo group. Most of these subjects were concurrently taking multiple other Parkinson's disease medications. ALT elevations were typically mild to moderate, asymptomatic, and self-limiting. Less than 1% of subjects experienced ALT elevations exceeding three times the upper limit of normal, and these rarely led to discontinuation of treatment. All transaminase elevations were asymptomatic or accompanied by jaundice. In premarket clinical trials and subsequent broader use, istrafenine has not been associated with clinically significant liver injury. However, its clinical application is limited. Probability Score: E (Unlikely to cause clinically significant liver injury). Protein Binding Istrafen has a protein binding rate of approximately 98% in plasma, primarily binding to serum albumin and α1-acid glycoprotein.

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Acute toxicity: Oral LD50 in rats and mice > 2000 mg/kg [6]
-Subchronic toxicity (oral administration in rats over 28 days): No significant adverse effects on liver, kidney or hematological parameters at doses up to 300 mg/kg/day [6]
-Chronic toxicity (oral administration in dogs over 1 year): Mild hepatocyte vacuolation occurred at doses ≥100 mg/kg/day, which was reversible upon discontinuation [6]
-No significant drug interaction with levodopa (a Parkinson's disease drug) or CYP3A4 substrates in preclinical studies [6]
-No genotoxicity was found in the Ames test, chromosomal aberration test or mouse lymphoma test [6]

[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.

Pharmacodynamics
Istradifenlin is a selective adenosine A2A receptor inhibitor. Because it only requires once-daily dosing, it has a long duration of action, with a half-life of 64-69 hours. Patients taking this medication should be monitored for symptoms such as motor disturbances, hallucinations, and impulse control disorders. A dose reduction may be considered for these patients.

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Istradifeneline (KW-6002) is a selective, orally effective adenosine A2A receptor antagonist used to treat Parkinson's disease (PD)[1][3][6]
- Its mechanism of action is to block striatal A2A receptors (co-localized with dopamine D2 receptors), thereby enhancing dopamine signaling and improving motor function in PD patients[4][6]
- It exerts neuroprotective effects by reducing microglial activation and neuroinflammation, which may delay disease progression[5][6]
- It has been approved as adjunctive therapy to levodopa for the treatment of PD patients with motor fluctuations (OFF phase)[6]
- Its high selectivity for A2A receptors minimizes off-target effects and contributes to its good tolerability in clinical use[2][6]
- It does not cause dopamine-related side effects (e.g., motor dysfunction at therapeutic doses[3][6]

C20H24N4O4

384.43

384.179

C, 62.49; H, 6.29; N, 14.57; O, 16.65

155270-99-8

Istradefylline-13C,d3; 2749234-46-4

5311037

Light green to green solid powder

1.2±0.1 g/cm3

601.0±65.0 °C at 760 mmHg

189-193

317.3±34.3 °C

0.0±1.7 mmHg at 25°C

1.598

2.84

0

5

6

28

613

0

O=C1C2=C(N=C(/C=C/C3=CC(OC)=C(OC)C=C3)N2C)N(CC)C(N1CC)=O

IQVRBWUUXZMOPW-PKNBQFBNSA-N

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+

8-[(E)-2-(3,4-dimethoxyphenyl)ethenyl]-1,3-diethyl-7-methylpurine-2,6-dione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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: ≥ 2.08 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.

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Solubility in Formulation 2: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30mg/mL

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