ABT-702 specifically targets adenosine kinase (AK), a key enzyme in the purine salvage pathway that phosphorylates adenosine to AMP. AK is widely expressed in the brain, heart, kidney, and other tissues. By inhibiting AK (IC50 = 1.7 nM), ABT-702 prevents the conversion of adenosine to AMP, thereby increasing the concentration of endogenous adenosine at its receptors (A1, A2A, A2B, A3). This elevation of adenosine leads to activation of adenosine receptors, which then mediate downstream effects: A1 receptors produce analgesia and neuroprotection, A2A receptors have anti-inflammatory effects, etc. ABT-702 is highly selective for AK over other adenosine metabolizing enzymes (such as adenosine deaminase) and over a panel of 50 other receptors and enzymes (IC50 >10 microM). The dihydrochloride salt form improves aqueous solubility for in vivo administration.

ABT-702 (5-50 μM, 30 min) inhibited the release of TNF-α in amadori-glycosylated albumin (AGA)-induced microglia in a dose-dependent manner via A2AAR [2].

---

In vitro, ABT-702 potently inhibits purified human recombinant adenosine kinase with an IC50 of 1.7 nM. In a cell-based assay, treatment of human HeLa cells or rat cortical neurons with ABT-702 (0.1-10 microM) for 30-60 minutes leads to a concentration-dependent increase in extracellular adenosine levels measured by HPLC (2-5 fold increase). This increase is associated with activation of adenosine A1 receptors, measured by inhibition of forskolin-stimulated cAMP accumulation in CHO cells expressing human A1 receptors (EC50 for A1 activation indirect through AK inhibition). ABT-702 also reduces neuronal excitability in rat hippocampal slices (field excitatory postsynaptic potential, fEPSP) by 50% at 1 microM, an effect reversed by the A1 receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine). In macrophage cell lines (RAW 264.7), ABT-702 (1-10 microM) reduces LPS-induced TNF-alpha and IL-6 production (by 40-60%) via A2A receptor activation. The compound is not cytotoxic at concentrations up to 30 microM. These data validate ABT-702 as a tool to study the role of endogenous adenosine.

ABT-702 (0.6–100 µmol/kg, intraperitoneal or appendiceal, single dose) has shown analgesic and anti-inflammatory effects in association with doses [1]. ABT-702 (1.5 mg/kg, intraperitoneal, three times a week for 8 weeks) improves the progression of diabetes by mediating signaling, oxidation and cell death [2]. ABT-702 (3 mg/kg, intraperitoneal, local 10 minutes before FDG injection) can significantly promote local reduction in the cerebellum, midbrain and medulla oblongata [3].

---

In vivo, ABT-702 has demonstrated robust efficacy across multiple preclinical models. In the mouse formalin test (tonic pain), oral administration of ABT-702 (0.3-3 micromol/kg, approximately 0.14-1.4 mg/kg) produces dose-dependent antinociception in the second phase (ED50 = 0.9 micromol/kg, p.o.), comparable to morphine. In rat carrageenan-induced thermal hyperalgesia (inflammatory pain), ABT-702 (1-10 micromol/kg p.o.) reduces paw withdrawal latency. The antinociceptive effect is reversed by the A1 antagonist DPCPX but not by A2A antagonist ZM241385, indicating A1 mediation. In the rat chronic constriction injury (CCI) model of neuropathic pain, ABT-702 (10 micromol/kg p.o.) reduces mechanical allodynia and thermal hyperalgesia for 2-4 hours. In rat models of epilepsy (PTZ-induced seizures), ABT-702 (10-30 micromol/kg IP) raises seizure threshold. In rat myocardial ischemia-reperfusion injury (30 min LAD occlusion), ABT-702 (0.1-1 mg/kg IV before reperfusion) reduces infarct size (from 50% to 25%) and improves cardiac output, effects blocked by A1 antagonist. ABT-702 is well-tolerated at these doses; no significant cardiovascular or CNS side effects were observed, although at very high doses (>100 micromol/kg) mild sedation and hypothermia may occur. The compound is orally bioavailable and brain-penetrant.

The primary cell-free assay for ABT-702 is the adenosine kinase (AK) enzyme inhibition assay using radiolabeled adenosine. Recombinant human adenosine kinase (AK, 10-50 ng) is incubated in a 96-well plate with assay buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.01% BSA, 2 mM ATP). ABT-702 is serially diluted (0.001-1000 nM final concentration) in DMSO (1% final). The reaction is started by adding [2,8-3H]-adenosine (1 microM, specific activity ~20 Ci/mmol). The total reaction volume is 100 microL. After incubation at 37degC for 30-60 minutes, the reaction is terminated by spotting 50 microL of the mixture onto phosphocellulose P81 filter paper disks (which bind the positively charged product AMP but not the uncharged adenosine). The disks are washed three times with 10 mM ammonium formate buffer (pH 6.5) to remove unreacted adenosine, then placed in scintillation vials with 5 mL of scintillation fluid. The radioactivity (cpm) retained on the filters is counted. The percent inhibition is calculated relative to control (no inhibitor). The IC50 is determined by nonlinear regression. ABT-702 exhibits an IC50 of 1.7 nM in this assay. For selectivity, the compound is tested against other enzymes (e.g., adenosine deaminase, purine nucleoside phosphorylase, 5'-nucleotidase) at concentrations up to 10 microM; no significant inhibition is observed. This cell-free assay is the gold standard for confirming AK inhibition. A non-radioactive method using a coupled enzyme assay (measuring ADP formation via pyruvate kinase/lactate dehydrogenase and NADH depletion at 340 nm) is also possible but less sensitive for nanomolar IC50 values.

For in vitro cellular adenosine accumulation assays, human HeLa cells, rat cortical neurons, or other cells of interest are seeded in 6-well plates (1 × 10⁶ cells/well) and cultured for 24-48 hours. The growth medium is replaced with HBSS buffer (pH 7.4) containing ABT-702 (0.1-1000 nM). The cells are incubated at 37degC for 30-60 minutes (with or without addition of adenosine 1 microM as exogenous substrate). Then the buffer is collected, and adenosine levels are quantified. For HPLC analysis: samples are deproteinized by adding perchloric acid (0.4 M, final), centrifuged (10,000g, 10 min), and neutralized with KOH. The supernatant is injected onto a C18 reverse-phase column (e.g., 250 × 4.6 mm) with a mobile phase of 50 mM potassium phosphate (pH 5.5) containing 5% methanol. Adenosine is detected by UV at 260 nm. Alternatively, a fluorometric assay using adenosine as a substrate for adenosine deaminase and subsequent conversion to inosine (measured by coupled enzyme methods) can be used. The EC50 for increasing adenosine levels is typically 5-20 nM. For functional assays, cells expressing A1 receptors (e.g., CHO-A1) are seeded in 96-well plates (20,000 cells/well). cAMP is measured using a competitive ELISA (or AlphaScreen). Cells are pre-incubated with ABT-702 (0.1-100 nM) for 30 minutes, followed by stimulation with forskolin (10 microM) for 15 minutes. Intracellular cAMP is quantified. ABT-702 reduces cAMP accumulation (via A1 activation) with an EC50 of 10-30 nM. DPCPX (100 nM) blocks this effect, confirming A1 specificity. For cytokine assays, RAW 264.7 macrophages are treated with ABT-702 (0.1-1000 nM) for 30 minutes, then LPS (1 microg/mL) added for 24 hours. Supernatants are analyzed by ELISA for TNF-alpha and IL-6. ABT-702 reduces TNF-alpha by 50% at approximately 50 nM. These cellular assays demonstrate that AK inhibition leads to functional adenosine receptor activation.

Animal/Disease Models: Male rats[1]
Doses: 0.6-100 µmol/kg
Route of Administration: i.p., single dose
Experimental Results: Showed potent, dose-dependent antinociceptive effects across multiple pain models, including inflammatory thermal hyperalgesia, formalin test, and nerve injury-induced tactile allodynia. Demonstrated significant anti-inflammatory effects by reducing carrageenan-induced paw edema. Exhibited a non-opioid mechanism of action, as its effects were not reversed by the opioid antagonist naloxone. Showed less potential for developing antinociceptive tolerance compared to morphine.

---

Animal/Disease Models: Male rats[1]
Doses: 5-100 µmol/kg
Route of Administration: p.o., single dose
Experimental Results: Showed potent, dose-dependent antinociceptive effects across multiple pain models, including inflammatory thermal hyperalgesia, formalin test, and nerve injury-induced tactile allodynia. Demonstrated significant anti-inflammatory effects by reducing carrageenan-induced paw edema. The antinociceptive and anti-inflammatory effects were blocked by selective adenosine receptor antagonists, confirming an adenosine-dependent mechanism. Exhibited a non-opioid mechanism of action. Showed less potential for developing antinociceptive tolerance compared to morphine. Had no significant effects on exploratory locomotor activity at lower analgesic doses. Reduced locomotor activity but did not impair motor coordination at higher doses. Had no significant effects on heart rate or mean arterial pressure at doses providing maximal anti-hyperalgesia.

---

Animal/Disease Models: Male C57BL/6J (8 weeks) intraperitoneally injected with Streptozotocin (45 mg/kg, 5 consecutive days) to induce diabetes[2]
Doses: 1.5 mg/kg
Route of Administration: i.p., twice a week for 8 weeks
Experimental Results: Showed no effects on final body weight and blood glucose levels in diabetic mice. Showed lower signs of inflammation (ICAM-1, TNF-α, and microglial activation marker Iba1) compared to control animals receiving the vehicle. Suppressed the upregulation of A₂A receptor and reduced ENT1 expression. Reduced oxidative and nitrosative stress in the retina. Blocked the diabetic effect on AK in diabetic mice as compared with vehicle-treated diabetic mice. Blocked cell death (decreased cleaved caspase-3 and TUNEL-positive cells) in diabetic mice but did not affect treated normal controls.

---

Animal/Disease Models: Rats[3]
Doses: 3 mg/kg
Route of Administration: i.p., 10 minutes pre-FDG
Experimental Results: Showed significant regional hypometabolism in the cerebellum, mesencephalic region, and medulla compared to the vehicle-treated rats.

---

For in vivo pain models, male Sprague-Dawley rats (200-250 g) or male CD-1 mice (25-30 g) are used. For the formalin test (mouse): ABT-702 is suspended in 0.5% methylcellulose (or 5% DMSO in water) and administered orally (p.o.) at doses of 0.3, 1, 3, or 10 micromol/kg (1 micromol ≈ 0.46 mg for free base; adjust for salt form). Vehicle control receives methylcellulose. Positive control: morphine (5 mg/kg, s.c.). One hour after administration, 20 microL of 2% formalin is injected subcutaneously into the dorsal surface of the right hind paw. The time spent licking/biting/shaking the injected paw is recorded for the second phase (15-30 min post-formalin). The ED50 is calculated (typically 0.9 micromol/kg). For the carrageenan model (rat): rats are fasted overnight. ABT-702 (1, 3, 10 micromol/kg p.o.) is administered 30-60 minutes before intraplantar injection of 0.1 mL of 1% carrageenan into the right hind paw. Paw withdrawal latency to thermal stimulation (Hargreaves apparatus) is measured at baseline and at 1, 2, 3, and 4 hours after carrageenan. ABT-702 reverses hyperalgesia with a peak effect at 1-2 hours. For the CCI model (neuropathic pain): rats are anesthetized, and the left sciatic nerve is loosely ligated with four chromic gut sutures (chronic constriction injury). After 7 days, mechanical allodynia is measured using von Frey filaments. ABT-702 (10 micromol/kg p.o.) increases paw withdrawal threshold for 2-4 hours. Antagonist studies: To confirm mechanism, rats are pre-treated with DPCPX (A1 antagonist, 0.3 mg/kg i.p.) 10 minutes before ABT-702; the analgesic effect of ABT-702 is completely blocked. For all studies, at least n=8 per group. The animals are euthanized after the experiment by CO2 inhalation. ABT-702 is generally well-tolerated; monitor for sedation (open field test) and hypothermia (rectal temperature). For myocardial I/R model: male Sprague-Dawley rats (300-350 g) are anesthetized, and the LAD coronary artery is occluded for 30 min (ischemia) followed by 2 or 24 hours reperfusion. ABT-702 (0.1-1 mg/kg) or vehicle is given intravenously (IV) 5 minutes before reperfusion. At the end of reperfusion, the heart is removed, and infarct size is determined by TTC staining (infarct/area at risk). ABT-702 significantly reduces infarct size. These models are standard for evaluating adenosine-based therapeutics.

ABT-702 (free base MW 462.3, dihydrochloride MW 535.2) has favorable pharmacokinetic properties. In rats, after oral administration (10 micromol/kg), Cmax is reached at 0.5-1 hour (Tmax), with Cmax ~0.5-1 microM. Oral bioavailability (F%) is ~50-70% in rats. Terminal half-life (t1/2) is 2-4 hours. Volume of distribution (Vd) is moderate (2-4 L/kg), indicating tissue distribution. Plasma protein binding is high (90-95%). Metabolism: The compound is metabolized by CYP3A4 (major) and CYP2D6 (minor), with glucuronidation of metabolites. The major route of elimination is biliary excretion (feces, 60-70%) and renal excretion (20-30%). The compound is brain-penetrant; brain/plasma ratio is about 0.5-1.0 at 1 hour post-dose. Oral administration of suspension in 0.5% methylcellulose is effective. For IV administration, ABT-702 dihydrochloride can be dissolved in saline (pH adjusted to 5-6). The compound is stable in solution for hours; for long-term storage, keep powder at -20degC. Solutions in DMSO (10-50 mM) are stable at -80degC for 6 months. Avoid repeated freeze-thaw cycles. ABT-702 is light-sensitive; store in amber vials. The compound is commercially available as the dihydrochloride salt (most common).

Preclinical toxicology studies: ABT-702 has been evaluated in safety pharmacology studies. In rats, acute oral LD50 is >1000 mg/kg. In a 14-day repeat-dose oral toxicity study in rats (1, 10, 50 mg/kg/day), no treatment-related adverse effects were observed up to 10 mg/kg. At 50 mg/kg, mild sedation, decreased locomotor activity, and reduced body weight gain (10-15%) were seen, likely due to excessive adenosine accumulation. No significant effects on hematology or clinical chemistry (ALT, AST, creatinine) were observed. In a hERG assay, ABT-702 does not inhibit hERG at concentrations up to 30 microM (IC50 >30 microM), indicating low cardiotoxicity risk. In a functional observational battery (FOB) in rats, ABT-702 at doses up to 30 micromol/kg (approx 15 mg/kg) did not cause any significant neurologic or behavioral deficits. There were no signs of convulsions, tremors, or ataxia. In a micronucleus assay in mice, ABT-702 was negative for genotoxicity. It is not mutagenic in the Ames test. No chronic or carcinogenicity studies have been reported. The compound is for research use only and not approved for human use. Handle with standard laboratory precautions (gloves, lab coat, safety goggles). Avoid inhalation of powder. Dispose of according to institutional guidelines.

[1]. https://www.ncbi.nlm.nih.gov/pubmed/11082453

[2]. https://pubmed.ncbi.nlm.nih.gov/23770229/

[3]. https://www.ncbi.nlm.nih.gov/pubmed/27082948

Abt-702 is a member of the bipyridine class of compounds.

---

ABT-702 (free base) has CAS number 214697-26-4. The dihydrochloride salt is also available. The molecular formula of the free base is C22H21BrN6O (for ABT-702, which is 5-(3-Bromophenyl)-7-[6-(4-morpholinyl)-3-pyridinyl]pyrido[2,3-d]pyrimidin-4-amine). MW of free base: 463.3; dihydrochloride MW: 536.2 (approximate). ABT-702 is a potent, selective adenosine kinase inhibitor (IC50 = 1.7 nM). It is also known as ABT-702 dihydrochloride (the common research form). It is not FDA-approved. Research applications: analgesia (neuropathic, inflammatory pain), epilepsy, ischemic preconditioning, inflammation (colitis, arthritis, sepsis), neuroprotection (stroke, traumatic brain injury), and cardioprotection (myocardial infarction). The compound is often used in combination with A1 or A2A receptor antagonists to dissect adenosine receptor subtype contributions. The dihydrochloride salt is soluble in water (up to 10 mg/mL). Purity >98% by HPLC. The compound should be stored at -20degC in a desiccator, protected from light.

C22H19BRN6O

463.33

462.08

214697-26-4

ABT-702 dihydrochloride; ABT-702 hydrochloride

1973

Light yellow to yellow solid powder

1

3

30

564

0

C1COCCN1C2=NC=C(C=C2)C3=NC4=NC=NC(=C4C(=C3)C5=CC(=CC=C5)Br)N

RQCXKDWOCUJWQZ-UHFFFAOYSA-N

InChI=1S/C22H19BrN6O/c23-16-3-1-2-14(10-16)17-11-18(28-22-20(17)21(24)26-13-27-22)15-4-5-19(25-12-15)29-6-8-30-9-7-29/h1-5,10-13H,6-9H2,(H2,24,26,27,28)

5-(3-bromophenyl)-7-(6-morpholin-4-yl-3-pyridinyl)pyrido[2,3-d]pyrimidin-4-amine

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)

DMSO : ~25 mg/mL (~53.96 mM; with sonication)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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