Table 3 Summary of preclinical studies

Arulmani et al. [1]

To investigate the effects of GR79236 on capsaicin-induced carotid hemodynamic changes and plasma CGRP release

Pigs (15)

Capsaicin infusion after treatment with adenosine A₁ receptor agonist, GR79236, or vehicle (saline)

GR79236 attenuated capsaicin-induced carotid hemodynamic changes dose-dependently, but not CGRP release

GR79236 might have potential as migraine treatment because of its vasoconstrictive effects rather than its inhibition of trigeminal CGRP release

Carruthers et al. [6]

To investigate the mechanisms of CGRP release and its potential modulation by adenosine agonists

Rats (n = NR)

1) Immunocytochemistry and western blotting

2) In vitro application of forskolin and adenosine A₁ receptor agonist (GR79236X), adenosine A₁ receptor antagonist (DPCPX), adenosine A_2A agonist (CGS21680) or A₃ receptor agonist (2-CI-IB-MECA) to trigeminal ganglion neuron cultures. CGRP was measured using ELISA

1) Adenosine A₁ receptor was expressed in the trigeminal ganglion

2) GR79236X inhibited forskolin stimulated CGRP release

3) DPCPX abolished inhibition of CGRP release mediated by GR79236X

4) CGS21680 and 2-CI-IB-MECA did not have effect on forskolin-induced CGRP secretion

A₁ adenosine receptors on CGRP-positive neurons can inhibit cAMP induced CGRP release from trigeminal neurons. Adenosine receptors has significant potential for development as therapeutic targets for pain

Faraci et al. [9]

To investigate the vascular responses of the dura mater to adenosine

Dogs (n = 9)^a

Intravenous infusion of 5 µM/kg/min adenosine while measuring blood flow with labelled, radioactive microspheres

Adenosine infusion showed

1) aortic pressure decreased

2) vascular resistance in the dura decreased

3) blood flow to the dura increased

4) no effect on cerebral blood flow

Adenosine did not affect cerebral blood flow but increased dural blood flow

Vasomotor responses of the dural circulation might contribute to some forms of vascular headache

Goadsby et al. [13]

To investigate the role of adenosine A₁ receptors in an animal model of nociceptive activation of the trigeminovascular system

Cats (n = 14)

Blood samples were taken at: baseline, after electrical stimulation of superior sagittal sinus (SSS), after electrical stimulation and GR79236 or GR190178.

A single dose of DPCPX was administered after highest dose of GR79236

1) GR79236 and GR190178 had a dose-dependent inhibitory effect on SSS stimulation-induced trigeminovascular activation in trigeminal nucleus caudalis (TNC)

2) GR79236 and GR190178 inhibited SSS stimulation-induced CGRP release in cranial circulation

3) DPCPX reversed the inhibitory effect of GR79236 on the trigeminal nucleus

Activation of adenosine A₁ receptor causes neuronal inhibition without concurrent vasoconstriction, proposing a novel avenue for treating migraine and cluster headache

Haanes et al. [15]

To investigate the vasomotor effects of purinergic receptor in the middle meningeal artery (MMA), using functional myograph with natural and designed agonists

Rats (n = NR)

1) In vitro application of adenosine, caffeine and a A_2A receptor antagonist, SCH58261

2) mRNA expression was measured using PCR

1) Adenosine caused vasodilation in pre-contracted MMA segments

2) Caffeine blocked adenosine-induced vasodilation and caused adenosine to elicit a contraction

3) SCH58261 partly mimic the effect of caffeine

4) All purinergic receptor mRNAs were present in the trigeminal ganglion at same base pair size as for MMA

Purinergic receptors might partly regulated blood flow through the MMA. Adenosine mainly binds to A_2A receptors, the strongest expressed adenosine receptor, to cause relaxation of MMAs. The relaxation is inhibited by SCH58261. Similar response is seen when adding physiological caffein concentrations (50 µM). This gives a putative molecular explanation for benefit and use of coffee/caffeine as a MMA vasoconstrictor potentially related to sensation of cranial pain

Haanes et al. [16]

To investigate the effects of five novel adenosine A_2A receptor antagonists on the vasodilation of the middle meningeal artery produced by an adenosine A₂ receptor agonist (CGS21680) or endogenous CGRP

Rats (n = 57)

1) Intravenous adenosine and caffeine

2) Periarterial electrical stimulation or intravenous CGS21680 followed by either i.v. bolus injections of

JNJ-41942914,

JNJ-39928122,

JNJ-40529749,

JNJ-40064440 and JNJ-41501798

Diameter was captured through closed cranial window

1) Adenosine caused dural arterial dilation and decreased blood pressure, which was inhibited by pre-treatment with caffeine

2) Caffeine increased blood pressure

3) JNJ antagonists did not affect electrical stimulated neurogenic vasodilation

4) CGS21680 caused dural arterial dilation and decrease in blood pressure

5) All JNJ-adenosine A_2A receptor antagonists blocked CGS21680-induced dural vasodilation with a more potent respond with A_2A over A₁ selectivity

Selective A_2A receptor antagonists may offer a novel approach to antimigraine therapy that still needs to be determined

Hardebo et al. [17]

To investigate whether adenosine and closely related adenine compounds (AMP, cyclic AMP, ADP, and ATP) may cause a sufficiently high degree of vasodilatation in vitro to account for a possible involvement in initiating the vasodilatory phase of a migraine attack

Cats (n = 24) & Humans (n = 3)

In vitro application of adenosine, cAMP,

ADP, and ATP to arteries (MCA, lingual and external maxillary artery), followed by measurement of tension and dilatory response

All adenine compounds caused

1) dilation of pial arteries before and after application of prostaglandin F_2a (PGF_2a)

2) did not affect the extracranial arteries in both cats and humans

3) a less pronounced

dilation of pial arteries when extracellular K⁺ concentration was increased

Adenine compounds might initiate the dilatory phase in an attack or reactive hyperemia in intracranial circulation since a marked dilated of intracranial dilation was caused by these compounds

Honey et al. [19]

To investigate the effect of a selective adenosine A₁ receptor agonist (GR79236) on neurogenic dural blood vessel dilation in anaesthetized rats

Rats (n = NR)

Electrically or CGRP evoked dural vasodilatation after treatment with an adenosine A₁ receptor agonist, GR79236, or both GR79236 and an adenosine A₁ receptor antagonist (DPCPX) through a cranial window

1) GR79236 dose-dependently inhibited electrically induced neurogenic vasodilation

2) DPCPX reversed the inhibitory effect of GR79236 on electrically evoked vasodilation

3) GR79236 did not inhibit CGRP induced vasodilation

It is possible that A₁ agonists might be clinically effective in migraine because of an inhibitory effect both in the brain and periphery. This mechanism might offer a novel approach to migraine therapy

Jenkins et al. [22]

To investigate the receptors and mechanisms involved in prostanoid-induced CGRP release in cultured rat trigeminal neurons

Rats (n = NR)

In vitro application of adenosine deaminase to trigeminal neuronal culture

Adenosine deaminase did not alter baseline CGRP level nor CGRP release evoked by 1 µM PGE₂

Not reported regarding adenosine

Lindquist et al. [31]

To investigate whether metabolic status could modulate adenosine accumulation in brain slices exposed to spreading depolarization (SD), and compare SD-associated adenosine release in vivo, under healthy, hypoglycemic, and ischemic conditions

Mice (n = NR)

Coronal slices were prepared at 250 μm, 350 μm, and 450 μm thicknesses. Adenosine measurements were done with amperometric recordings in brain slices in vivo

1) SD caused adenosine accumulation in vivo

2) Adenosine signals triggered by SD could reliably report underlying metabolic status in brain slices

Adenosine or adenosine derivates might be useful as biomarkers of SD incidence in different clinical conditions

Lu et al. [32]

To investigate whether CGRP, A_2AR and A₁R are involved in migraine pain information transmission in the electrical stimulation of the trigeminal ganglion (ESTG) in migraine rat model and exploring the mechanisms of Tianshi capsule (TSC) as migraine treatment

Male rats (n = 40)

ESTG for 30 min. in one group, sham-operation without ESTG in another group and Tianshu capsule (TSC) followed by electrical stimulation of TG in the last group. The TNC and ipsilateral TG were removed for western blot analysis or RT-qPCR to evaluate CGRP, A₁R and A_2AR expression

1) Electrical stimulation increased CGRP and A_2AR expression, and decreased A₁R expression in the TNC and ipsilateral TG compared with the blank groups and sham-operated groups

2) Treatment with TSC caused:

- decreased CGRP and A_2AR expression,

- increased A₁R expression in the TNC and ipsilateral TG compared to ESTG group

CGRP, A₁R and A_2AR mediates pain transmission and regulates the process during migraine. TSC regulates the expression of the three proteins

Wei et al. [46]

To investigate the possibility that nitric oxide donors, nitroglycerin and/or sodium nitroprusside activate trigeminovascular fibers by promoting neuropeptide release and vasodilation within the pial vasculature. Additionally, it was examined whether LY83583, a drug that lowers cyclic GMP, blocks the relaxation mediated by the topical application of the released neuropeptide CGRP or by sodium nitroprusside

Cats (n = 10)^a

1) Application of adenosine before and after the CGRP antagonist (CGRP (8–37))

2) Application of adenosine before and after application of guanylate cyclase inhibitor (LY83583)

Cranial window over the parietal cortex was used to observe arteries

1) CGRP-induced dilation was completely blocked by the CGRP antagonist, but the adenosine-induced vasodilation was not affected by the CGRP antagonist

2) Adenosine-induced vasodilation was not affected by guanylate cyclase inhibitor

Not reported regarding adenosine

Yegutkin et al. [48]

To investigate pro-nociceptive effects of adenine nucleotides in control and in migraine-like conditions modeled with the neuropeptide CGRP

Male rats (n = NR)

1) Bioluminescent and fluorometric techniques to measure purine levels in trigeminal ganglion cells, before and after pre-treatment with CGRP

2) Electro-physiological recordings of nociceptive spikes in trigeminal nerves in meningeal tissues

1) Basal ATP and ADP levels in trigeminal cultures were maintained at very low level, meanwhile basal adenosine and AMP levels were almost one-two orders higher. CGRP pretreatment led to decreased adenosine levels by ∼ 50% in trigeminal cultures, but no changes in CGRP-treated meninges

2) Adenosine could not activate nociceptive firing in meningeal nerves

Data are consistent with the purinergic hypothesis of migraine and proposes new targets against trigeminal pain