PAMAM dendrimers are biocompatible polymers with extensive and advantageous applications in drug delivery in vivo
. Previous data have shown GLiDe conjugates and other nanoconjugates to acquire beneficial pharmacological characteristics, such as increased receptor affinities, increased potency and/or increased selectivity in comparison to monomeric ligands [1
]. These nanocarriers may also provide pharmacokinetic and pharmacodynamic advantages, such as impeded metabolic degradation or tissue selectivity [1
]. In our case, the drug is not intended for cleavage from the carrier at the site of action. The extracellular intact conjugate holds the relevant biological activity and can benefit from the versatility of the PAMAM dendrimers for further derivatization or structural modification as needed for the biological application.
For the present studies, an adenosine analogue (MRS5233) selective for the hA3
AR, but modified to possess improved selectivity for the A3
AR in mouse, was characterized in mouse model experiments [27
]. This compound was also investigated as a mA3
AR agonist pharmacophore for conjugation to a G4 PAMAM dendrimer for the synthesis of the GLiDe conjugate, MRS5246.
It was determined that both agonists, MRS5246 and MRS5233, bind with high affinity to the mA3AR with Ki values of 0.04 and 3.94 nM, respectively. In comparison to other AR subtypes, they are highly selective for the mA3AR. This includes high selectivity over the A1AR, the subtype to which these analogues might be more likely to bind compared to A2A or A2BARs. It was further determined that MRS5233 and MRS5246 are highly potent in in vitro functional assays to inhibit cAMP production via A3AR-induced activation of Gi proteins compared to that of the A1AR. MRS5246 showed an IC50 of 0.02 nM at the mA3AR compared to 577.6 nM at the mA1AR (ratio of 28,880), while MRS5233 resulted in an IC50 of 4.0 nM at the mA3AR and lacked inhibition at the A1AR up to 0.1 mM. Therefore, these compounds promised to be excellent candidates for the study of cardioprotection by A3AR activation in mouse models.
In comparing the activities of the two compounds, MRS5246 possessed a much higher affinity to the mA3
AR compared to the monomer, MRS5233, by 98.5-fold. The conjugated MRS5246 was also 200-fold more potent A3
AR agonist than the monomer, MRS5233, in an in vitro
cAMP assay. These findings are comparable to previous findings with other GLiDe conjugates, which significantly improved affinity and potency over the unconjugated monomeric agonists [13
]. The Ki
values of these agonists in binding to hARs have been reported and indicate that 3
, and 6
are all A3
]. For example, the triazole derivative 3
values of 22.3 ± 1.6 nM and 2440 ± 320 nM at the hA3
AR and hA2A
AR, respectively, and at 10 μM inhibited only 12 ± 4% of the radioligand binding at the hA1
]. Thus, the model compound 3
binds to the mA3
AR with 5.7-fold greater affinity than to the hA3
AR. The conjugate 6
binds to the mA3
AR with 3.5-fold greater affinity than to the hA3
AR. In comparison to the A1
AR, the A3
AR selectivity of 6
is slightly greater in the mouse (359-fold) than human (161-fold).
The binding and in vitro
cAMP assays were performed under unrestricted solution conditions, with no intervening biological barriers such as would be encountered in vivo
. There is limited hindrance in these experiments for the PAMAM dendrimers, which range in size of around ~45 Å in diameter for a G4 dendrimer [29
]. This size estimate does not include the addition of the covalently conjugated agonist moieties, which would further increase the diameter. However, in vivo
, the GLiDe conjugates must reach the myocardial targets by extravasation from the coronary microvasculature. It has been determined that the extravasation process of PAMAMs alone is dependent on the size and maybe even charge of the compounds [29
]. These authors found that with each size-increasing generation of PAMAM dendrimers, up to the G4, there was a weight- and size-dependent decrease in the extravasation of the dendrimers. There was a steady decrease in the rate of extravasation from the G1 to G3 dendrimers, but an especially high decrease between G3 and G4 [29
However, in light of these intuitive results that greater sizes offer hindrances, the Langendorff isolated heart experiments showed that the GLiDe conjugate, MRS5246, was just as, maybe even more, effective than the monomeric agonist in generating cardioprotection after I/R. MRS5246 and MRS5233 were effective in a concentration-dependent manner, where both showed significant protection starting at one nanomolar, and the effects were greater at 10 nM. The percentage of improvements after 45 min of reperfusion was marginally greater for the GLiDe conjugate at these concentrations. However, the multivalent conjugate MRS5246 (1 nM) was more effective than the monomer MRS5233 in improving LVDP over control when observing the hearts throughout reperfusion. Since we did not obtain a direct measure of cell death, it should be noted that we do not know whether the improvements in functional recovery with agonist treatment observed in the isolated mouse heart studies resulted from protection against reversible injury (i.e., myocardial "stunning") and/or necrotic cell injury (i.e., infarction).