Phosphodiesterase (PDE5)

In transfectants coexpressing FLAG-hGPR35 and four exogenous Gα proteins, Zaprinast (0.1, 0.3, 1, 3, 10, 30 μM) induces intracellular excitation in a concentration-dependent manner in HEK293 cells [2]. Zaprinast (100 μM; 5 minutes) can promote the phosphorylation of five different cells in the C-terminal tail of human GPR35a in HEK293T cells [4].

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Proinflammatory cytokines produced by activated glial cells may in turn augment the immune/inflammatory reactions of glial cells through autocrine and paracrine routes. The NO/cGMP signaling represents one of the reactions of activated glial cells. We investigated whether the production of proinflammatory cytokines by glial cells is affected by NO-dependent downstream cGMP signaling. In primary cultures of mixed astrocytes and microglial cells, Zaprinast (0.1 mM), an inhibitor of cGMP-selective phosphodiesterases, enhanced the basal and LPS (1.0 microg/ml)-induced secretion of TNF-alpha and IL-1beta. Zaprinast also enhanced NO production induced by LPS or IFN-gamma (100 U/ml), and in microglial cell cultures, but not in astrocyte cultures, zaprinast enhanced the basal and the IFN-gamma-induced production of the cytokines, TNF-alpha and IL-1beta, and of NO. This upregulation by zaprinast was partially inhibited by KT5823 (1.0 microM), an inhibitor of protein kinase G. The LPS-induced production of TNF-alpha, IL-1beta, and NO was inhibited by ODQ (50 microM), an inhibitor of soluble guanylyl cyclase, and by KT5823. Immunohistochemical analysis of mixed glial cell cultures showed that LPS/IFN-gamma-induced iNOS expression and the enhanced expression of iNOS by zaprinast were restricted to microglial cells. Zaprinast enhanced the IFN-gamma (200 U/ml)-induced expression of MHC Class II molecules in astrocytes and microglial cells in mixed cultures, but did not enhance this IFN-gamma-induced expression in pure astrocytes, which lacked paracrine TNF-alpha from microglial cells. Summarizing, zaprinast, which is associated with cGMP/protein kinase G signaling, may augment central immune/inflammatory reactions, possibly via the increased production of TNF-alpha and IL-1beta by activated microglial cells. [1]

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We found that Zaprinast, a well-known cyclic guanosine monophosphate-specific phosphodiesterase inhibitor, acted as an agonist for a G protein-coupled receptor, GPR35. In our intracellular calcium mobilization assay, zaprinast activated rat GPR35 strongly (geometric mean EC(50) value of 16nM), whereas it activated human GPR35 moderately (geometric mean EC(50) value of 840nM). We also demonstrated that GPR35 acted as a Galpha(i/o)- and Galpha(16)-coupled receptor for zaprinast when heterologously expressed in human embryonic kidney 293 (HEK 293) cells. These findings will facilitate the research on GPR35 and the drug discovery of the GPR35 modulators. [2]

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Zaprinast can promote phosphorylation of five distinct amino acids in the C-terminal tail of human GPR35a. Elimination of hydroxy-amino acids from the C-terminal tail of human and mouse GPR35 prevents zaprinast-induced phosphorylation. Phosphorylation sites in the C-terminal tail of hGPR35a and mGPR35 are required for arrestin-3 recruitment induced by Zaprinast. Phosphorylation sites in the C-terminal tail of rat GPR35 are required for arrestin-3 recruitment induced by zaprinast [4].

Zaprinast (3 and 10 mg/kg; i.p.) decreases exploratory activity in the Hughes box test and increases spatial memory in the elevated plus maze (EPM) [5].

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Guanosine-specific cyclic nucleotide signaling is suggested to serve protective actions in the vasculature; however, the influence of selective pharmacologic modulation of cyclic guanosine monophosphate- synthesizing soluble guanylate cyclase or cyclic guanosine monophosphate-degrading phosphodiesterase on vessel remodeling has not been thoroughly examined. In this study, rat carotid artery balloon injury was performed and the growth-modulating effects of the soluble guanylate cyclase stimulator YC-1 or the cyclic guanosine monophosphate-dependent phosphodiesterase-V inhibitor Zaprinast were examined. YC-1 or zaprinast elevated vessel cyclic guanosine monophosphate content, reduced medial wall and neointimal cell proliferation, stimulated medial and neointimal cellular apoptosis, and markedly attenuated neointimal remodeling in comparable fashion. Interestingly, soluble guanylate cyclase inhibition by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one failed to noticeably alter neointimal growth, and concomitant zaprinast with YC-1 did not modify any parameter compared to individual treatments. These results provide novel in vivo evidence that YC-1 and zaprinast inhibit injury-induced vascular remodeling through antimitogenic and proapoptotic actions and may offer promising therapeutic approaches against vasoproliferative disorders. [3]

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Rat carotid artery balloon injury model [3]
Representative photomicrographs of rat balloon-injured LCAs 14 days post-injury are shown in Figure 1. An injured LCA treated with empty hydrogel (Fig. 1A) exhibits a concentric and significant neointima with a clearly defined medial wall and elastic laminae. Figure 1B shows an injured Zaprinast-treated LCA with a significantly attenuated neointima, appearing sporadically as a thin non-concentric layer adjacent to the internal lamina. Figure 1C illustrates an injured YC-1-treated LCA and similarly shows reduced neointimal development. Figure 1D shows an injured LCA treated with combined zaprinast and YC-1, and a significantly diminished neointima is observed yet no additive effect is seen compared to individual zaprinast- or YC-1-treated sections. Figure 1E shows an ODQ-treated vessel with a significant and concentric neointima (similar to controls), while Figure 1F shows an ODQ and YC-1-treated vessel. Corresponding histomorphometric data obtained 14 days post-injury are shown in Figure 2. Figures 2A, 2C, and 2D clearly show significant and comparable attenuation of neointimal growth in injured LCAs treated with zaprinast, YC-1, or zaprinast plus YC-1 compared to vehicle controls. Separate cohorts of injured arteries treated with ODQ failed to show observable changes in neointima formation relative to vehicle controls, and arteries exposed to combined ODQ and YC-1 failed to fully reverse the YC-1-inhibitory effects on neointimal growth. Interestingly, LCAs exposed to zaprinast, either alone or in combination with YC-1, displayed significant medial wall enlargement compared to vehicle controls (Fig. 2B). Analyses of the circumferences of LCA internal and external elastic laminae between all treatment groups revealed no significant differences after 14 days (data not shown).

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Both Zaprinast (10 mg/kg) and rolipram (0.1 mg/kg) significantly decreased second-day latency compared to the control group in the EPM test, while only rolipram (0.1 mg/kg) significantly increased second-day latency in the PA test. Both zaprinast (10 mg/kg) and rolipram (0.1 mg/kg) significantly decreased the number of entries to new areas and time spent in new areas in the Hughes box test. Conclusions: Our study revealed that both Zaprinast and rolipram enhanced spatial memory in EPM, while rolipram seemed to have more emotional memory-enhancing effects in the PA test compared to zaprinast. Both zaprinast and rolipram diminished exploratory activity in the Hughes box test, which can be attributed to the drugs' anxiogenic effects. [5]

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Effects of Zaprinast and rolipram on learning and memory in the mEPM test [5]
When zaprinast (3 and 10 mg/kg) and rolipram (0.05 and 0.01 mg/kg) were administered before the acquisition session (training; Day 1), there was no significant difference in first-day latency (TL1) among the groups (H=7.12; p=0.12, Figure 1). Zaprinast (10 mg/kg) and rolipram (0.1 mg/kg) significantly shortened latency (TL2) on the second day compared to the control group when the drug was administered before the acquisition session (Kruskal-Wallis H=16.36; p<0.05 and p<0.01, respectively) (Figure 1). In the comparison of TL1 and TL2 for each drug-treated group, TL2 was significantly decreased in the control, zaprinast 10 mg/kg and rolipram (0.05 and 0.1 mg/kg) groups (p<0.05), but this measure was not significantly different between the zaprinast 3 mg/kg groups (p>0.05) (Figure 1).

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Effects of Zaprinast and rolipram on learning and memory in the passive avoidance test [5]
When zaprinast (3 and 10 mg/kg) and rolipram (0.05 and 0.1 mg/kg) were administered before the acquisition session of passive avoidance test, there was no significant difference in first-day latency among the groups [F(4.34)=2.12, p>0.05, Figure 2]. Rolipram (0.1 mg/kg) significantly prolonged retention latency compared to the control group and zaprinast had a partial effect; however, this effect did not reach significance when the drugs were administered before the acquisition session [F(4.34)=4.64; p<0.01 Figure 2]. In the comparison of first- and second-day latencies for each drug-treated group, retention latency was significantly prolonged in the control, zaprinast 10 mg/kg, and rolipram (0.05 and 0.1 mg/kg) groups (p<0.05), but this measure was not significantly different between the zaprinast 3 mg/kg groups (p>0.05; Figure 2).

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Effects of Zaprinast and rolipram on exploratory activity in the Hughes box [5]
There was a significant difference between groups after evaluating the total number of entries to the novel side [F(4.29)=4.86, p=0.0049; Figure 3A] and total time spent in the novel side [F(4.29)=4.25, p=0.009, Figure 3B] in the Hughes box. Both zaprinast (10 mg/kg) and rolipram (0.1 mg/kg) significantly decreased entries to the novel side compared to the control group in the Hughes box when administered before the test (p<0.01 and p<0.05; respectively; Figure 3A). Zaprinast (3 and 10 mg/kg) and rolipram (0.1 mg/kg) also significantly shortened the time spent in the novel side in the Hughes box (p<0.05; Figure 3B). [5]
This study revealed that the PDE5 inhibitor, Zaprinast (10 mg/kg), and the PDE4 inhibitor, rolipram (0.1 mg/kg), significantly decreased second-day latency in the EPM test, but only rolipram (0.1 mg/kg) significantly increased second-day latency in the PA test compared to the control mice. Both zaprinast (10 mg/kg) and rolipram (0.1 mg/kg) significantly decreased entry to new areas and time spent in new areas in the Hughes box test.

Confluent cultures of mixed glial cells or pure astrocytes were treated with agents in serum-free media, and microglial cells, separated from astrocyte layer, were seeded at 1.0 × 105 cells/cm2, allowed to settled down for 24 hr, and treated with agents in DMEM containing 10% FBS. These mixed and pure glial cell cultures were pretreated with Zaprinast for 24 hr, and then stimulated with LPS or IFN-γ. To inhibit LPS-induced NO production and NO-dependent cGMP production, L-NMMA (NG-monomethyl-L-arginine) and ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) were added. KT5823 was used to examine the involvement of protein kinase G activity upon the effect of zaprinast. Control cultures contained 0.1% (v/v) of vehicles. [1]

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Discovery of Zaprinast as an agonist for hGPR35. (A) Expression of FLAG-hGPR35 in HEK293 cells confirmed by Western blotting. (B) HEK293 cells transiently coexpressing the receptor (FLAG-hGPR35 or wild-type hGPR35) and/or Gα proteins (G qs5, G qi5, G qo5, and Gα16) were loaded with Fura-2, and then were exposed to zaprinast (broken line; addition of zaprinast). Dose–response curves are shown in parallel. EC50 values for FLAG-hGPR35 and wild-type hGPR35 were 5.2 and 1.9 μM, respectively. (C) GPR35 acted as a Gαi/o- and Gα16-coupled receptor for zaprinast in HEK293 cells. The Fura-2-loaded HEK293 cells transiently coexpressing FLAG-hGPR35 and a Gα protein (G qs5, G qi5, G qo5, or Gα16) were exposed to zaprinast (broken line; addition of zaprinast). [2]
Effect of Zaprinaston rat GPR35. Fura-2-loaded HEK293 cells transiently coexpressing rat or human GPR35 and G qi5 were exposed to zaprinast and intracellular calcium mobilization was measured. Concentration–response curves are from three independents experiments with each point determined in quadruplicate.[2]

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Cells were treated with Zaprinast (Zap) or pamoic acid (Pam) for 5 min prior to lysis. CID-2745687-treated cells (Zap+CID) were preincubated with CID-2745687 for 15 min prior to addition of Zap. Quantification (lower panels) shows mean fold change of [32P] incorporation over vehicle-treated cells, measured by densitometric analysis of autoradiographs from n = 3 independent experiments ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with vehicle; #p < 0.05, ##p < 0.01, ###p <0.001 compared with zaprinast in one-way ANOVA with Tukey’s multiple comparisons posttest. (dox = doxycycline; Zap = 100 μM zaprinast; CID = 10 μM CID-2745687; Pam = 100 nM pamoic acid). GPR35, G protein-coupled receptor 35. [4]

Zaprinast can promote phosphorylation of five distinct amino acids in the C-terminal tail of human GPR35a. LC-MS/MS identified five phosphorylation sites in the hGPR35a C-terminal tail following zaprinast stimulation. [4]

Elimination of hydroxy-amino acids from the C-terminal tail of human and mouse GPR35 prevents Zaprinast-induced phosphorylation. C-terminal sequences of human (upper) and mouse (lower) GPR35 are presented in the one letter amino acid code. Amino acids highlighted and boxed in gray were mutated to alanine. A, residues marked with an asterisk are those found to be phosphorylated in LC-MS/MS performed on hGPR35a-HA. Representative autoradiographs showing [32P] incorporation into (B) human or (C) mouse WT or potentially phosphorylation-deficient mutants of GPR35 (PDM) are shown. Cells were stimulated with 100 μM zaprinast (Zap) for 5 min prior to lysis. [4]

Phosphorylation sites in the C-terminal tail of hGPR35a and mGPR35 are required for arrestin-3 recruitment induced by Zaprinast.A, amino acid residues as highlighted (colors) were mutated to alanine in hGPR35a-eYFP and mGPR35-eYFP either individually or in the indicated combinations. B, zaprinast concentration–response curves for hGPR35a-YFP WT (gray circles); Ser287Ala (red circles); Ser294Ala (purple circles); Ser300Ala (blue circles); Ser303Ala (green circles); Thr307Ala (orange circles); Ser300Ala/Ser303Ala (blue/green circles); Ser303Ala/Thr307Ala (green/orange circles); and the phosphorylation-deficient mutant (black circles) in arrestin-3 interaction assay. C, maximal BRET stimulated by zaprinast treatment. [4]

Phosphorylation sites in the C-terminal tail of rat GPR35 are required for arrestin-3 recruitment induced by Zaprinast.A, the C-terminal sequence of rat GPR35 is shown. Amino acid residues as highlighted (colors) were mutated to alanine in rGPR35-eYFP in the indicated combinations (A). B, zaprinast concentration–response curves for rGPR35-eYFP (black circles); a fully phospho-deficient form (PDM) (black squares) and the combinations of alanine mutations corresponding to A–D in panel (A), (red, purple, blue, orange, as noted in arrestin-3 interaction assays. C, maximal BRET stimulated by zaprinast treatment of the forms in (B). [4]

Agonist-induced internalization of hGPR35a requires phosphorylation and an arrestin.A, hGPR35a-eYFP and hGPR35a-PDM-eYFP internalization was measured after treatment for 45 min with varying concentration of Zaprinast in parental HEK293 cells (parental) stably expressing each construct or of hGPR35a-eYFP in HEK293 cells genome-edited to lack expression of both arrestin-2 and arrestin-3 (Arr null). [4]

Production and characterization of GPR35 phospho-site–specific antisera. Peptides as described in the text and experimental procedures were used to generate immune responses in rabbits. These antisera were then used in immunoblots of lysates of HEK293T cells either nontransfected or transfected to express (A) hGPR35a-eYFP or (B) mGPR35-eYFP. Prior to production of lysates, cells were treated with the same range of ligands and combinations as in Figure 1. Antisera used were hGPR35a-pSer300/pSer303 (A) or mGPR35-pSer298/pSer301 (B). Further studies were performed on lysates of cells expressing hGPR35a-eYFP (C–E), hGPR35a-PDM-eYFP (C), mGPR35-eYFP (C and E), or mGPR35-PDM-eYFP (C) in which cells were pretreated with vehicle, Zaprinast (Zap), compound 101 (101), or a combination of zaprinast and compound 101. [4]

GPR35 phospho-site–specific antisera function as biosensors of agonist activated, fully mature GPR35. Cells as in Figure 1A able to express hGPR35a-HA (A) or mGPR35-HA (B) were either uninduced (−dox) or induced by treatment with doxycycline (+dox) and then treated with either vehicle (veh) or Zaprinast (zap). Such cells were then used in immunocytochemical studies employing hGPR35a-pSer300/pSer303 or mGPR35-pSer298/pSer301 (left panels) (Alexa Fluor 488). Samples were counterstained with the nuclear dye DAPI (center panels). Brightfield images (right panels) are also shown. Scale bar = 10 μm. Representative images are shown. mGPR35, mouse GPR35 [4].

Animal/Disease Models: 7-year-old male inbred BALB/c ByJ mice [5]
Doses: 3 and 10 mg/kg
Route of Administration: IP; 60 minutes before first treatment
Experimental Results: In the EPM test at 10 mg/kg , the incubation period on the second day was Dramatically shortened compared with the control group. 10 mg/kg Dramatically shortened the time spent in the Hughes box on the new side.

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Dosing protocol [3]
Topical administration of pharmacologic agents immediately followed balloon injury. Two hundred μl of a 25% copolymer gel solution (Pluronic F-127; BASF) containing 1 mg Zaprinast, YC-1, or the specific sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), or a combination thereof, in 50 μl DMSO was administered to the exposed LCA adventitia. Control animals received 200 μl gel containing 50 μl DMSO. Animals were closed and allowed to recover until euthanasia. Drug administration [5]
Zaprinast and rolipram were dissolved in saline supplemented with small amounts of DMSO. All drugs were freshly prepared and administered in a volume of 0.1 ml per 10 g body weight. The control groups received the same volume of vehicle. Zaprinast (3 and 10 mg/kg), rolipram (0.05 and 0.1 mg/kg), or vehicle was administered intraperitoneally (i.p.) 60 and 30 min, respectively, before the first session (acquisition session, Day 1) of the mEPM (n=6) and PA tests (n=7). Zaprinast (3 and 10 mg/kg), rolipram (0.05 and 0.1 mg/kg), or vehicle was administered intraperitoneally (i.p.) 60 and 30 min, respectively, before the exploratory activity test (n=6). The animals were administered a single injection before the start of the behavioral tests. The number of animals per group ranged from 6 to 7. Different animals were used for each test. The effective dose of each drug was selected according to previous behavioral and neurochemical studies

[1]. Zaprinast, an inhibitor of cGMP-selective phosphodiesterases, enhances the secretion of TNF-alpha and IL-1beta and the expression of iNOS and MHC class II molecules in rat microglial cells. J Neurosci Res. 2002 Feb 1;67(3):411-21.

[2]. Zaprinast, a well-known cyclic guanosine monophosphate-specific phosphodiesterase inhibitor, is an agonist for GPR35. FEBS Lett. 2006 Sep 18;580(21):5003-8. Epub 2006 Aug 17.

[3]. Keswani AN , et al The cyclic GMP modulators YC-1 and zaprinast reduce vessel remodeling through antiproliferative and proapoptotic effects. J Cardiovasc Pharmacol Ther. 2009 Jun;14(2):116-24.

[4]. Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor. J Biol Chem. 2022 Mar;298(3):101655.

[5]. Zaprinast and rolipram enhances spatial and emotional memory in the elevated plus maze and passive avoidance tests and diminishes exploratory activity in naive mice. Med Sci Monit Basic Res. 2014 Jul 24:20:105-11.

5-(2-propoxyphenyl)-2,3-dihydrotriazolo[4,5-d]pyrimidin-7-one is a member of triazolopyrimidines.

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Moreover, the present study demonstrates the Zaprinast-induced amplification of glial cell activities to express iNOS and MHC Class II possibly via the production of proinflammatory cytokines. As we demonstrated by triple immunohistochemistry, iNOS expression is restricted to microglial cells. This finding is consistent with the undetectable production of NO by stimulated pure astrocytes. In contrast to our results, other studies have reported that iNOS expression may occur in astrocytes (Galea et al., 1992; Vincent et al., 1998; Akama and Van Eldik, 2000). Possel et al. (2000) also showed the exclusive expression of iNOS in microglial cells, and suggested that experimental designs and protocols, culture conditions, and microglial contaminations may have caused iNOS expression and NO production by cultured or in vivo astrocytes. In the present study, zaprinast enhanced LPS/IFN-γ-induced iNOS expression only in microglial cells, and this upregulation might be mediated by the zaprinast-induced increase of TNF-α and IL-1β. Though astrocytes in mixed cultures have been stimulated by LPS/IFN-γ and microglial cytokines, astrocytes have not expressed iNOS to date.

Astrocytes and microglial cells can be induced to perform antigen presenting cell functions (Shrikant and Benveniste, 1996), and IFN-γ has been shown to be the most potent factor for the induction of MHC Class II expression in most cell types (Rohn et al., 1996). In the present study, astrocytes in mixed cultures may have been affected by paracrine factors secreted by microglial cells. IFN-γ induced the expression of MHC Class II in astrocytes and microglial cells, both in mixed cultures and in pure cultures, and Zaprinast enhanced the IFN-γ-induced expression of MHC Class II in astrocytes and microglial cells in mixed cultures and in pure microglial cell cultures. The expression of MHC Class II in pure astrocytes, however, which lack the paracrine communications from microglial cells, was not enhanced by zaprinast. TNF-α alone had no effect on the expression of MHC Class II, but acted to enhance the expression initially induced by IFN-γ (Benveniste et al., 1989; Vidovic et al., 1990). Therefore, microglial TNF-α secretion increased by zaprinast seems to contribute to the enhancement of MHC Class II expression initiated by IFN-γ through autocrine and paracrine mechanisms. [1]

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In this study, we demonstrated that Zaprinast (a cGMP-PDE inhibitor) induced the intracellular calcium mobilization in the cells coexpressing GPR35 and G qi5, G qo5, or Gα16. Induction of intracellular calcium mobilization by zaprinast in the GPR35-expressing transfectants was due to selective activation of the GPR35-Gα (G qi5, G qo5, or Gα16) signaling pathway, rather than inhibition of phosphodiesterases (PDE5, PDE6, PDE9, PDE10, and PDE11), direct activation of Gα proteins, or non-selective stimulation of endogenous GPCR-Gα signaling pathway, because expression of both GPR35 and Gα proteins (G qi5, G qo5, or Gα16) was necessary preconditions for this action of zaprinast (1, 3). Thus, these observations strongly suggest that zaprinast acts as an agonist for GPR35. Zaprinast potently induced intracellular calcium mobilization in the transfectant coexpressing rGPR35 and G qi5 with an EC50 value of 16 nM (Fig. 3), while effects of zaprinast on PDEs are moderate or weak (see Section 1). In addition, the selective PDE5/PDE6 inhibitors T-0156 and T-1032 did not have any effect on the transfectants coexpressing GPR35 and G qi5 (Fig. 4B). Furthermore, 8Bromo-cGMP did not induce intracellular calcium mobilization in our assay (Fig. 4B). These facts also support our conclusion.

Several recent reports have suggested that Zaprinast may possess pharmacological activities other than PDEs inhibition. For example, Wibberley et al. demonstrated a nitric oxide (NO)-independent role for zaprinast in the regulation of urethral sphincter tone. Yoon et al. reported that intrathecal zaprinast had an antinociceptive effect in the rat formalin test, and that this effect was not related to the NO-cGMP-potassium channel pathway. Because cGMP PDEs are deeply involved in the NO-cGMP signaling pathway (NO activates soluble guanylyl cyclase to increase the intracellular cGMP level, while cGMP PDE terminates NO/cGMP-dependent signals by degrading cGMP), these NO/cGMP-independent effects of zaprinast may be mediated by GPR35 activation. Numerous reports about pharmacological studies of PDEs inhibition by zaprinast have been published. However, it may be necessary to repeat those experiments with different structural classes of selective PDE inhibitors since the GPR35 agonist activity of zaprinast was revealed in this study.

It is important to note that zaprinast is a lead compound for sildenafil (Viagra™), indicating that Zaprinast has favorable chemical properties for drug design. It may be feasible to identify potent and selective agonists and/or antagonists for hGPR35 without phosphodiesterase inhibitory activity from among compounds related to zaprinast. In fact, we have already identified a number of GPR35 agonists with different potency and species selectivity among zaprinast derivatives (Taniguchi et al, Patent Application WO2005085867(A2)), suggesting that zaprinast may serve as a lead compound to develop drugs that modulate GPR35 activity.

During the review process of this manuscript, Wang et al. reported that kynurenic acid was a natural ligand for GPR35. They independently showed that GPR35 acted as a Gαi/o- and Gα16-coupled receptor using kynurenic acid, which was consistent with our results obtained with Zaprinast. However, affinity of kynurenic acid for GPR35 is relatively low (EC50 values of 7.4–39.2 μM). Thus, other chemical classes of natural ligand with higher affinity may be expected. Because GPR35 shares homology with GPCRs belonging to the P2Y family and zaprinast is a xanthine derivative, our findings may provide a hint to discover the natural ligand for GPR35. [2]

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Herein we demonstrate that the cyclic GMP-dependent PDE-V inhibitor Zaprinast provides protection against vessel growth after injury through inhibition of vascular cell proliferation and induction of apoptosis. Analogous results with the sGC stimulator YC-1 suggest that YC-1 and zaprinast operate through parallel pathways in the vascular growth response following trauma. Also, lack of noticeable effects from concomitant YC-1 and zaprinast suggest that these agents operate in sGC-independent fashion under these experimental conditions. This new evidence provides insights into the mechanisms and potential therapeutic applicability of the cyclic GMP modulators YC-1 and zaprinast for the treatment of vasculoproliferative disorders. [3]

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G protein-coupled receptor 35 (GPR35) is poorly characterized but nevertheless has been revealed to have diverse roles in areas including lower gut inflammation and pain. The development of novel reagents and tools will greatly enhance analysis of GPR35 functions in health and disease. Here, we used mass spectrometry, mutagenesis, and [32P] orthophosphate labeling to identify that all five hydroxy-amino acids in the C-terminal tail of human GPR35a became phosphorylated in response to agonist occupancy of the receptor and that, apart from Ser294, each of these contributed to interactions with arretin-3, which inhibits further G protein-coupled receptor signaling. We found that Ser303 was key to such interactions; the serine corresponding to human GPR35a residue 303 also played a dominant role in arrestin-3 interactions for both mouse and rat GPR35. We also demonstrated that fully phospho-site-deficient mutants of human GPR35a and mouse GPR35 failed to interact effectively with arrestin-3, and the human phospho-deficient variant was not internalized from the surface of cells in response to agonist treatment. Even in cells stably expressing species orthologues of GPR35, a substantial proportion of the expressed protein(s) was determined to be immature. Finally, phospho-site-specific antisera targeting the region encompassing Ser303 in human (Ser301 in mouse) GPR35a identified only the mature forms of GPR35 and provided effective sensors of the activation status of the receptors both in immunoblotting and immunocytochemical studies. Such antisera may be useful tools to evaluate target engagement in drug discovery and target validation programs. [4]

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Zaprinast has been used to inhibit PDE5, and when given immediately after training at a dose of 10 mg/kg (i.p.), zaprinast improved the long-term memory performance of rats in the object recognition task. Previous studies have also shown that zaprinast reversed the object memory deficits induced by the NOS inhibitor 7-nitroindazole in rats in the object recognition task [16]. However, zaprinast was unable to reverse memory deficits in aged rats in this task. Animal studies indicate that PDE5 inhibitors have the potential to improve the early consolidation processes of long-term memory, although PDE5 inhibitors may not affect spatial information. This memory improvement might be mediated by elevations in central cGMP levels.

Our literature search found a few studies investigating the effects of Zaprinast and rolipram on memory in the passive avoidance test (PA), although there were no studies investigating the effects of zaprinast and rolipram on memory in the elevated plus maze test (EPM) or on exploratory activity in the Hughes box test. The aim of this study was to investigate the effects of the phosphodiesterase-5 inhibitor, zaprinast, and the phosphodiesterase-4 inhibitor, rolipram, on spatial memory in the EPM, on emotional memory in the PA, and also on exploratory activity in the Hughes box test in naive mice. [5]

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The present study demonstrates that both the PDE 5 inhibitor Zaprinast and the PDE 4 inhibitor rolipram enhanced spatial memory in the EPM test, and rolipram enhanced emotional memory in the PA test compared to zaprinast. Both zaprinast and rolipram diminished exploratory activity in the Hughes box test, which can be attributed to their anxiogenic effects. Our results confirm that the effects of zaprinast and rolipram on learning and memory seem to be test-dependent, and future studies using different PDE inhibitors with different cognition methods should be performed to verify our findings. [5]

C13H13N5O2

271.27462

271.106

C, 57.56; H, 4.83; N, 25.82; O, 11.80

37762-06-4

135399235

Off-white to pink solid powder

1.484g/cm3

406.3±47.0 °C at 760 mmHg

237-238ºC dec.

199.5±29.3 °C

0.0±0.9 mmHg at 25°C

1.719

0.17

2

5

4

20

400

0

CCCOC1=CC=CC=C1C1=NC(=O)C2=NNNC2=N1

REZGGXNDEMKIQB-UHFFFAOYSA-N

InChI=1S/C13H13N5O2/c1-2-7-20-9-6-4-3-5-8(9)11-14-12-10(13(19)15-11)16-18-17-12/h3-6H,2,7H2,1H3,(H2,14,15,16,17,18,19)

5-(2-propoxyphenyl)-2,6-dihydrotriazolo[4,5-d]pyrimidin-7-one

zaprinast; 37762-06-4; Zaprinastum; M&B 22948; Zaprinastum [INN-Latin]; M and B 22948; M&B 22,948; Zaprinast [INN:BAN];

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~62.5 mg/mL (~230.40 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.67 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.67 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.67 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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 (Please use freshly prepared in vivo formulations for optimal results.)