Conscious Rabbit Model Research Articles

Design, synthesis and biological evaluation of pyrazolopyrimidinone based potent and selective PDE5 inhibitors for treatment of erectile dysfunction

Our previous discovery of series of pyrazolopyrimidinone based PDE5 inhibitors led to find potent leads but with low aqueous solubility and poor bioavailability, and low selectivity. Now, a new series of same pyrazolopyrimidinone scaffold is designed, synthesized and evaluated for its PDE5 inhibitory potential. In this study, some of the molecules are found more potent and selective PDE5 inhibitors in vitro than sildenafil. The studies revealed that compound 5 is 20 fold selective to PDE5 against PDE6. As PDE6 enzyme is involved in the phototransduction pathway in the retina and creates distortion problem, the selectivity for PDE5 specifically against PDE6 enzyme is preferred for any development candidate and in present study, compound 5 has been found to be devoid of this liability of selectivity issue. Moreover, compound 5 has shown excellent in vivo efficacy in conscious rabbit model, it's almost comparable to sildenafil. The preclinical pharmacology including pharmacokinetic and physicochemical parameter studies were also performed for compound 5, it was found to have good PK properties and other physicochemical parameters. The development of these selective PDE5 inhibitors can further lead to draw strategies for the novel preclinical and/or clinical candidates based on pyrazolopyrimidinone scaffold.

Melt-Cast Films Significantly Enhance Triamcinolone Acetonide Delivery to the Deeper Ocular Tissues.

Delivering an effective drug load to the posterior section of the ocular tissues, while using a non-invasive technique, has always been a challenge. In this regard, the goal of the present study was to develop sustained release triamcinolone acetonide (TA) loaded polymeric matrix films for ocular delivery. The TA-films were prepared in two different polymer matrices, with drug loadings of 10% and 20% w/w, and they were evaluated for ocular distribution in vivo in a conscious rabbit model. A 4% w/v TA suspension (TA-C) was used as a control for in vitro and in vivo studies. The TA-films, prepared with melt-cast technology, used polyethylene oxide (PEO) and Soluplus® as the polymer matrix. The films were evaluated with respect to assay, content uniformity, excipient interaction, and permeability across isolated rabbit sclera. The distribution of TA in the ocular tissues, post topical administration, was determined in New Zealand male albino rabbits as a function of dose, and was compared against TA-C. The assay of the 10% and 20% w/w film was in the range from 70–79% and 92–94% for the Soluplus® and PEO films, respectively, and content uniformity was in the range of 95–103% for both the films. The assay of the TA from Soluplus® films was less compared with the PEO films and showed an interaction with TA, as revealed by Differential Scanning Calorimetry (DSC). Hence, Soluplus® films were not selected for further studies. No interaction was observed between the drug and PEO polymer matrix. The enhancement of trans-scleral flux and permeability of TA was about 1.16 and 1.33-folds, respectively, from the 10% w/w PEO and 3.5 and 2.12-folds, respectively, from the 20% w/w PEO films, as compared with TA-C formulations. The in vivo studies demonstrate that significantly higher TA levels were observed in the anterior and posterior segments of the eye at the end of 6h with the PEO films. Therefore, the PEO based polymeric films were able to deliver TA into the back of the eye efficiently and for prolonged periods.

Rapamycin Alters MicroRNA Signature Profile in Diabetic Rabbit following Myocardial Ischemia Reperfusion Injury: A Preclinical Approach for Cardioprotection.

BackgroundMyocardial ischemia reperfusion (I/R) injury associated with cardiovascular risk factors and comorbidities, including Diabetes (DM), is a major complication for development of heart failure. Mammalian target of Rapamycin (mTOR) is robustly active in DM and further exacerbated during I/R. mTOR inhibition with rapamycin at reperfusion preserves cardiac function with reduction of myocardial infarction in diabetic mice. Recent advances in non‐coding RNA analysis demonstrates microRNA (miR) plays an important role in cardiovascular diseases. We examined the effect of rapamycin treatment at the onset of reperfusion in a conscious diabetic rabbit model of I/R and identified differentially regulated miRs.Methods and ResultsTo induce diabetes, alloxan monohydrate (125 mg/kg, i.v.) was administered in New Zealand male rabbits (n=26; average 3 kg, 4 month old). Blood glucose levels increased to 345±66 mg/dL (n=10) following 2 weeks of alloxan administration. The animals were categorized to three groups: Diabetes (DM), Diabetes subjected to I/R (DM+I/R) and Diabetes underwent I/R and treated with Rapamycin (DM+I/R+Rapa). Post 7 days of implantation of balloon occluders on the top of the coronary artery, the conscious rabbits were subjected to 45‐min ischemia and 3 days of reperfusion by inflating/deflating the balloon. Rapamycin (0.25 mg/kg, i.v.) or DMSO (vehicle) was infused 5 min before reperfusion. Plasma was collected at baseline, 1, 2, 4 and 24 hours during the reperfusion and cardiac Troponin I (cTnI) release was measured by ELISA. The results show that rapamycin significantly reduced myocardial infarction in DM rabbits following I/R (Figure A) as assessed by triphenyltetrazolium chloride staining. Rapamycin also reduced cTnI at 1 hr and 2 hr of reperfusion as compared to vehicle alone (Figure B). RNA isolated from risk area of left ventricle heart tissue from different groups (n=3) was subjected to miRNA‐Array analysis using μParaflo® Microfluidic Biochip Technology (Figure C). The results show 34 out of 761 miRs were differentially regulated with Rapamycin treatment (p<0.01) (Figure D). Enrichment Analysis and Annotation of selected miRs were performed using miEAA and DAVID tools to identify potential targets and its interaction with mTOR signaling pathway.ConclusionThis Preclinical translational study demonstrates that rapamycin could be a promising drug in attenuation of reperfusion injury in diabetic patients following acute MI by altering novel miRs.Support or Funding InformationNIH RO1HL134366 (AD & RCK) & CCTR Endowment Fund UL1RR031990 (AD)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstracts From the 36th Annual Scientific Meeting of the HBPRCA

### CONTRIBUTION OF THE AREA POSTREMA TO THE INCREASED CARDIAC SYMPATHETIC NERVE ACTIVITY IN OVINE HEART FAILURE Abukar Y, Ramchandra R, May CN The Florey Institute of Neuroscience and Mental Health, Melbourne, Victoria, Australia Background : Heart failure (HF) is associated with an increase in cardiac sympathetic nerve activity (CSNA), which is directly linked to mortality in HF patients. The mechanisms responsible for the elevated CSNA remain unclear. Previous studies indicate that the area postrema (AP), a circumventricular organ in the brainstem, plays a role in the control of sympathetic nerve activity. We hypothesized that the elevated CSNA in HF is mediated by the AP and lesioning this region would reduce the increased CSNA in sheep with HF. Aims : To determine the effect of sham lesion or lesion of the AP on CSNA and hemodynamics in conscious sheep with HF. Methods : Studies were conducted in 2 groups of sheep with pacing-induced HF: sham (n=6) and AP lesion (n=6) sheep. Mean arterial blood pressure (MAP), heart rate (HR) and CSNA were recorded simultaneously in conscious sheep at least 4 days after surgery. Results : Heart failure was associated with a significant decrease in ejection fraction (from 74±2 % to 38±1 %; P<0.001), which was similar in both groups. There was a significant reduction in CSNA burst incidence in the AP lesion group compared with the sham group (45±10 and 89±3 bursts/100 heartbeats, respectively; P<0.01). Conclusions : In sheep with HF, the group with lesion of the AP had a significantly lower CSNA compared with the sham group. These data suggest that the AP plays a role in setting the detrimental high levels of CSNA in HF. ### G PROTEIN-COUPLED ESTROGEN RECEPTOR SIGNALING IMPROVES STROKE OUTCOME IN FEMALE MICE Broughton BRS, Jansen GL, Sobey CG Department of Pharmacology, Monash University, Clayton, Victoria, Australia Background: Estrogen has been assumed to provide neuroprotection following stroke entirely via classical estrogen receptors. Interestingly, there is recent evidence that activation of a novel G …

Role of the Renal Nerves in a Conscious Rabbit Model of Chronic Kidney Disease

Chronic kidney disease (CKD) contributes substantially to the global burden of cardiovascular disease. The renal nerves are likely to play a major role in CKD-induced hypertension since renal injury stimulates afferent sensory nerve fibers which modulate central neuronal circuits and thus sympathetic outflow. We investigated a novel conscious rabbit model of CKD by lesioning 5/6th of the renal cortex in one kidney and removing the contralateral kidney. We examined the role of the renal nerves by denervating the kidneys. Blood pressure (BP) and renal sympathetic nerve activity (RSNA), autonomic reflexes and noradrenaline spillover were examined after 3 weeks CKD. Plasma creatinine and urea were 44% and 38% higher in CKD than controls. BP and RSNA were 8% and 33% higher but total noradrenaline spillover was 32% lower in CKD rabbits (P<0.05). Responses to hypoxia were attenuated by CKD and RSNA baroreflexes were shifted to the higher BP level. Renal denervation reduced RSNA in both CKD and control rabbits (30%) but only lowered BP in controls. Noradrenaline spillover was unaltered by denervation but RSNA baroreflexes in both groups were shifted downwards. The hypertension to airjet was enhanced in both groups after denervation but the hypertension to hypoxia was attenuated in the control animals only. Blood parameters were not altered by renal denervation. Thus, while RSNA is elevated in CKD, there is sympathoinhibition in other beds. Renal denervation did not alter the renal impairment or hypertension suggesting that the renal nerves play a minor role in maintaining hypertension in this model of CKD.

Discovery of novel pyrazolopyrimidinone analogs as potent inhibitors of phosphodiesterase type-5

Cyclic guanosine monophosphate (cGMP) specific phosphodiesterase type-5 (PDE5), a clinically proven target to treat erectile dysfunction and diseases associated with lower cGMP levels in humans, is present in corpus cavernosum, heart, lung, platelets, prostate, urethra, bladder, liver, brain, and stomach. Sildenafil, vardenafil, tadalafil and avanafil are FDA approved drugs in market as PDE5 inhibitors for treating erectile dysfunction. In the present study a lead molecule 4-ethoxy-N-(6-hydroxyhexyl)-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)benzenesulfonamide, that is, compound-4a, an analog of pyrazolopyrimidinone scaffold has been identified as selective PDE5 inhibitor. A series of compounds was synthesized by replacing N-methylpiperazine moiety (ring-C) of sildenafil structure with different N-substitutions towards sulfonamide end. Compound-4a showed lower IC50 value (1.5nM) against PDE5 than parent sildenafil (5.6nM) in in vitro enzyme assay. The isoform selectivity of the compound-4a against other PDE isoforms was similar to that of the Sildenafil. In corroboration with the in vitro data, this molecule showed better efficacy in in vivo studies using the conscious rabbit model. Also compound-4a exhibited good physicochemical properties like solubility, caco-2 permeability, cLogP along with optimal PK profile having no significant CYP enzyme inhibitory liabilities. Discovery of these novel bioactive compounds may open a new alternative for developing novel preclinical candidates based on this drugable scaffold.

Oxytocin does not directly alter cardiac repolarization in rabbit or human cardiac myocytes.

Oxytocin, a nine amino acid peptide, is highly conserved in placental mammals, including humans. Oxytocin has a physiological role in parturition and parenteral administration of the synthetic peptide is used to induce labor and control postpartum hemorrhage. Endogenous levels of oxytocin before labor are ∼20 pg/mL, but pharmacological administration of the peptide can achieve levels of 110 pg/mL (0.1 nmol/L) following intravenous administration. Cardiac arrhythmia and premature ventricular contractions have been associated with oxytocin administration in addition to QTc interval prolongation. In the conscious rabbit model, intravenous oxytocin produced QT and QTc prolongation. The mechanism of oxytocin-induced QTc prolongation is uncertain but could be the result of indirect changes in autonomic nervous tone, or a direct effect on the duration of cardiomyocyte repolarization. The purpose of this study was to examine the ability of oxytocin to alter cardiac repolarization directly. Two conventional models were used: QTc interval evaluation in the isolated rabbit heart (IRH) and assessment of action potential duration (APD) in human ventricular myocytes (HVM). Oxytocin did not prolong QTc intervals in IRH or APD in HVM when tested at suprapharmacological concentrations, for example, up to 1 μmol/L. The results indicate that oxytocin has very low risk for eliciting QTc and APD prolongation directly, and infer that the QTc changes observed in vivo may be attributed to an indirect mechanism.

Vitreal Pharmacokinetics of Peptide-Transporter-Targeted Prodrugs of Ganciclovir in Conscious Animals

To delineate the vitreal pharmacokinetics of dipeptide monoester prodrugs of ganciclovir (GCV) with conscious rabbit model using ocular microdialysis and to compare with published results from anesthetized model. New Zealand albino male rabbit was selected as the animal model. Conscious animal ocular microdialysis technique with permanently implanted probes was employed to delineate the pharmacokinetics of GCV, L-valine-GCV (Val-GCV), and dipeptide monoester GCV prodrugs [val-val and L-glycine-val (Gly-Val)] after intravitreal administration. This work employs conscious model to evaluate vitreal pharmacokinetic parameters and compares the results with previously published data from anesthetized animal, thereby demonstrating the effect of anesthesia on the vitreal disposition of dipeptide prodrugs of GCV. Results have revealed that area under curve (AUC), clearance, and last measured plasma concentration (C(last)) for all 4 compounds were significantly altered in a conscious animal relative to the anesthetized model, while mean residence time (MRT) was significantly reduced. However, the AUCs of regenerated Val-GCV and GCV from Gly-Val-GCV and Val-Val-GCV were found to be unchanged, suggesting higher ocular metabolism in conscious animals. This study for the first time delineates the vitreal pharmacokinetics of a GCV prodrug in conscious animals and compares the data with anesthetized animals. Lower vitreal exposure levels were obtained in case of conscious animal model; however, the elimination rates were not influenced by anesthesia.

Discovery of potent, selective, and orally bioavailable PDE5 inhibitor: Methyl-4-(3-chloro-4-methoxybenzylamino)-8-(2-hydroxyethyl)-7-methoxyquinazolin-6-ylmethylcarbamate (CKD 533)

In a continuing effort to discover novel PDE5 inhibitors, we have successfully found quinazolines with 4-benzylamino substitution as potent and selective PDE5 inhibitors. Initial lead compound (1) was found to be easily metabolized when incubated with human liver microsomes mainly through C6 amide hydrolysis. Blocking of this metabolic hot spot led to discovery of 10 (CKD533) which is highly potent, selective and orally efficacious in conscious rabbit model for erectile dysfunction and now is undergoing preclinical toxicology study.

DA-8159 has erectile potentials much longer than the plasma half-life in a conscious rabbit model

DA-8159 is a pyrazolopyrimidinone derivative which is a potent and selective phosphodiesterase type 5 inhibitor. The efficacy of oral DA-8159 has been demonstrated in conscious and spinalized rabbits by its enhancement of nitric oxide-induced erections. The aim of this study was to investigate the time dependency of this efficacy on its plasma concentration in rabbits. DA-8159 was given orally to normal rabbits at a dose of 10 or 30mg/kg in order to determine its pharmacokinetic parameters. After then, to investigate the relationship between penile erectile activity and plasma half-life, a dose of 10mg/kg DA-8159 was administered and the erectile response was examined in a time-course manner by measuring the length of the uncovered penile mucosa after the intravenous administration of sodium nitroprusside, which was administered 1, 3, 6, 8, 24 hours after administering DA-8159. DA-8159 was absorbed rapidly with a Tmax of 0.6 hours in 30mg/kg and 1.0 hour in the 10mg/kg group, and T 1/2 of 1.23 hours in 30mg/kg and 1.17 hours in 10mg/kg, respectively. DA-8159 was not detected in the blood plasma 3 hours (10mg/kg) or 6 hours (30mg/kg) after administration. In an erection test, DA-8159 alone (10mg/kg) induced a penile erection for approximately 2 hours but there was no significant erection thereafter. Although the DA-8159-induced penile erection disappeared, an intravenous injection of sodium nitroprusside significantly induced a penile erection for 6 hours, when the plasma drug concentration was below the detection limit and a no longer visible erection was noted. These results demonstrate that DA-8159 is absorbed and rapidly cleared in rabbits. In addition, it can enhance a sodium nitroprusside-induced penile erection even after 6 hours, which is approximately five times longer than the plasma half-life in the rabbits. These results suggest that DA-8159 may have an erectile potential for much longer than its measured half-life.

DA-8159 has erectile potentials much longer than the plasma half-life in a conscious rabbit model

DA-8159 is a pyrazolopyrimidinone derivative which is a potent and selective phosphodiesterase type 5 inhibitor. The efficacy of oral DA-8159 has been demonstrated in conscious and spinalized rabbits by its enhancement of nitric oxide-induced erections. The aim of this study was to investigate the time dependency of this efficacy on its plasma concentration in rabbits. DA-8159 was given orally to normal rabbits at a dose of 10 or 30mg/kg in order to determine its pharmacokinetic parameters. After then, to investigate the relationship between penile erectile activity and plasma half-life, a dose of 10mg/kg DA-8159 was administered and the erectile response was examined in a time-course manner by measuring the length of the uncovered penile mucosa after the intravenous administration of sodium nitroprusside, which was administered 1, 3, 6, 8, 24 hours after administering DA-8159. DA-8159 was absorbed rapidly with a Tmax of 0.6 hours in 30mg/kg and 1.0 hour in the 10mg/kg group, and T1/2 of 1.23 hours in 30mg/kg and 1.17 hours in 10mg/kg, respectively. DA-8159 was not detected in the blood plasma 3 hours (10mg/kg) or 6 hours (30mg/kg) after administration. In an erection test, DA-8159 alone (10mg/kg) induced a penile erection for approximately 2 hours but there was no significant erection thereafter. Although the DA-8159-induced penile erection disappeared, an intravenous injection of sodium nitroprusside significantly induced a penile erection for 6 hours, when the plasma drug concentration was below the detection limit and a no longer visible erection was noted. These results demonstrate that DA-8159 is absorbed and rapidly cleared in rabbits. In addition, it can enhance a sodium nitroprusside-induced penile erection even after 6 hours, which is approximately five times longer than the plasma half-life in the rabbits. These results suggest that DA-8159 may have an erectile potential for much longer than its measured half-life.

BAY 41-2272: a stimulator of soluble guanylyl cyclase induces nitric oxide-dependent penile erection in vivo

Objectives To determine the effectiveness of BAY 41-2272 on penile erections in an in vivo rabbit model. The nitric oxide (NO)-dependent increase of intracellular cyclic guanosine monophosphate (cGMP) by cGMP-phosphodiesterase (PDE5) inhibition has been shown to be an effective mechanism in the treatment of erectile dysfunction. Direct, NO-independent stimulation of soluble guanylyl cyclase should also lead to elevated cGMP levels in tissues and could be an attractive alternative therapeutic option for the treatment of erectile dysfunction. BAY 41-2272 is a novel non-NO-based direct stimulator of soluble guanylyl cyclase that activates purified enzyme in a synergistic fashion with NO. Methods BAY 41-2272 was administered to conscious rabbits intravenously (IV) and orally (PO). Erection was assessed in a time-dependent manner by measuring the length of the uncovered penile mucosa. Erections were evaluated in the absence and presence of NO (with intravenous sodium nitroprusside [SNP] as the NO donor). Results BAY 41-2272 only induced weak penile erections in conscious rabbits after IV (1 mg/kg) and PO (10 mg/kg) administration in the absence of an NO donor. However, the efficacy of BAY 41-2272 was potentiated by the simultaneous administration of SNP. Through simultaneous SNP administration, the effective doses of BAY 41-2272 were reduced significantly (minimal effective dose 0.1 mg/kg IV and 1 mg/kg PO). Conclusions The results of this study clearly demonstrated the effect of BAY 41-2272 on penile erection in the conscious rabbit model after PO and IV administration. The time-course and onset of erection was concurrent with the stimulation by exogenous NO (SNP), suggesting that this new pharmacologic mechanism of soluble guanylyl cyclase stimulation could be used in the treatment of erectile dysfunction. Because the effect is increased by SNP, it can be expected that BAY 41-2272 would have enhanced activity during sexual arousal, when NO is produced endogenously.

Posterior segment ocular pharmacokinetics using microdialysis in a conscious rabbit model.

To develop and establish a conscious rabbit model for ocular pharmacokinetic studies and to delineate the effects of anesthesia and probe implantation on the ocular disposition of ganciclovir. A conscious rabbit model was developed for microdialysis of the posterior ocular segment. Rabbits were divided into three groups. Group I consisted of rabbits with no recovery period after probe implantation and were anesthetized throughout the experiment. Group II consisted of rabbits that had a more than 5-day recovery period and were conscious during the experiment. Group III consisted of rabbits that had a more than 5-day recovery period and were anesthetized during the experiment. (3)[H] ganciclovir was administered (50 microL) intravitreously in all groups, and ocular levels were determined for 10 hours at appropriate time intervals. Data obtained were subjected to noncompartmental modeling. Probe calibration studies indicated that the probes were functional for at least 14 days. The anesthetized groups, regardless of the period of recovery from probe implantation, exhibited higher areas under the curve than did the conscious group. The vitreous half-life of ganciclovir was significantly shorter in the groups with a recovery period of more than 5 days compared with the group with no recovery period. The conscious rabbit model was developed and can be used for a period of at least 14 days. Anesthesia increased ocular bioavailability of intravitreously administered ganciclovir, whereas probe implantation led to increased protein efflux into the vitreous, which may be the reason for the increased half-life of ganciclovir in group I.

Pharmacological delayed preconditioning against ischaemia-induced ventricular arrhythmias: effect of an adenosine A(1)-receptor agonist.

1. The goal of this study was to investigate the effects of the delayed pharmacological preconditioning produced by an adenosine A(1)-receptor agonist (A(1)-DPC) against ventricular arrhythmias induced by ischaemia and reperfusion, compared to those of ischaemia-induced delayed preconditioning (I-DPC). 2. Eighty-nine instrumented conscious rabbits underwent a 2 consecutive days protocol. On day 1, rabbits were randomly divided into four groups: 'Control' (saline, i.v.), 'I-DPC' (six 4-min coronary artery occlusion/4-min reperfusion cycles), 'A(1)-DPC(100)' (N(6)-cyclopentyladenosine, 100 microg kg(-1), i.v.), and 'A(1)-DPC(400)' (N(6)-cyclopentyladenosine, 400 microg kg(-1), i.v.). On day 2, i.e., 24 h later, the incidence and severity of ventricular arrhythmias during a 30-min coronary artery occlusion and subsequent reperfusion were analysed in all animals, using an arrhythmia score. 3. I-DPC, A(1)-DPC(100) and A(1)-DPC(400) significantly reduced the infarct size (34+/-5, 42+/-3 and 43+/-7% of the area at risk, respectively) as compared to Control (55+/-3% of the area at risk). 4. During both ischaemia and reperfusion, neither the incidence nor the severity of ventricular arrhythmias were altered by A(1)-DPC(100), A(1)-DPC(400) or I-DPC as compared to Control. 5. Thus, despite reduction of infarct size induced by delayed preconditioning, A(1)-DPC as well as I-DPC failed to exert any anti-arrhythmic effect in the conscious rabbit model of ischaemia-reperfusion.

A conscious-rabbit model to study vardenafil hydrochloride and other agents that influence penile erection.

Experimental models to study the effect of agents on penile erection usually include electrical stimulation of peripheral nerves in anesthetized animals combined with systemic or intracavernous injection of drugs. The objective of this study was to demonstrate that conscious rabbits can be used as a simple and quantitative model for the assessment of compounds that show potential for the treatment of erectile dysfunction. Erection was assessed by measuring the length of uncovered penile mucosa before and after the intravenous (i.v.) administration of agents. Animals did not require anesthesia during the course of the study. The phosphodiesterase 5 (PDE5) inhibitors vardenafil x HCl (hereafter called vardenafil) and sildenafil were given intravenously, and measurements were taken for 0-5 h. The effects of phentolamine and milrinone were also evaluated. Vardenafil (0.1-3 mg/kg) induced dose-dependent penile erections in conscious rabbits following i.v. administration. The efficacy of vardenafil was potentiated, and the minimal effective dose was reduced significantly to 0.01 mg/kg by simultaneous administration of the nitric oxide (NO) donor sodium nitroprusside (SNP). Administration of the NO-synthase inhibitor L-NAME abolished the effect. Sildenafil was effective in this model after i.v. administration. The alpha-adrenergic receptor antagonist phentolamine (0.1, 0.3 and 1 mg/kg i.v.) induced erections with a slower t(max) compared with vardenafil and sildenafil. Intravenous administration of the PDE3 inhibitor milrinone (1 mg/kg i.v.) was less effective than the PDE5 inhibitor vardenafil. The conscious rabbit is a suitable and reliable model for the evaluation of compounds with potential for the treatment of erectile dysfunction. This was demonstrated using compounds that target different signaling pathways that induce smooth muscle relaxation in the penis.

Protein kinase C epsilon-Src modules direct signal transduction in nitric oxide-induced cardioprotection: complex formation as a means for cardioprotective signaling.

An essential role for protein kinase C epsilon (PKCepsilon) has been shown in multiple forms of cardioprotection; however, there is a distinct paucity of information concerning the signaling architecture that is responsible for the manifestation of a protective phenotype. We and others have recently shown that signal transduction may proceed via the formation of signaling complexes (Circ Res. 2001;88:59-62). In order to understand if the assembly of multiprotein complexes is the manner by which signaling is conducted in cardioprotection, we designed a series of experiments to characterize the associations of Src tyrosine kinase with PKCepsilon in a conscious rabbit model of nitric oxide (NO)-induced late preconditioning. Our data demonstrate that PKCepsilon and Src can form functional signaling modules in vitro: PKCepsilon interacts with Src; the association with PKCepsilon activates Src; and adult cardiac cells receiving recombinant adenoviruses encoding PKCepsilon exhibit increased Src activity. Furthermore, our results show that NO-induced late preconditioning involved PKCepsilon-Src module formation and enhanced the enzymatic activity of PKCepsilon-associated Src. Inhibition of PKC blocked cardioprotection, module formation, and PKCepsilon-associated Src activity, providing direct evidence for a functional role of the PKCepsilon-Src module in the orchestration of NO-induced cardioprotection in conscious rabbits.

Rabbits as models for impotence research.

The anesthetized rabbit model is useful and has many advantages: ability to perform neurophysiological studies; more administration routes, including intracavernous injection; haemodynamic measurements in parallel to measurements of intracavernous pressure or penile volume; and direct measurement of intracavernosal pressure and blood flow. This model has been evaluated with many different types of drugs. The conscious rabbit model is a simple and valid model for the assessment of compounds with potential for treatment of ED. It offers several methodological advantages as a screening model for compounds with erection stimulating properties. It was clearly successful in demonstrating the efficacy and the mechanism of the new potent and selective PDE5 inhibitor vardenafil. The model was also effective in demonstrating erection-generating properties through other mechanisms, eg PDE3 inhibitors and alpha-receptor blockers. In conclusion, both anaesthetized rabbit model and the newly developed conscious-rabbit models are well-suited for studies in impotence research.

THE ORAL EFFICACY OF VARDENAFIL HYDROCHLORIDE FOR INDUCING PENILE ERECTION IN A CONSCIOUS RABBIT MODEL

Inhibiting cyclic guanosine monophosphate metabolism may induce penile erection during concomitant nitric oxide production. Vardenafil hydrochloride is a new, highly selective, potent cyclic guanosine monophosphate phosphodiesterase 5 inhibitor. We determined the oral effectiveness of vardenafil in a simple and quantitative conscious rabbit model. Vardenafil was given orally to conscious rabbits. Erection was assessed in a time dependent manner by measuring the length of the uncovered penile mucosa. Erection was evaluated in the absence and presence of intravenous sodium nitroprusside, a nitric oxide donor. Vardenafil induced dose dependent penile erection in conscious rabbits after the oral administration of 1 to 30 mg./kg. The efficacy of vardenafil was potentiated and effective doses were significantly reduced by the simultaneous administration of sodium nitroprusside. The effect of vardenafil on penile erection after oral administration was clearly demonstrated in the conscious rabbit model. The time course and early onset of activity indicate that it may be useful for treating erectile dysfunction. Potentiation of the effect by the nitric oxide donor sodium nitroprusside implies that it would have enhanced activity during sexual arousal, when nitric oxide is produced endogenously. The clinical development of this product for erectile dysfunction is proceeding.

Pharmacoydnamics of Beta-Blocker Modulation of Aqueous Humor Production

A conscious rabbit model with microdialysis sampling of endogenous aqueous humor ascorbate was developed in order to assess the pharmacodynamics of beta-blocker modulation of aqueous humor production. CMA/20 microdialysis probes were implanted in the anterior chamber of each eye of rabbits (n =6). After a 2 week recovery period, an i.v. bolus of14C-ascorbate (20 μCi) was administered. Blood samples and aqueous humor microdialysis probe effluent were collected and analysed for endogenous and14C-ascorbate to estimate the basal rate of ascorbate blood to aqueous humor secretion (Ro). After a 1 hr washout, each rabbit received a series of three doses of3H-propranolol (750–3000 μ g, 16.5 μCi mg−1) every 60 min into the lower cul-de-sac of each eye. Probe effluent was analysed for endogenous ascorbate and3H-propranolol; ascorbate and propranolol in the iris/ciliary body, vitreous and aqueous was determined at the end of the experiment. Nonlinear least-squares regression analysis of the concentration-time profiles for aqueous humor ascorbate was performed to estimate the change in aqueous humor flow. The average basal aqueous humor ascorbate secretion rate was ∼48 μg hr−1. Propranolol (1500 μg) produced significant increases in aqueous humor ascorbate, this observation is consistent with a reduction in aqueous humor production (∼47%). Analysis of intraocular tissue ascorbate indicated that propranolol inhibited ascorbate secretion at the 3000 μg dose, the highest dose examined in this study; this inhibition was not observed at the 750 μg or 1500 μg doses. Changes in aqueous humor production precipitated by the administration of beta-adrenergic antagonists can be estimated by measuring changes in aqueous humor ascorbate concentrations in the conscious rabbit. Microdialysis sampling of aqueous humor for endogenous ascorbate provides a relevant analytic tool to estimate modulatory effects of anti-glaucoma drugs on aqueous humor production.

PKC-dependent activation of p46/p54 JNKs during ischemic preconditioning in conscious rabbits.

A conscious rabbit model was used to study the effect of ischemic preconditioning (PC) on stress-activated kinases [c-Jun NH(2)-terminal kinases (JNKs) and p38 mitogen-activated protein kinase (MAPK)] in an environment free of surgical trauma and attending external stress. Ischemic PC (6 cycles of 4-min ischemia/4-min reperfusion) induced significant activation of protein kinase C (PKC)-epsilon in the particulate fraction, which was associated with activation of p46 JNK in the nuclear fraction and p54 JNK in the cytosolic fraction; all of these changes were completely abolised by the PKC inhibitor chelerythrine. Selective enhancement of PKC-epsilon activity in adult rabbit cardiac myocytes resulted in enhanced activity of p46/p54 JNKs, providing direct in vitro evidence that PKC-epsilon is coupled to both kinases. Studies in rabbits showed that the activation of p46 JNK occurred during ischemia, whereas that of p54 JNK occurred after reperfusion. A single 4-min period of ischemia induced a robust activation of the p38 MAPK cascade, which, however, was attenuated after 5 min of reperfusion and disappeared after six cycles of 4-min ischemia/reperfusion. Overexpression of PKC-epsilon in cardiac myocytes failed to increase the p38 MAPK activity. These results demonstrate that ischemic PC activates p46 and p54 JNKs via a PKC-epsilon-dependent signaling pathway and that there are important differences between p46 and p54 JNKs with respect to the subcellular compartment (cytosolic vs. nuclear) and the mechanism (ischemia vs. reperfusion) of their activation after ischemic PC.