Toxins active on GPCR are only found in two venomous animals. Marine cone snails produce toxins acting on vasopressin, neurotensin and adrenoceptors (Nielsen et al., 1994; Craig et al., 1999; Sharpe et al., 2001). Snakes produce sarafotoxins, which are active at endothelin receptors, muscarinic toxins, which act on muscarinic receptors or β-cardiotoxin, which acts on β-adrenoceptors (Servent and Fruchart-Gaillard, 2009). Since animal venoms are likely to represent a novel source of new GPCRs ligands, we screened mamba snake venom for peptides binding to α1-adrenoceptors. Using a combination of liquid chromatography and binding experiments, we identified ρ-Da1a (previously known as AdTx1) the first peptide to show high affinity and selectivity for the human α1A-adrenoceptor subtype (Quinton et al., 2010). Adrenoceptors are divided into three classes (α1, α2 and β), and α1-adrenoceptors into three subtypes (α1A, α1B and α1D). These receptors are involved in the regulation of urogenital tissue muscle tone (α1A), myocardial contractility (α1B) and vascular tone (α1D; Koshimizu et al., 2003). Due to its potency and selectivity, ρ-Da1a is a new pharmacological tool, which could be useful to clarify the physiological functions of α1-adrenoceptors in animal models. Moreover, this peptide could be the basis for the synthesis of new α1-adrenoceptor antagonists that could be used to treat lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH).
Following an in depth biochemical characterization and a limited functional study on rabbit isolated prostatic smooth muscle (Quinton et al., 2010), the first aim of this study was to test the antagonistic potency of ρ-Da1a on human isolated prostatic adenoma contractility. Moreover, we compared the in vivo activity of ρ-Da1a in anaesthetized rats on intra-urethral (IUP) and arterial blood pressure (AP) increases induced by i.v. administration of phenylephrine (PHE). As a reference compound, we used tamsulosin, one of the most potent α1A-adrenoceptor antagonists known and the gold standard for the symptomatic treatment of lower urinary tract symptoms secondary to BPH in humans (Chapple and Andersson, 2002).
Safety panel of ρ-Da1a
ρ-Da1a (1 μM) was tested as an agonist and as an antagonist on 78 human GPCRs and on eight ion channels implicated in cardiac activity (hERG, hHCN4, hKCNQ1/minK, hKir2.1, hKv1.5, hKv4.3/hKChIP2, Human L-type Calcium, hNav1.5). These tests were performed in duplicate by Millipore (Dundee, UK).
Functional tests on recombinant α1-adrenoceptor subtypes
Functional tests were performed in 96-well plates on COS cells (100,000 cells per well) transfected using calcium phosphate precipitation by α1A-adrenoceptor receptor plasmid (pRK5, 0.1 μg per well, kindly provided by Michael Brownstein, Craig Venter Institute, Rockville, MD, USA). Forty-eight hours after transfection, cells were incubated for 1 h at 37°C with 10 μL of 10X solutions of the various compounds tested and 100 μL of dye solution (FLIPR Calcium Kit buffer, Molecular Devices, Sunnyvale, CA, USA) supplemented with 2.5 mM probenecid. Changes in fluorescence induced by adrenaline were measured at room temperature using a Flexstation II (Molecular Devices). The resulting activation curves obtained were analysed using Softmax Pro software (Molecular Devices, Sunnyvale, CA, USA). Receptor activation for the different experimental conditions was evaluated using maximum and minimum fluorescence values and analysed with GraphPad Prism® (GraphPad Software, La Jolla, CA, USA).
Antagonism of NA-induced contractions on human isolated prostatic smooth muscle
Human prostatic adenoma specimens were obtained from four male patients (age range 68–76 years). These patients (negative for HIV1-2, HTLV1-2, hepatitis B-C and syphilis) had undergone transvesical prostatectomy for BPH in the Urology Department of Rangueil Hospital (Toulouse, France) or Foch Hospital (Suresnes, France). These tissues were donated following patients written informed consent. UROsphere obtained, from the Urology Department, an anonymous patient sheet containing information on patient's age, sex, body weight, height, anaesthetics used during surgery, nature of drugs administered in the 1 month period before hospitalization.
The specimens, which appeared macroscopically healthy, were placed in a cold storage solution (Custodiol®, OPi-France, Limonest, France) immediately after surgery and transported to UROsphere's laboratories in a box maintained at 4°C. The shipment between Hospital and UROsphere's laboratories was carried out by a French courier (Trans-Medical-Services, Montreuil, France). Upon receipt, tissues were stored at 4°C until the start of the experiment (maximum 24 h after surgery).
Prostatic strips (approximate size 8 mm × 3 mm) from the transition zone (12 from each patient) were dissected and mounted, under 1.0 g of initial tension, in organ baths containing a Krebs–Henseleit solution of the following composition (in mM): NaCl 114, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 11.7 and ascorbic acid 1.1 (pH 7.4, gassed with 95%O2 and 5%CO2 at 37°C). The Krebs also contained propranolol (1 μM), desipramine (0.1 μM), deoxycorticosterone (3 μM) and normetanephrine (1 μM) in order to block β-adrenoceptors, neuronal and extraneuronal uptake and catechol-O-methyltransferase respectively.
Five experimental groups (n = 5–10 per group) in four patients were performed, one for the vehicle and four for ρ-Da1a-treated strips, at four different concentrations. A total of 43 strips were used for the experiments.
After 60 min of equilibration, smooth muscle strips were exposed to 30 μM NA to measure their viability. Strips having a contractile response <0.3 g were discarded (n = 5). After washout and 60 min of re-equilibration, ρ-Da1a at four concentrations (10, 30, 100 and 300 nM) or its vehicle (distilled water) were incubated for 3 h, then a cumulative concentration-response curve (CRC) to NA (0.1–1000 μM) was performed. Only one concentration of ρ-Da1a was tested on each single strip.
In separate experiments, the strips were pre-incubated with tamsulosin (10 nM) or its solvent for 1 h before obtaining a cumulative CRC to NA.
Effects of ρ-Da1a and tamsulosin on the increase in IUP and AP induced by PHE in anaesthetized rats
All experimental protocols were carried out in accordance with guidelines for animal care published by the European Community Council Directive 86/609/EEC and the French veterinary service. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010).
Sixty-seven adult male Wistar rats (Charles River Laboratories, L'Arbresle, France), weighing 275–375 g at the beginning of the experiments, were used. They were delivered to the laboratory at least 5 days before the experiments in order to be acclimatized to laboratory conditions. They were housed five per cage and given food (Teklad Global 16% Protein Rodent Diet, Harlan, Gannat, France) and water ad libitum. Sawdust (Lignocel 3–4, Harlan, Gannat, France) bedding in rodent cages was changed twice weekly. The animal room was maintained with a 12/12 h alternating light-dark cycle (light phase 07:00–19:00 h).
The experimental protocol for testing ρ-Da1a and tamsulosin was derived from a paper describing the effects of some α1-adrenoceptors antagonists on IUP in anaesthetized rats (Akiyama et al., 1999). Rats were anaesthetized with pentobarbital 40 mg kg−1, i.p. (Centravet, Lapalisse, France), with a supplementary dose of 10 mg kg−1, s.c., to maintain anaesthesia as necessary during the experiment. The depth of anaesthesia was assessed by pinching the rat hindpaw and by visual control of respiration rate during the experiment. A jugular catheter was inserted for drug administration and another into the carotid artery to record arterial pressure. After a midline incision of the abdomen, a polyethylene catheter was inserted into the urethra via an incision in the bladder. The urethral catheter was held in place by a suture on the bladder wall.
Measurements of urethral and arterial pressures
The urethral catheter was connected via a T-tube to a strain gauge and a syringe injection pump. Saline at room temperature was continuously infused into the urethra at a flow rate of 0.5 mL·h−1.
I.v. administration (protocol 1)
After a stabilization period of at least 10 min, ρ-Da1a (0.1, 0.3 or 1 mg·kg−1), tamsulosin (0.01 mg·kg−1) or the common vehicle (physiological saline) was administered i.v. and 30, 60 or 90 min later, the first dose of PHE was administered.
The different doses of PHE (from 10 to 3000 μg·kg−1) were administered i.v. in a volume of 1 mL·kg−1 (bolus) with a 3 min interval between each dose. Baseline values were measured 1 min before administration of the test compounds.
Oral administration (protocol 2)
Rats were deprived of food (fasted) for at least 12 h before the start of this experiment. Vehicle, ρ-Da1a (10 mg·kg−1) or tamsulosin (0.3 mg·kg−1) was administered p.o. before anaesthesia. Ninety minutes after gavage, the first dose of PHE was administered; the different doses of PHE (10 to 3000 μg·kg−1) were administered i.v. in a volume of 1 mL·kg−1 (bolus) with a 3 min interval between each dose. Baseline values were measured after a stabilization period.
Expression of data and statistical analysis
In vitro studies
In COS cells, results are expressed as mean ± SD with n, number of independent experiments. In the presence of non-competitive antagonism (depression of maximal response to adrenaline but without change in pEC50), the Gaddum method for non-competitive antagonist was used (see Kenakin, 2009). Briefly, equi-active concentrations of adrenaline in the absence or in the presence of 10 nM ρ-Da1a were compared in a linear regression. The slope of this regression was used to estimate the KB for the antagonist with the equation KB = [antagonist]/(slope − 1).
On human isolated prostatic adenoma, contractile responses to NA were expressed as % of the maximal effect (Emax) obtained with the initial contraction to 30 μM NA. CRCs were fitted, using mean results, by non-linear regressions (GraphPad Prism® version 4.0) to obtain the following parameters: Emax, maximal contraction induced by NA; EC50, NA concentration, which induces 50% of the maximum effect, expressed as pEC50 (-log EC50). Mean CRCs for controls and treated strips were fitted in parallel and statistically compared. The first fitting was used to compare Emax values and when these values were not statistically significantly different, a second fit was performed, sharing Emax, in order to obtain pEC50 values for each pair of curves. Differences were considered statistically significant when the null hypothesis could be rejected at a risk α of less than 0.05.
Finally, concentration-ratios (ratios of pEC50 values) for NA in the absence and presence of various concentrations of ρ-Da1a were calculated and used to estimate antagonist potency.
Due to non-parallel shifts of the CRCs to NA, ρ-Da1a antagonist potency was calculated, using the data obtained following incubation with 100 nM ρ-Da1a, from the following equation: pKB = −log[antagonist] + log(dose ratio − 1).
In vivo studies
Increases in IUP and AP (mmHg) were calculated for each animal and each dose of PHE (10, 30, 100, 300, 1000 and 3000 μg·kg−1, i.v.). Increases in IUP and AP (Δ-IUP and Δ-AP) corresponded to the maximal values for IUP and AP after PHE administration minus basal value measured at the end of the stabilization period (1 min before PHE administration).
One-way anova followed by Dunnett's test was used to compare different treated groups to vehicle group. A P < 0.05 was accepted for statistical significance.
In order to facilitate comparison of ρ-Da1a and tamsulosin potencies in in vivo experiments, PHE doses were expressed as mol·kg−1 and data plotted on a logarithmic scale.
In the presence of competitive antagonism, pKB was calculated from the mean pEC50 values of PHE in the presence and absence of antagonist using the following equation:
where antagonist concentration is expressed as mol·kg−1 and dose ratio (DR) = EC50(with antagonist)/EC50(without antagonist).
In the present paper, this equation was used for the calculation of ρ-Da1a potencies (0.1 mg·kg−1, i.v., 30 min) and tamsulosin (0.01 mg·kg−1, i.v., 30 or 90 min post-administration; Figures 4 and 7).
When antagonism was considered as non-competitive (depression of maximal response to PHE but without change in pEC50), equi-active concentrations of PHE were compared in a linear regression. The slope of this regression was used to estimate the KB for the antagonist from the equation KB = [antagonist]/(slope − 1).
This equation was used to calculate antagonist potency values for ρ-Da1a (0.3 mg·kg−1, i.v., 90 min post-administration; Figure 7).
When antagonism was considered as insurmountable (depression of maximal response to agonist with an associated shift to the right of the curve), an equi-active DR for PHE concentrations was calculated at a level of 30% of the maximal response of the dose-response curve. The pA2 was calculated from the following equation:
This equation was used to calculate antagonist potency values for tamsulosin (0.3 mg·kg−1, p.o., 90 min post-administration; Figure 9).
In vitro studies
Screening of ρ-Da1a versus 78 human GPCR and eight ion channels revealed an impressive selectivity of this peptide for α1A-adrenoceptors. Unfortunately, as far as we know, the selectivity of the non-peptidic α1A-adrenoceptors antagonists was not demonstrated on such large panels of targets, making a direct comparison with ρ-Da1a unfeasible.
The pEC50 value for NA-induced contractions in the human isolated prostate was equal to 5.26 ± 0.09, which is similar to the value previously obtained for NA in the human prostatic adenoma (5.48 ± 0.11; Palea et al., 2000). The antagonist potency of ρ-Da1a versus NA in human isolated prostate (pKB = 7.54) was smaller than the potency exhibited in functional studies on COS cells expressing the human α1A-adrenoceptors (pKB = 9.04). The hypothesis that the contractility of human prostate is essentially mediated by activation of α1A-adrenoceptors was advanced, leading to the clinical development of silodosin, an extremely selective α1A-adrenoceptor antagonist (Marks et al., 2009). However, the antagonistic potency of ρ-Da1a versus NA in the human isolated prostate is quite lower with respect to the corresponding value obtained on recombinant human α1A-adrenoceptors, which is in sharp contrast with this hypothesis. This discrepancy could be due to a variety of factors, the more straightforward being related to the abnormal pharmacology of α1A-adrenoceptors in prostatic and urethral tissues. In the past, observations such as these justified the hypothesis for the existence of the α1L-adrenoceptor subtype which, however, is now recognized to be a product of the α1A-adrenoceptor gene (Gray et al., 2008; Muramatsu et al., 2008). The α1L-adrenoceptor subtype is characterized by a low affinity for prazosin and other α1A-adrenoceptor antagonists (with the exception of tamsulosin) when compared with recombinant α1A-adrenoceptors. Another potential explanation for the differences in potencies values between recombinant receptors and native receptors in the human prostate could be the presence of a heterogeneous population of α1-adrenoceptor subtypes in the hyperplastic tissue.
Nevertheless, we have shown here that ρ-Da1a is an insurmountable antagonist of α1A-adrenoceptors in human prostatic tissue, and this accords with results obtained in rabbit isolated prostate (Quinton et al., 2010). Tamsulosin, the most potent α1A-adrenoceptor antagonist on the market, was a potent antagonist in our hands, having a pKB value of 10.3. This value is similar to the potency previously calculated on human isolated prostate by another group (Noble et al., 1997). Therefore, ρ-Da1a is approximately 100 times less potent than tamsulosin on human isolated prostate. Nevertheless, it should be noted that the potency of ρ-Da1a on human prostate (pKB = 7.54) is quite similar to potencies displayed by drugs frequently used to treat lower urinary tract symptoms, such as alfuzosin (pKB = 7.37; Palea et al., 2000), terazosin (pA2 = 8.17; Chueh et al., 2002) and doxazosin (pA2 = 8.43; Hatano et al., 1996). However, unlike ρ-Da1a, alfuzosin, terazosin and doxazosin are competitive antagonists in human isolated prostate. Tamsulosin, in our hands, strongly depressed the maximal response to NA in human tissue but, unlike ρ-Da1a, was not able to inhibit completely the agonist response (see Figure 2 but also Noble et al., 1997). Therefore, we presume that the nature of the antagonism induced by ρ-Da1a differs from that of tamsulosin, which is supported by the strong difference in chemical structure between these two compounds as well as by the half-life of almost 4 h of the ρ-Da1a/α1A-adrenoceptor complex (Quinton et al., 2010).
In vivo studies
Pretreatment with ρ-Da1a and tamsulosin (as well as the vehicle) was ineffective at reducing basal values of AP, suggesting that these molecules are not likely to produce hypotension when administered alone in animals or humans. This finding is consistent with the lack of effect of tamsulosin on AP in a large cohort of patients suffering from BPH (Chapple et al., 1997).
ρ-Da1a (0.1, 0.3 and 1 mg·kg−1, i.v.), administered 30 min before PHE, dose-dependently antagonized the effects of PHE on IUP. In the presence of 1 mg·kg−1 ρ-Da1a, the effect of PHE on IUP was almost totally abolished. This result is consistent with the non-competitive antagonism observed on the rabbit isolated prostate (Quinton et al., 2010) and human prostatic tissue (present study). Hence, we suggest that ρ-Da1a is an insurmountable antagonist not only in vitro but also in vivo.
The antagonist effect of ρ-Da1a on IUP at 0.3 mg·kg−1 (i.v.) was found to be similar to that of tamsulosin at 0.01 mg·kg−1, i.v. Therefore, expressed in mg·kg−1, tamsulosin seems to be approximately 30 times more potent than ρ-Da1a. However, in order to obtain a better comparison of the relaxant effects of ρ-Da1a and tamsulosin on rat urethra in vivo, we decided to express their potency as pKB, a method classically used to quantify antagonist potency in vitro but can also be applied to in vivo pharmacological results when an exogenous agonist of a particular receptor is administered systemically in the absence and presence of a selective antagonist for the same receptor (Dykstra et al., 1988; Negus et al., 1993).
The antagonism induced by 0.1 mg·kg−1 ρ-Da1a was competitive in nature and its antagonistic potency (pKB = 8.17) was slightly less than that of tamsulosin (pKB = 8.89). Interestingly, the potency of ρ-Da1a in vivo was similar to its potency on rabbit isolated prostate (pA2 = 8.38; Quinton et al., 2010), whereas tamsulosin potency in vivo was similar to the potency value calculated in the rat isolated prostate (pKB = 9.23; Pulito et al., 2000). Based on these pKB values calculated in vivo, we conclude that tamsulosin is only three times more potent than ρ-Da1a when administered by the i.v. route in anaesthetized rats.
Importantly, we found that ρ-Da1a has a long duration of action since its effect on IUP was statistically significant 60 min after its i.v. administration. Similar to tamsulosin, a small inhibitory effect was even observed 90 min post-administration of ρ-Da1a. However, the potency of tamsulosin declined over time, pKB being 8.89 and 7.74 at 30 and 90 min post-administration respectively, whereas the potency of ρ-Da1a was only slightly reduced between 30 and 90 post-administration (from 8.17 to 7.73). Hence, we suggest that ρ-Da1a has a longer duration of action than tamsulosin when administered by the i.v. route.
With regard to the effects of the two antagonists on AP 30 min post-administration, we found that ρ-Da1a, even at 1 mg·kg−1 (i.v.), was ineffective at blocking the effects of PHE on AP, whereas tamsulosin at 0.01 mg·kg−1 (i.v.) markedly antagonized the effects of PHE on AP. This suggests that tamsulosin is at least 100 times more potent than ρ-Da1a on α1-adrenoceptors activated by PHE in the resistance arteries (probably the α1B-adrenoceptor subtype). These findings also confirm results obtained in binding studies where it was shown that the affinity of ρ-Da1a for α1B-adrenoceptors is 100 times less than for α1A-adrenoceptors (Quinton et al., 2010).
When administered p.o., ρ-Da1a at 10 mg·kg−1 was ineffective, as expected. However, it was not possible to test the effect of ρ-Da1a at 30 mg·kg−1 because of limited amounts of the peptide available. Tamsulosin, on the other hand, at 0.3 mg·kg−1, p.o., was able to antagonize the effect of PHE on IUP but was ineffective on AP, confirming its uroselectivity when administered orally; an effect previously observed in anaesthetized dogs (Sato et al., 2001).
The ability of ρ-Da1a to act as an insurmountable antagonist on α1A-adrenoceptors represents an advantage compared to classical α1-blockers on the market, because it is able to exert a stronger antagonism of the receptor even in the presence of high concentrations of the agonist. In addition, the long duration of action of ρ-Da1a, linked to the half-life of almost 4 h of the complex ρ-Da1a/α1A-adrenoceptor (Quinton et al., 2010), is a real advantage for chronic diseases. Coupled with ad hoc depot formulations, administration of the drug can be envisaged to be compatible with the long duration of the treatment. Moreover, ρ-Da1a displayed an impressive selectivity for the recombinant α1A-adrenoceptor and a clear uroselectivity in vivo, therefore decreasing the chance of potential cardiovascular side effects (hypotension). Finally, due to its completely new chemical structure, ρ-Da1a could lack some of the side effects associated with the use of tamsulosin, in particular intra-operative floppy iris syndrome (Chang and Campbell, 2005). This syndrome is specific to tamsulosin and not related to a class effect of α1-adrenoceptors (Bell et al., 2009).
At present, all therapeutic peptides are administered by the parenteral route, because of insufficient absorption from the gastrointestinal tract. Peptide drugs are usually indicated for chronic conditions, and the use of injections on a daily basis has obvious drawbacks, whereas the oral route offers the advantages of self-administration. The main reasons for the low oral bioavailability of peptide drugs are enzymatic degradation and poor penetration of the intestinal mucosa. A considerable amount of research has focused on overcoming the challenges presented by absorption barriers to provide effective oral delivery of peptides (Dünnhaupt et al., 2012). Therefore, if the problems of oral absorption could be solved by new technologies, ρ-Da1a has the potential to be a new drug for the pharmacological treatment of urinary tract dysfunctions in humans.
Apart from the classical use of α1A-adrenoceptor antagonists for the pharmacological treatment of BPH, this class of compound could be used for other urological disorders. α1A-Adrenoceptors are functionally expressed by capsaicin-sensitive, nociceptive, primary sensory neurons of the rat urinary tract, and their activation may contribute to the signalling of irritative and nociceptive responses arising from the urinary tract (Trevisani et al., 2007). Interestingly pretreatment with alfuzosin (10 mg·kg−1day−1) was able to suppress bladder overactivity induced by cyclophosphamide in conscious rats (Lluel et al., 2008) suggesting that ρ-Da1a has the potential to decrease bladder inflammation. This property could be useful for controlling the symptoms of pain and urinary frequency in patients suffering from painful bladder syndrome.
Furthermore, tamsulosin and other α1A-adrenoceptor antagonists have been successfully used in recent years to facilitate the passage of renal stones from the ureter to the urinary bladder (Yilmaz et al., 2005; Porpiglia et al., 2006). A clinical investigation monitoring ureteric pressure during ureteroscopy for stone disease showed that oral administration of tamsulosin significantly reduced ureteric pressure (Davenport et al., 2007). Therefore, administration of ρ-Da1a could be very effective in facilitating expulsion of ureteral stones. The advantage of using ρ-Da1a rather than tamsulosin would be its longer duration of action of α1A-adrenoceptor blockade in vivo, as demonstrated in the present study in rats (by i.v. route).
We conclude that ρ-Da1a could be a new treatment for lower urinary tract symptoms, painful bladder syndrome and urolithiasis. Local administration of ρ-Da1a (i.e. by intraprostatic, intravesical or intra-ureteral routes) could be used to circumvent its lack of oral bioavailability.
This work was supported by CEA (France). ρ-Da1a was synthesized by CEA/DSV/iBiTec-S/SIMOPRO Toxins, Receptors and Channels laboratory.
Conflict of interest
The authors declare no conflict of interest.