α1A-adrenoceptor (pKi = 9.6); α1D-adrenoceptor (pKi = 7.9); (α1B-adrenoceptor (pKi = 7.5)

Human lower urinary tract (LUT) tissue, specifically RS100329 hydrochloride, inhibits norepinephrine contraction in a concentration-dependent and surmountable manner [2].

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In competition studies Ro 70-0004, RS100329 hydrochloride, prazosin and tamsulosin all competed to the same extent for [3H]-prazosin binding: calculated affinity estimates (pKi) are shown in Table 1. Prazosin and tamsulosin demonstrated limited α1-AR subtype selectivity (<10 fold), whereas significant selectivities for the α1A- over the α1B- and α1D-AR subtypes were evident for Ro 70-0004 (60 and 50 fold respectively) and RS100329 hydrochloride (126 and 50 fold respectively). [2]
Prazosin showed some selectivity for the α1B- and α1D-AR subtypes over the α1A-AR subtype, whereas tamsulosin showed approximately 10 fold selectivity for the α1A-AR subtype over the α1B-, but not the α1D-AR subtype. Ro 70-0004 (Figure 2a) was 30 and 80 fold selective for the α1A-subtype over the α1B- and α1D-subtypes respectively, and RS100329 hydrochloride (Figure 2b) also showed significant α1A-adrenoceptor selectivity (60 and 50 fold respectively). [2]
In human LUT tissues, contractions to NA were antagonized in a surmountable and concentration-dependent manner by Ro 70-0004, RS100329 hydrochloride and prazosin with similar affinity (pA2 ∼9). [2]
In contrast, both Ro 70-0004 and RS100329 hydrochloride were approximately 100 fold weaker in antagonizing the contractile response to NA in human renal artery. The affinities of prazosin and tamsulosin did not differ from those observed in studies using human LUT tissues (Table 2). In studies using rat aortic rings, Ro 70-0004, RS100329 hydrochloride and tamsulosin antagonized contractile responses to NA with affinities close to those observed for human renal artery. Prazosin however, was approximately 10 fold more potent in rat aorta when compared to human renal artery. [2]

Sprague Dawley rats administered with RS100329 hydrochloride (0.01-0.1 mg/kg) intravenously have lower baseline urethral pressure and less reflex urethral contractions [1].

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The effects of the α1-adrenoceptor antagonists doxazosin (0.1 – 2 mg kg−1), RS100329 hydrochloride (α1A; 0.01 – 1 mg kg−1), RS-513815 (Ro 151-3815, α1B; 0.3 – 3 mg kg−1) and BMY 7378 (α1D; 0.1 – 1 mg kg−1), the 5-HT1A receptor agonist, 8-OH-DPAT (0.03 – 0.3 mg kg−1) and antagonist WAY-100635 (0.03 – 0.3 mg kg−1) were investigated (i.v.) on the ‘micturition reflex' in the urethane anaesthetized male rat. [1]
Reflex-evoked urethra contractions were most sensitive to the inhibitory action of RS100329 hydrochloride, followed by doxazosin, BMY 7378 and WAY-100635 and then RS-513815. The maximum inhibition was 66, 63, 54, 46 and 22% at doses of 0.3, 0.5, 0.3, 0.3 and 3 mg kg−1 respectively. [1]
BMY 7378 and 8-OH-DPAT decreased, while WAY-100635 increased, the pressure threshold to induce bladder contraction. WAY-100635 (0.01 mg kg−1) blocked the effects of BMY 7378 (1 mg kg−1) on bladder pressure and volume threshold. [1]
Doxazosin, RS100329 hydrochloride and BMY 7378 had a similar potency in inducing a fall in arterial blood pressure while WAY-100635 only caused a fall at the highest dose. [1]
Therefore, reflex-evoked urethral contraction involves the activation of α1A/1D-adrenoceptors, as BMY 7378 and RS100329 hydrochloride are similarly potent in attenuating this effect. The ability of WAY-100635 to attenuate this contraction may suggest that 5-HT1A receptors are also involved. However, as this inhibition occurred at the highest dose of WAY-100635, which also caused a fall in arterial blood pressure; this effect is considered to be due to blockade of α1-adrenoceptors not 5-HT1A receptors. Nevertheless the initiation of the ‘micturition reflex' involves the activation of 5-HT1A receptors.

Radioligand binding studies [2]
Affinity estimates (pKi) were made from competition curves (using ten concentrations of displacing agent) using intact CHO-K1 cells stably expressing human cloned α1A-, α1B- and α1D-adrenoceptors. Cells were grown as described above and harvested by incubating with Dulbecco's phosphate buffered saline (PBS) containing EDTA (30 μM) for 10 min at 37°C. Harvested cells were washed twice by centrifugation and resuspension in Ham's medium, and finally resuspended in Ham's medium at approximately 0.2×106 cells ml−1. [2]
[3H]-Prazosin (0.3–0.4 nM; specific activity 82 Ci mmole−1) was used as the radioligand and specific binding was defined using 10 μM phentolamine. Assay tubes contained 100 μl competing compound, 100 μl [3H]-prazosin and 300 μl cell suspension. All equilibrations were carried out for 30 min at 37°C in Ham's culture medium (pH 7.4), and were terminated by vacuum filtration through glass fibre filters. Bound radioactivity was determined using liquid scintillation spectroscopy. Concentrations of competing agent producing 50% reduction of specific [3H]-prazosin binding (IC50) were calculated using non-linear iterative curve-fitting methodologies, and affinity estimates (pKi) were estimated according to Cheng & Prusoff (1973). Estimates of pKD were made by saturation analysis using 10–12 concentrations of [3H]-prazosin (1 pM–3 nM).

In vitro tissue bath studies [2]
Tissue bath studies were conducted at 37°C in 10 ml organ baths and used Krebs' buffer (mM: Na+ 143.5, K+ 6.0, Ca2+ 2.5, Mg2+ 1.2, Cl− 126, HCO3− 25, H2PO4− 1.2, SO42− 1.2), pH 7.4, gassed with 95% O2, 5% CO2, and supplemented with cocaine (30 μM), corticosterone (30 μM), propranolol (1 μM), idazoxan (0.3 μM), ascorbate (100 μM) and nitrendipine (1 μM: rat aorta only, see Blue et al., 1995) to ensure equilibrium conditions and α1-adrenoceptor isolation.[2]
Human lower urinary tract tissues (LUT) were obtained from patients undergoing transurethral resection of the prostate (TURP). Samples were kept in cold Krebs' solution until use in functional studies, which were performed later the same day or early the following day. Human renal artery sections (3 mm×6 mm) were obtained from a transplant donor bank, and were usually received within 24–36 h of removal. Rabbit bladder neck strips (2 mm×6 mm) were from male New Zealand White rabbits (2.5–3.5 kg) and rat aortic rings (2 mm width) were from male Sprague-Dawley rats (350–500 g), both euthanized with carbon dioxide. Vascular tissues were endothelium-denuded prior to study.[2]
Tissues were mounted under 0.5 g (human LUT, renal artery) or 1.0 g (rabbit bladder neck, rat aorta) resting tension and allowed to equilibrate for at least 1 h. Cumulative concentration-response curves to NA were constructed in the absence and presence (following a 1 h equilibration) of various concentrations of antagonist. Responses were measured as changes in isometric tension. Iterative curve fitting was used to determine EC50 values for the agonist in the absence and presence of various concentrations of each antagonist. Schild plots (Arunlakshana & Schild, 1959) were constructed in order to estimate antagonist affinity estimates.

Animal/Disease Models: Sprague Dawley rat (300-390 g) [1]
Doses: 0.01-0.1 mg/kg
Route of Administration: intravenous (iv) (iv)injection
Experimental Results: Caused a decrease in baseline urethral pressure up to 23%.

[1]. The role of alpha(1)-adrenoceptors and 5-HT(1A) receptors in the control of the micturition reflex in male anaesthetized rats. Br J Pharmacol. 2001 May;133(1):61-72.

[2]. In vitro alpha1-adrenoceptor pharmacology of Ro 70-0004 and RS-100329, novel alpha1A-adrenoceptor selective antagonists. Br J Pharmacol. 1999;127(1):252-258.

It has been hypothesized that in patients with benign prostatic hyperplasia, selective antagonism of the alpha1A-adrenoceptor-mediated contraction of lower urinary tract tissues may, via a selective relief of outlet obstruction, lead to an improvement in symptoms. The present study describes the alpha1-adrenoceptor (alpha1-AR) subtype selectivities of two novel alpha1-AR antagonists, Ro 70-0004 (aka RS-100975) and a structurally-related compound RS-100329, and compares them with those of prazosin and tamsulosin. Radioligand binding and second-messenger studies in intact CHO-K1 cells expressing human cloned alpha1A-, alpha1B- and alpha1D-AR showed nanomolar affinity and significant alpha1A-AR subtype selectivity for both Ro 70-0004 (pKi 8.9: 60 and 50 fold selectivity) and RS-100329 (pKi 9.6: 126 and 50 fold selectivity) over the alpha1B- and alpha1D-AR subtypes respectively. In contrast, prazosin and tamsulosin showed little subtype selectivity. Noradrenaline-induced contractions of human lower urinary tract (LUT) tissues or rabbit bladder neck were competitively antagonized by Ro 70-0004 (pA2 8.8 and 8.9), RS-100329 (pA2 9.2 and 9.2), tamsulosin (pA2 10.4 and 9.8) and prazosin (pA2 8.7 and 8.3 respectively). Affinity estimates for tamsulosin and prazosin in antagonizing alpha1-AR-mediated contractions of human renal artery (HRA) and rat aorta (RA) were similar to those observed in LUT tissues, whereas Ro 70-0004 and RS-100329 were approximately 100 fold less potent (pA2 values of 6.8/6.8 and 7.3/7.9 in HRA/RA respectively). The alpha1A-AR subtype selectivity of Ro 70-0004 and RS-100329, demonstrated in both cloned and native systems, should allow for an evaluation of the clinical utility of a 'uroselective' agent for the treatment of symptoms associated with benign prostatic hyperplasia.[2]

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The fact that both RS-100329 and BMY 7378 have a similar potency in lowering arterial pressure suggests that the maintenance of α1-adrenoceptor-mediated sympathetic vasoconstrictor tone is mediated by both α1A- and α1D-adrenoceptors and a comparison of the potency differences between the antagonists at these receptors (see above) suggests that α1D-adrenoceptors are predominant in this effect. In this respect α1D-adrenoceptors have been shown to be involved in the contraction of rat aorta, mesenteric and pulmonary artery (Hussain & Marshall, 1997; Williams et al., 1999), while α1A-adrenoceptors have been shown to mediate contraction of the rat caudal artery (Lachnit et al., 1997). However, the failure of RS-513815 to affect arterial pressure indicates that α1B-adrenoceptors, at least in the present model of urethane anaesthetized rats, play no role in the maintenance of baseline arterial blood pressure. Interestingly, at high doses, RS-100329 caused a profound fall in arterial blood pressure and this is greater than that observed for high doses of doxazosin, implying that RS-100329 is interfering with a mechanism other than α1-adrenoceptors that is also important in the maintenance of arterial blood pressure. In addition, and somewhat surprisingly, 8-OH-DPAT failed to cause a fall in arterial blood pressure in this model. However, in the rat, activation of central 5-HT1A receptors can evoke both sympathoinhibition (Gradin et al., 1985; Fozard et al., 1987) and excitation (Anderson et al., 1995; 1996). [1]

C20H26CLF3N4O3

462.9

462.164

C, 51.89; H, 5.66; Cl, 7.66; F, 12.31; N, 12.10; O, 10.37

1215654-26-4

11340200

White to off-white solid powder

2

8

7

31

648

0

Cl.FC(COC1C=CC=CC=1N1CCN(CCCN2C(NC=C(C)C2=O)=O)CC1)(F)F

CWVABCXVOAVUJL-UHFFFAOYSA-N

InChI=1S/C20H25F3N4O3.ClH/c1-15-13-24-19(29)27(18(15)28)8-4-7-25-9-11-26(12-10-25)16-5-2-3-6-17(16)30-14-20(21,22)23;/h2-3,5-6,13H,4,7-12,14H2,1H3,(H,24,29);1H

5-methyl-3-[3-[4-[2-(2,2,2-trifluoroethoxy)phenyl]piperazin-1-yl]propyl]-1H-pyrimidine-2,4-dione;hydrochloride

RS100329; RS 100329; RS 100329 hydrochloride; 1215654-26-4; 232953-52-5; RS 100329 HCl; RS100329 (hydrochloride); 5-METHYL-3-[3-[3-[4-[2-(2,2,2-TRIFLUOROETHOXY)PHENYL]-1-PIPERAZINYL]PROPYL]-2,4-(1H,3H)-PYRIMIDINEDIONE] HYDROCHLORIDE; 5-methyl-3-[3-[4-[2-(2,2,2-trifluoroethoxy)phenyl]piperazin-1-yl]propyl]-1H-pyrimidine-2,4-dione;hydrochloride; 5-Methyl-3-[3-[3-[4-[2-(2,2,2,-trifluroethoxy)phenyl]-1-piperazinyl]propyl]-2,4-(1H,3H)-pyrimidinedionehydrochloride; RS-100329

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~31.25 mg/mL (~67.51 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)