nAChR

The results of the binding and in vitro dopamine (DA) release experiments with 1, 2, 5, and 7 are presented in Table 1. The data indicate that all the compounds tested exhibited a high degree of selectivity for the [3H]Nic (nicotine) binding site compared with the [3H]QNB (muscarinic) binding site in rat cortical membranes. Differences were observed, however, in the ability of the compounds to release DA from rat striatal slices. Whereas 2 tended toward a greater efficacy than 1 in this bioassay, 7 was significantly less active. Furthermore, the data demonstrate that SIB-1508Y/(S)-2 was the enantiomer exhibiting the highest affinity in the binding assay and was primarily responsible for the DA release stimulated by 2, an effect which is blocked by the NAChR antagonists mecamylamine and dihydro-β-erythroidine. The results illustrate the utility of a functional assay for the differentiation of two closely related analogues (2 and 7) and led to the decision to more fully evaluate 2 and (S)-2. [1]

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Electrophysiological recording of current responses in Xenopus oocytes expressing recombinant human NAChRs is a sensitive assay for detecting the activity of agonists or antagonists. Thus voltage clamp recordings of oocytes expressing human α2β2, α2β4, α3β2, α3β4, α4β2, α4β4, or α7 NAChR subtypes were obtained (Figure 1). The inward current elicited by SIB-1508Y/2 was normalized to the response produced by a prior application of acetylcholine (ACh; 10 μM) to the same cell. At a concentration of 10 μM, 2 produced currents that ranged between 20% and 50% of the response elicited by ACh (10 μM) in oocytes expressing the α2β2, α2β4, α3β2, α4β2, and α4β4 NAChR subtypes. No detectable inward currents were observed at the human α7 receptor subtype,25 and only a minimal response at the α3β4 subtype was detected. This contrasts with 1 (data not shown) which is a potent agonist at both the α3β4 and α7 receptor subtypes in this assay. [1]

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A functional assay employing cell lines stably expressing human recombinant NAChR subtypes provided further data comparing 1, 2 SIB-1508Y/(S)-2, and (R)-2. In this assay, the activation of recombinant receptors by agonists stimulates calcium entry into cells. The subsequent elevation of cytosolic Ca2+ concentration ([Ca2+]i) is determined by measuring the changes in fluorescence of the Ca2+-sensitive dye 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N‘,N‘-tetraacetic acid (fluo-3).30 The compounds were tested in cell lines expressing recombinant human α2β4, α3β4, α4β4, and α4β2 NAChR subtypes and a cell line expressing the fetal form (α1β1γδ) of human neuromuscular NAChRs (Tables 2 and 3). In each cell line 1, 2, and (S)-2 elicited increases in [Ca2+]i, and these effects were blocked by the NAChR antagonists d-tubocurarine and mecamylamine (data not shown). While (S)-2 showed its greatest potency at the α4β2 NAChR subtype (Table 2), (R)-2 exhibited low activity in the β4-containing cell lines and was inactive in the cell line expressing the α4β2 subtype. Furthermore, at a maximally efficacious concentration, SIB-1508Y/(S)-2 was 26−52% as efficacious as 1 in each cell line, whereas (R)-2 displayed weak efficacy (Table 3). The data establish that (S)-2 is the more active enantiomer which is in agreement with the binding and DA release results (Table 1) [1].

Since 2 stimulated the release of DA from rat striatal slices, it was evaluated in an animal model of Parkinson's disease. The effect of 2 on ipsilateral rotations of rats with unilateral nigrostriatal 6-hydroxydopamine (6-OHDA) lesions32 (rat-turning model) is shown in Table 4. At a dose of 25 mg/kg given subcutaneously (sc), 2 produced a significant increase in ipsilateral rotations compared with saline-treated animals, an effect which is blocked by mecamylamine. Futhermore, 2 was more efficacious than 1 at the maximum effective dose withminimal adverse side effects. These studies led to the preclinical development of SIB-1508Y/(S)-2 for the treatment of PD. [1]

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The potential antiparkinsonian effects of the centrally acting, subtype-selective neuronal nicotinic acetylcholine receptor agonist (S)-(-)-5-ethynyl-3-(1-methyl-2-pyrrolidinyl)-pyridine (SIB-1508Y) was assessed on motor symptoms and disability scale ratings in three monkeys previously made parkinsonian by chronic exposure to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Compared with levodopa (L-dopa), SIB-1508Y exerted only mild antiparkinsonian effects when administered alone. Emetic effects of this drug interfered with potential therapeutic effects at higher doses. However, when a low, ineffective dose of SIB-1508Y was combined with low, ineffective doses of L-dopa, a significant clinical effect was observed. These data suggest that subtype-selective nicotinic acetylcholine receptor agonists may hold promise as antiparkinsonian agents, and when administered in combination with L-dopa may allow a reduction in the dose of L-dopa needed to achieve a significant clinical effect [2].

Xenopus oocytes. [1]
Preparation and Electrophysiology Xenopus laevis frogs were anaesthetized by immersion in a 0.15% tricaine methane-sulfonate solution and the ovaries were surgically removed. Oocytes were injected with 10-50 nL containing 10-100 ng of mRNA encoding a, + x in a 1:1 ratio (50 ng for a7). Following injection, oocytes were incubated at 16-19 OC in a solution containing (in mM): NaCl (77.5), KCl (2), CaCl 2 (1.8), MgCl 2 (1), HEPES (5), Napyruvate (5); pH 7.3, adjusted with NaOH, and supplemented with 100 U/mL of penicillin and 100 ptg/mL of streptomycin. Oocytes were examined for functional expression two to five days following mRNA injection using the two-electrode voltage clamp technique (holding membrane potential: -80 mV). The recording solution contains (in mM): NaCl (115), KCl (2.5), CaC12 (1.8), HEPES (10), atropine (0.001), pH 7.3. Recording electrodes were filled with 3 M KCl. Perfusion solutions were gravity fed into the recording chamber (capacity: 100 pL) at a rate of about 6-10 ml/min. All recordings were performed at room temperature (19-23 OC). Data were amplified, digitized (100-500 Hz) and filtered (at 40-200 Hz). Sham injected oocytes (50 nL distilled water) or oocytes injected with either ax or Px mRNA (except for a7) did not display any detectable currents (<1 nA) when challenged with standard NAChR agonists (nicotine or ACh, 100-300 iiM).

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Measurement of Cytosolic Ca2+ Concentration by the Plate-based Fluo-3 Assay. [1]
Cells stably transfected with expression plasmids encoding human NAChR subunits were plated at 2 x 105 cells/well on a 96-well poly-L-lysine coated microtiter plate. Twenty four hours later, the cells were washed with 200 pIL of HEPES buffered saline (HBS: 125 mM NaCl, 5 mM KC1, 0.62 mM MgCl 2, 1.8 mM CaCl2 , 20 mM glucose, 20 mM Hepes, pH 7.4) and incubated with 30 [iL of 20 mM fluo-3-acetoxymethyl ester (fluo-3/AM) for 2 h at 20 oC in the dark. The fluo3/AM stock solution was prepared in HBS containing 2% dimethyl sulfoxide (DMSO) and 0.2% pluronic F127. Residual unloaded dye was washed with 200 gL HBS, and subsequently, 180 pL HBS was added to each well. The fluorescence measurements were performed using a plate reading fluorimeter. First, the basal fluorescence (Fb) was determined before drug addition. Next, 20 pL of drug solution was added to each well and the fluorescence was recorded for 60 s at 0.33 s intervals to determine the peak response (Fp). The maximal fluorescence (Fma, was determined after lysing the cells with Triton X-100 (final concentration of 0.20%). To record the minimum fluorescence (Fmin), MnCl 2 was then added to a final concentration of 10 mM. All fluorescence determinations were performed in quadruplicate. The peak and basal cytosolic Ca concentrations [Ca ]i were calculated according to the method of Daggett et al.2

6-Hydroxydopamine (6-OHDA) Rotation Model Animals. [1]
Unilaterally 6-OHDA lesioned rats (male Sprague-Dawley rats) weighing between 250 to 350 g at the start of the experiment were used for these studies. The animals were housed three per cage and maintained in a humidity and temperature (22 oC) controlled facility on a 12 h: 12 h light/dark cycle (lights on at 6:00 a.m.) with free access to food (Harlan-Teklad 4% rat diet 7001) and water. After arrival at SIBIA, each rat was allowed a 2-week period for habituation to the animal room. For all treatment groups 8 rats were tested.

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Compounds. [1]
(-)-Nicotine hydrogen tartrate was used. Fumarate 2/SIB-1508Y was synthesized as described above. Nicotine (1) and SIB-1508Y fumarate were dissolved in saline and the pH was adjusted to 7.0 by addition of 1OM NaOH. Doses of drugs are expressed in terms of their free amine concentrations. All compounds were administered subcutaneously into the dorsal neck region in a volume of 1 ml/kg body weight. Saline was used as control.

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Lesioning. [1]
The ascending nigrostriatal DA pathway was lesioned by unilateral stereotaxic injection of 6-OHDA hydrobromide (16 [tg/ L administered at a rate of 0.34 ul/min for 6 min) slightly anterior and to the left substantia nigra. All injections of 6-OHDA were preceded (30 min) by desipramine (25 mg/kg, i.p.) and pargyline (75 mg/kg, i.p.) in order to protect noradrenergic neurons from being destroyed. The rats were lesioned.

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Behavioral Testing. [1]
After 7-10 days of recovery from the surgery, the effectiveness of the lesion was verified by determining the response of the animals to apomorphine (0.2 mg/kg, sc). Only rats with a minimum response of 100 contralateral turns/30 min were used further in this study. Two weeks later, the selected rats were tested with the drugs using an automated rotometer system to record number and direction of rotations. In order to distinguish spontaneous rotations (non-specific) from induced rotations (specific to the effect of the drug), each rat served as its own control.
The procedure was as follows: the rats were placed in the rotometer system (bowl: 45.5 cm internal diameter) for a 15-min acclimation period, after which they were administered the vehicle subcutaneously and were monitored for one hour. They then received the test compound subcutaneously and were monitored for an additional two hours. The rats were first administered with amphetamine (1 mg/kg free amine) then SIB-1508Y/2 fumarate and finally (-) nicotine. Washout periods of 4 days and 9 days separated the injection between amphetamine/2 fumarate and 2 fumarate/(-) nicotine, respectively. Six animals were tested at one time. Testing was carried out between 9:00 a.m. and 4:00 p.m. each day (light cycle).

[1]. (S)-(-)-5-ethynyl-3-(1-methyl-2-pyrrolidinyl)pyridine maleate (SIB-1508Y): a novel anti-parkinsonian agent with selectivity for neuronal nicotinic acetylcholine receptors. J Med Chem. 1996 Aug 16;39(17):3235-7.

[2]. Effects of SIB-1508Y, a novel neuronal nicotinic acetylcholine receptor agonist, on motor behavior in parkinsonian monkeys. Mov Disord. 1998 Jul;13(4):637-42.

In conclusion, the novel NAChR agonist SIB-1508Y/(S)-2 has been synthesized and evaluated in a range of in vitro and in vivo assays. Significantly, the differentiation of (S)-2 from structurally related agonists with quite similar binding affinities for endogenous NAChRs was accomplished on the basis of results derived from functional assays, including a novel functional assay employing cell lines stably expressing recombinant human NAChR subtypes. This assay provides a powerful method for the discovery of subtype selective NAChR agonists and antagonists, acting either at the ACh binding site or at novel allosteric sites on the receptor complex [1].

C16H18N2O4

302.33

292.142

C, 63.56; H, 6.00; N, 9.27; O, 21.17

192231-16-6

192231-16-6; 179120-92-4

11954277

Typically exists as solid at room temperature

152.5-153.5 °C

2

6

4

22

359

1

C(O)(=O)/C=C/C(O)=O.C(C1=CC(C2CCCN2C)=CN=C1)#C

BCPPKHPWLRPWBJ-OURKGEEVSA-N

InChI=1S/C12H14N2.C4H4O4/c1-3-10-7-11(9-13-8-10)12-5-4-6-14(12)2;5-3(6)1-2-4(7)8/h1,7-9,12H,4-6H2,2H3;1-2H,(H,5,6)(H,7,8)/b;2-1-/t12-;/m0./s1

(Z)-but-2-enedioic acid;3-ethynyl-5-[(2S)-1-methylpyrrolidin-2-yl]pyridine

Altinicline maleate; 192231-16-6; SIB-1508Y; Altinicline maleate [USAN]; UNII-PCL7Q9Q62Q; 5-ethynyl-3-(1-methyl-2-pyrrolidinyl)pyridine maleate; PCL7Q9Q62Q; Altinicline maleate (USAN);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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.)