D₃ Receptor ( Ki = 0.71 nM ); D₂ Receptor ( Ki = 13.5 nM ); D₄ Receptor ( Ki = 3.9-15 nM ); D₅ Receptor ( Ki = 5.4 nM ); D₁ Receptor ( Ki = 83 nM nM ); 5-HT_1A Receptor ( Ki = 30 nM ); 5-HT_2B Receptor ( Ki = 27 nM ); Alpha-2B adrenergic receptor
Dopamine D1 receptor (pKi using [³H]rotigotine = 9.2 ± 0.1; pKi using [³H]SCH23390 = 6.6 ± 0.1; pEC50 = 9.6 ± 0.1). [1]
Dopamine D2 receptor (pKi using [³H]rotigotine = 7.8 ± 0.2; pKi using [³H]raclopride = 7.4 ± 0.1; pEC50 = 10.4 ± 0.1). [1]
Dopamine D3 receptor (pKi using [³H]rotigotine = 9.9 ± 0.1; pKi using [³H]spiperone = 10.0 ± 0.2; pEC50 = 8.2 ± 0.2). [1]
Dopamine D4 receptor (pKi using [³H]rotigotine = 8.5 ± 0.1; pKi using [³H]spiperone = 7.7 ± 0.5; pEC50 = 7.7 ± 0.1; Emax = 62 ± 2%). [1]
Dopamine D5 receptor (pKi using [³H]rotigotine = 6.5 ± 1.85; pKi using [³H]SCH23390 = 1.08 ± 0.44; pEC50 = 7.7 ± 0.2; Emax = 64 ± 5). [1]
In vivo, rotigotine acts as a potent and effective agonist of dopamine D1 and D2 receptors. [2]

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rotigotine can be used as an agonist radioligand, [³H]rotigotine, to label all dopamine receptor subtypes. At the dopamine D1 and D2 receptors, the saturation binding of [³H]rotigotine was best described by a two-site model. For the D1 receptor, the high-affinity Kd was 0.19 ± 0.13 nM (Bmax 3440 ± 420 fmol·mg⁻¹ protein) and low-affinity Kd was 6.80 ± 3.23 nM (Bmax 2970 ± 4000 fmol·mg⁻¹ protein). For the D2 receptor, the high-affinity Kd was 0.09 ± 0.02 nM (Bmax 6390 ± 3600 fmol·mg⁻¹ protein) and low-affinity Kd was 4.57 ± 4.46 nM (Bmax 20530 ± 4600 fmol·mg⁻¹ protein). The binding to D3, D4, and D5 receptors was best described by a one-site model, with Kd values of 0.21 ± 0.06 nM (Bmax 22700 ± 5600 fmol·mg⁻¹ protein), 0.83 ± 0.06 nM (Bmax 1550 ± 640 fmol·mg⁻¹ protein), and 6.50 ± 1.85 nM (Bmax 1300 ± 290 fmol·mg⁻¹ protein), respectively. The presence of GppNHp (10 µM) attenuated the high-affinity [³H]rotigotine binding site at the D1 receptor but had no significant effect on D2 and D3 receptor binding. [1]
In competition binding studies, rotigotine displaced [³H]rotigotine binding at human dopamine receptors with pKi values of 10.2 ± 0.2 (D2), 9.9 ± 0.1 (D3), 9.2 ± 0.1 (D1), 8.5 ± 0.1 (D4), and 6.5 ± 1.85 (D5). [1]
In functional studies using cellular dielectric spectroscopy (CDS), rotigotine acted as a potent agonist at dopamine D1, D2, and D3 receptors, producing maximal responses similar to dopamine. The pEC50 values were 9.6 ± 0.1 (D1), 10.4 ± 0.1 (D2), and 8.2 ± 0.2 (D3). At the D4 receptor, rotigotine acted as a partial agonist (Emax 62 ± 2% compared to dopamine) with a pEC50 of 7.7 ± 0.1. At the D5 receptor, it also acted as a partial agonist (Emax 64 ± 5%) with a pEC50 of 7.7 ± 0.2. [1]
In primary mesencephalic cell culture, treatment with rotigotine alone (0.01, 0.1, 1 µM for 8 days) did not affect the number of tyrosine hydroxylase immunoreactive (THir) neurons compared to untreated controls. However, at a concentration of 10 µM, it significantly decreased the number of THir neurons by 40%. [4]
Co-treatment with rotigotine (0.01 µM) rescued 10% of THir neurons from MPP⁺ (10 µM, 48h)-induced cell death, although this effect was not statistically significant. [4]
Co-treatment with rotigotine (0.01 µM) significantly rescued 20% of THir neurons from rotenone (20 nM, 48h)-induced cell death. Furthermore, rotigotine (0.01 µM) significantly attenuated reactive oxygen species (ROS) production induced by rotenone, reducing it by 17% compared to rotenone-treated cultures. [4]

In vitro activity: Rotigotine exhibits a 100-fold selectivity when compared to D1 receptors (pKi=7.2) and a 10-fold selectivity for D3 (pKi=9.2) receptors when compared with D2, D4, and D5 (pKi=8.5-8.0). Rotigotine exhibits full agonistic behavior at all dopamine receptors in functional studies; however, it is noteworthy that the potency of D1 receptor stimulation is comparable to that of D2 and D3 receptors (pEC50: 9.0, 9.4-8.6, 9.7)[1]. In primary mesencephalic cell culture, rotigotine (10 µM) reduces the number of THir neurons by forty percent. Rotigotine (0.01 µM) significantly inhibits rotenone-induced ROS production, significantly protects dopaminergic neurons against MPP+ toxicity, and mildly protects dopaminergic neurons against rotenone-induced cell death[4].

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In unilaterally 6-OHDA-lesioned rats primed with apomorphine, rotigotine (0.035, 0.1, and 0.35 mg/kg, s.c.) induced contralateral turning behavior in a dose-dependent manner. The dopamine D1 receptor antagonist SCH 39166 (0.1 mg/kg, s.c.) significantly reduced the contralateral turning induced by 0.1 mg/kg of rotigotine. The D2 receptor antagonist eticlopride (0.1 mg/kg, s.c.) reduced turning behavior induced by 0.1 mg/kg rotigotine. In drug-naive rats, SCH 39166 also reduced contralateral turning induced by 0.35 mg/kg rotigotine. Priming with apomorphine significantly enhanced the turning behavior induced by 0.35 mg/kg rotigotine compared to drug-naive rats. [2]
In apomorphine-primed, 6-OHDA-lesioned rats, rotigotine (0.035, 0.1, and 0.35 mg/kg, s.c.) increased the number of Fos-positive nuclei in the lesioned striatum in a dose-dependent manner. This effect was fully counteracted by pre-treatment with the D1 antagonist SCH 39166 (0.1 mg/kg, s.c.). No difference in the number of Fos-positive nuclei was observed between primed and drug-naive rats at the 0.35 mg/kg dose of rotigotine. [2]

Rotigotine (N-0437; N-0923; 0.035, 0.1, and 0.35 mg/kg) dose-dependently produces contralateral turning behavior in primed rats. Compared to primed rats, drug-naive rats exhibit less turning behavior when Rotigotine, either by itself or in conjunction with SCH 39166, is administered.

In 96-well polypropylene tubes, binding assays are carried out with a final volume of 2 mL for D1 and D4 membranes and 1 mL for D2, D3, and D5 membranes. These tubes contain the following materials: 50 μL radioligand, 10 μL drug/buffer/non-specific binding, buffer (final concentration 50 mM Tris-HCl pH 7.4, MgCl2 2 mM), and membranes (5 μg protein for D2 and D3 and 25 μg protein for D1 and D5). Rapid vacuum filtration through A/C glass fiber filters presoaked in 0.1% polyethylenimine is used to determine bound radioligand after 120 minutes of incubation at 25°C. Liquid scintillation counting is used to determine the retained radioactivity after the filters are four times cleaned with 2 mL of ice-cold ishing buffer (Tris-HCl 50 mM, pH 7.4 at 4°C).

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For radioligand binding assays, membranes from cells expressing human dopamine receptors were incubated in a buffer (50 mM Tris-HCl pH 7.4, 2 mM MgCl₂) with radioligand and test compounds. The incubation lasted 120 minutes at 25°C. Bound radioligand was determined by rapid vacuum filtration through presoaked glass fibre filters, followed by four washes with ice-cold washing buffer (50 mM Tris-HCl pH 7.4). Retained radioactivity was determined by liquid scintillation counting. For saturation studies, the concentration of [³H]rotigotine used was typically 0.02 - 5 nM. For competition and kinetic studies, [³H]rotigotine was used at 0.8 nM for D1 and 0.2 nM for D2, D3, D4, and D5 receptors. [1]
Kinetic binding studies of [³H]rotigotine were performed to determine association and dissociation rates. Dissociation was induced by an excess (10 µM) of chlorpromazine for D2, D3, and D4 receptors or apomorphine for D1 and D5 receptors, followed at different times up to 180 minutes. The association of [³H]rotigotine with D2, D4, and D5 receptors was best described by a two-phase model, while D1 and D3 receptor association data were best described by a one-phase model. Dissociation data at all receptors were best described by a one-phase model. [1]
For the [³⁵S]GTPγS binding assay at the D4 receptor, membranes (20 µg per assay) were incubated with Tris-MgCl₂ buffer containing 50 mM NaCl, 3 mM MgCl₂, 1 µM GDP, and 10 µg·mL⁻¹ saponin for 15 minutes at 25°C. Then, [³⁵S]GTPγS (0.15 - 0.20 nM) was added and incubated for 60 minutes at 25°C. The plates were then filtered, dried, and retained radioactivity was determined. [1]

Cellular dielectric spectroscopy (CDS) measurements were performed to assess functional activity. Cells (LMtk D1, CHO D2, CHO D3) were seeded into 96-well microplates and incubated overnight. The growth medium was then exchanged for an incubation buffer (Hank's balanced salt solution containing 20 mM HEPES, pH 7.4). Baseline measurements were taken for 5 minutes, followed by the addition of varying concentrations of test agonist (ranging from 0.1 pM to 10 µM). Impedance measurements were then collected for 30 minutes. The optimal cell density was 50,000 cells/well for LMtk D1, 30,000 cells/well for CHO D2, and 35,000 cells/well for CHO D3 cells. [1]
For intracellular calcium measurements at the D5 receptor, cells stably expressing the human D5 receptor and G-protein Gα₁₅ were pre-incubated for 60 minutes with 4 µM Fluo-4 AM and pluronic acid in the presence of probenecid (0.8 mM final). Cells (20,000 per well) were washed and incubated in HBSS with 20 mM HEPES (pH 7.4). After basal readings, drugs were added and changes in intracellular calcium were determined. [1]
Primary mesencephalic cell cultures were prepared from embryonic mouse mesencephalon at gestation day 14. Cells were plated into multi-dishes pre-coated with poly-D-lysine and grown at 37°C in 5% CO₂. The medium was exchanged on day 1 and day 3 in vitro (DIV). On DIV 5, half the medium was replaced with serum-free medium containing B-27. From DIV 6 onwards, serum-free supplemented medium was used and replaced every second day. To identify dopaminergic neurons, cultures were fixed with 4% paraformaldehyde, permeabilized with 0.4% Triton X-100, and blocked with 5% horse serum. Cells were then incubated with anti-TH primary antibody overnight at 4°C, followed by a biotinylated secondary antibody and an avidin-biotin-horseradish peroxidase complex. The reaction was developed with diaminobenzidine and H₂O₂, and stained cells were counted. [4]
For ROS measurement, cultures were incubated with 5 µM of the fluorescence indicator CM-H₂DCFDA for 30 minutes. After rinsing, cultures were photographed under an epifluorescence microscope. The average fluorescence density in individual cell bodies was determined using software. [4]

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For the 6-OHDA lesion model, male Sprague-Dawley rats (275-300 g) were anesthetized with Equitesin (i.p.) and secured in a stereotaxic frame. 6-OHDA (8 µg/4 µl in 0.05% ascorbic acid in saline) was injected unilaterally into the left medial forebrain bundle at coordinates A.P. = -2.2, M.L. = +1.5, D.V. = -7.8. Rats were pretreated with 10 mg/kg i.p. of desipramine to prevent damage to noradrenergic neurons. The extent of the lesion was assessed using a cylinder test and spontaneous ipsilateral turning behavior. Only rats with >80% striatal dopamine depletion were used. For priming, rats were injected with apomorphine (0.5 mg/kg s.c.) two weeks after the lesion, and those showing less than 150 contralateral rotations in 1 hour were excluded. [2]
For turning behavior studies, rotigotine (0.035, 0.1, 0.35 mg/kg s.c.), the D1 antagonist SCH 39166 (0.1 mg/kg s.c.), and the D2 antagonist eticlopride (0.1 mg/kg s.c.) were dissolved in saline and injected s.c. in a volume of 0.1 ml/100 g body weight. For antagonist studies, SCH 39166 was administered 30 minutes before rotigotine, and eticlopride was administered 30 minutes before rotigotine. Rats were fitted with a harness fastened to an automated rotameter, and contralateral turns were recorded for 2 hours (for rotigotine) or 2.5 hours (for pramipexole) after agonist injection. [2]
For immunohistochemistry, rats were deeply anesthetized and transcardially perfused with ice-cold saline followed by ice-cold 4% paraformaldehyde. Brains were postfixed, and 40 µm thick coronal sections were cut using a vibratome. Free-floating sections were incubated with primary antibodies against Fos (1:2000) and TH (1:1000). The reaction was visualized using biotinylated secondary antibodies and the standard avidin-biotin-peroxidase method with DAB as the chromogen. Fos immunoreactivity was quantified as counts/mm² of positive nuclei in the dorso-lateral striatum. [2]

Two weeks following the 6-OHDA lesions, rats receive a 0.5 mg/kg s.c. apomorphine priming. Rats that perform fewer than 150 contralateral rotations in the course of the one-hour testing session are not included in the research. After priming, rats are split into three experimental groups and given varying dosages of dopamine receptor agonists (pramipexole or rotigotine), either by themselves or in conjunction with dopamine D1 (SCH 39166) or D2 (eticlopride) receptor antagonists, as previously reported: saline+Rotigotine (0.035 mg/kg s.c., n=9; 0.1 mg/kg s.c., n=9; 0.35 mg/kg s.c., n=8); SCH 39166 (0.1 mg/kg s.c.)+Rotigotine (0.035 mg/kg s.c., n=5; 0.1 mg/kg s.c., n=7; 0.35 mg/kg s.c., n=5); eticlopride (0.1 mg/kg s.c.) + Rotigotine (0.1 mg/kg s.c., n=5; 0.35 mg/kg s.c., n=5); Saline+pramipexole (0.035 mg/kg s.c., n=5; 0.1 mg/kg s.c., n=12; 0.35 mg/kg s.c., n=7); SCH 39166 (0.1 mg/kg s.c.)+pramipexole (0.035 mg/kg s.c., n=5; 0.1 mg/kg s.c., n=6; 0.35 mg/kg s.c., n=6); eticlopride (0.1 mg/kg s.c.)+pramipexole (0.1 mg/kg s.c., n=7; 0.35 mg/kg s.c., n=5).

Rats

In primary mesencephalic cell culture, treatment with an unphysiologically high concentration of rotigotine (10 µM) for 8 consecutive days significantly decreased the number of THir neurons by 40% compared to untreated controls. No toxicity was observed at lower concentrations (0.01, 0.1, 1 µM). [4]

[1]. Rotigotine is a potent agonist at dopamine D1 receptors as well as at dopamine D2 and D3 receptors. Br J Pharmacol. 2015 Feb;172(4):1124-35.

[2]. The in vitro receptor profile of rotigotine: a new agent for the treatment of Parkinson's disease. Naunyn Schmiedebergs Arch Pharmacol. 2009 Jan;379(1):73-86.

[3]. In vivo dopamine agonist properties of rotigotine: Role of D1 and D2 receptors. Eur J Pharmacol. 2016 Oct 5;788:183-91.

[4]. Neuroprotective effect of rotigotine against complex I inhibitors, MPP+ and rotenone, in primary mesencephalic cell culture. Folia Neuropathol. 2014;52(2):179-86.

rotigotine is a non-ergoline dopamine receptor agonist used for the treatment of idiopathic Parkinson's disease and restless legs syndrome. It is administered via a 24-hour transdermal delivery system (patch) to provide continuous dopaminergic stimulation and reduce motor fluctuations. [1][4]
Unlike conventional D2 agonists like ropinirole and pramipexole, which lack activity at D1 and D5 receptors, rotigotine is a potent agonist at all dopamine receptors, resembling the profile of apomorphine. Apomorphine has greater efficacy in Parkinson's disease than other dopamine agonists but has suboptimal pharmacokinetic properties. [1]
In a rat model of Parkinson's disease, rotigotine demonstrated D1 agonist properties in vivo, as its effects were counteracted by a selective D1 antagonist. This makes it distinct from pramipexole, which acts as a pure D2/D3 agonist. The full D1 agonist nature of rotigotine might be clinically relevant, as full D1 receptor agonists have been shown to be more effective antiparkinsonian agents than partial agonists in primate models. This property makes it similar to apomorphine and levodopa. [2]
rotigotine provided minor protection against MPP⁺ and significant protection against rotenone-induced cell death in primary mesencephalic cell cultures, which is attributed to a significant suppression of rotenone-induced reactive oxygen species (ROS) production. [4]

C19H26CLNOS

351.93

351.142

102120-99-0

(Rac)-Rotigotine-d3 hydrochloride; 1215846-20-0; (Rac)-Rotigotine; 92206-54-7; (Rac)-Rotigotine-d7 hydrochloride

6917969

Solid powder

5.067

2

3

6

23

337

0

CEXBONHIOKGWNU-UHFFFAOYSA-N

InChI=1S/C19H25NOS.ClH/c1-2-11-20(12-10-17-6-4-13-22-17)16-8-9-18-15(14-16)5-3-7-19(18)21;/h3-7,13,16,21H,2,8-12,14H2,1H3;1H

6-[propyl(2-thiophen-2-ylethyl)amino]-5,6,7,8-tetrahydronaphthalen-1-ol;hydrochloride

N 0923; N-0923; N-0924 Rotigotine Hydrochloride; Rotigotine HCl Neupro

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: ≥ 50 mg/mL (~142.1 mM)
H2O: < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.10 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.10 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.10 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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