Dopamine D1 receptor (K0.5 = 4 nM)

Compute-aided conformational analysis was used to characterize the agonist pharmacophore for D1 dopamine receptor recognition and activation. Dihydrexidine (DHX), a high-affinity full agonist with limited conformational flexibility, served as a structural template that aided in determining a molecular geometry that would be common for other more flexible, biologically active agonists. The intrinsic activity of the drugs at D1 receptors was assessed by their ability to stimulate adenylate cyclase activity in rat striatal homogenates (the accepted measure of D1 receptor activation). In addition, affinity data on 12 agonists including six purported full agonists (dopamine, dihydrexidine, SKF89626, SKF82958, A70108, and A77636), as well as six less efficacious structural analogs, were obtained from D1 dopamine radioreceptor-binding assays. The active analog approach to pharmacophore building was applied as implemented in the SYBYL software package. Conformational analysis and molecular mechanics calculations were used to determine the lowest energy conformation of the active analogs (i.e., full agonists), as well as the conformations of each compound that displayed a common pharmacophoric geometry. It is hypothesized that DHX and other full agonists may share a D1 pharmacophore made up of two hydroxy groups, the nitrogen atom (ca. 7 A from the oxygen of m-hydroxyl) and the accessory ring system characterized by the angle between its plane and that of the catechol ring (except for dopamine and A77636). For all full agonists (DHX, SKF89626, SKF82958, A70108, A77636, and dopamine), the energy difference between the lowest energy conformer and those that displayed a common pharmacophore geometry was relatively small (< 5 kcal/mol). The pharmacophoric conformations of the full agonists were also used to infer the shape of the receptor binding site. Based on the union of the van der Waals density maps of the active analogs, the excluded receptor volume was calculated. Various inactive analogs (partial agonists with D1 K0.5 > 300 nM) subsequently were used to define the receptor essential volume (i.e., sterically intolerable receptor regions). These volumes, together with the pharmacophore results, were integrated into a three-dimensional model estimating the D1 receptor active site topography. [1]

After a training dose of 0.03 mg/kg, SKF-82958 ((±)-SKF 82958) (0.003-0.1 mg/kg; ip) completely replaced SKF-82958, causing a dose-dependent increase in responses on the relevant lever of SKF-82958. Significantly reduced response rates were also obtained by increasing the dose of SKF-82958 [2]. Pilocarpine-induced mandibular movements are considerably inhibited by SKF-82958 (0.5-2.0 mg/kg; intraperitoneal injection) [3].

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We examined the discriminative stimulus effects of the high-efficacy dopamine D(1) receptor agonist (+/-)6-chloro-7, 8-dihydroxy-3-ally1-phenyl-2,3,4,5-tetrahydro-1H-3benzazepine++ + hydrobromide (SKF-82958) in rats trained to discriminate SKF-82958 (0.03 mg/kg) from vehicle in a two-lever food-reinforced drug discrimination task. SKF-82958 produced dose-related increases in responding to the SKF-82958 appropriate lever with full substitution occurring at the training dose. Pretreatment with the dopamine D(1)/D(5) receptor antagonist (-)-trans-6,7,7a,8,9, 13b-hexahydro-3-chloro-2hydroxy-N-methyl-5H-benzo-[d]naphtho -¿2, 1-b¿azepine (SCH-39166) (0.01 mg/kg) attenuated the discriminative stimulus effects of SKF-82958. Pretreatment with the dopamine D(2) receptor antagonist raclopride (0.03 mg/kg) had no effect. The high-efficacy dopamine D(1) receptor agonist R(+)6chloro-7, 8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-81297) fully substituted for SKF-82958, whereas the low-efficacy dopamine D(1) receptor agonist (+/-)1-phenyl-2,3,4, 5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF-38393) produced only partial substitution. The dopamine D(2) receptor agonist trans-(+/-)-4,4a,5,6,7,8,8a, 9-octahydro-5-propyl-1H-propyl-1H-pyrazolo[3,4-g]quinoline dihydrochloride (quinpirole) and the indirect dopamine agonist cocaine did not substitute fully for the SKF-82958 discriminative stimulus cue. These results demonstrate that the high-efficacy dopamine D(1) receptor agonist SKF-82958 can serve as an effective discriminative stimulus in the rat, and that these effects are mediated by a dopamine D(1)-like receptor mechanism. [2]

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SKF 82958 (0.5-2.0 mg/kg IP) reduced the tremulous jaw movements induced by pilocarpine. The suppressive effects of SKF 82958 on jaw movements were dose-dependently reversed by systemic pretreatment with the selective D1 dopamine receptor antagonist SCH 23390 (0.025-0.2 mg/kg IP); SCH 23390 was about 16 times more potent than the D2 antagonist raclopride at reversing the effects of SKF 82958. Intracranial injection of SCH 23390 (0.5-2.0 micrograms/side) into the ventrolateral striatum, the rodent homologue of the human ventral putamen, dose-dependently reversed the reduction of pilocarpine-induced jaw movements produced by SKF 82958. Intracranial injection of SCH 23390 (0.5-2.0 micrograms/side) into the substantia nigra pars reticulata also dose-dependently reversed the reduction by SKF 82958 of pilocarpine-induced jaw movements. Injections of SCH 23390 (2.0 micrograms/side) into control sites dorsal to the striatum or substantia nigra had no effects on the action of SKF 82958. Intranigral (SNr) injections of the GABA-A antagonist bicuculline blocked the suppressive effect of systemically administered SKF 82958 on jaw movement activity. Conclusions: These data suggest that the antiparkinsonian actions of SKF 82958 may be due to stimulation of D1 receptors in the ventrolateral striatum and substantia nigra pars reticulata. In addition, these results indicate that GABA mechanisms in the substantia nigra pars reticulata may be important for the antiparkinsonian effects of D1 agonists. [3]

Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rats [3]
Doses: 0.5-2.0 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Dramatically diminished the number of mandibular tremors caused by 4.0 mg/kg pilocarpine. Drug discrimination training [2]
Procedures that were used to train rats to discriminate injections of SKF-82958 (0.03 mg/kg) from saline were similar to those described by Kosten et al. (1999). Rats were first food restricted and maintained at approximately 85% of their free feeding body weight. During initial training, rats were placed into the operant chambers, where the stimulus light above one of the levers was illuminated to signal the beginning of the session. Lever pressing was shaped progressively until 30 responses [fixed ratio 30 (FR30)] were emitted to obtain one food pellet reinforcer. Once stable responding under the FR30 schedule was achieved on either lever, SKF-82958 discrimination training began. For half of the rats, the left lever was designated as the SKF-82958 appropriate lever and for the other half, the right lever as the SKF-82958 appropriate. On SKF-82958 training days, the drug was administered intraperitoneally (i.p.) and the rats were placed in the operant chamber. The session began 15 min later when the stimulus lights above the levers were illuminated. Every thirtieth response on the SKF-82958-appropriate lever produced a food pellet. On vehicle training days, vehicle was administered i.p. and 15 min later, every 30th response on the vehicle-appropriate lever produced a food pellet. Training sessions ended after 30 min of responding. During training, a double alternation sequence of drug or vehicle injection (SKF, SKF, saline, saline, SKF, etc.) was utilized, with training sessions occurring 5 days per week. Initially, 1.0 mg/kg SKF-82958 was used for the training dose, however, this dose profoundly disrupted lever pressing as shown by rate and response records. The training dose was then lowered to 0.3 mg/kg, where response disruption was also observed. Eventually, the training dose was adjusted to 0.03 mg/kg. At this dose, no overt rate disruption was observed. Discrimination training with this dose of SKF-82958 continued until rats met the following criteria: (1) the first completed FR30 was on the injection-appropriate lever and (2) ≥85% of total responses made were on the injection-appropriate lever for six consecutive sessions.

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Discrimination testing [2]
Once the above criteria were met, substitution tests began. Tests were usually performed twice a week with the discrimination training occurring on the intervening days. If discrimination performance fell below 85% on these training days, testing was not performed, and rats were given further discrimination training. If rats maintained ≥85% condition-appropriate responding on training days, tests were performed on the following day, otherwise at least two training sessions separated each testing session. Test sessions were identical to training sessions with the exception that rats could obtain food reinforcement by completing 30 responses on either lever. Substitution tests were performed with different doses of SKF-82958 (0.003–0.1 mg/kg), SKF-81297 (0.1–1.0 mg/kg), SKF-38393 (0.3–5.6 mg/kg), trans-(±)-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-1H-propyl-1H-pyrazolo[3,4-g]quinoline dihydrochloride (quinpirole) (0.01–0.1 mg/kg) and cocaine (0.3–5.6 mg/kg). In antagonism studies, combinations of different doses of SKF-82958 and the dopamine D1/D5 receptor antagonist SCH-39166 (0.01 mg/kg) or the dopamine D2 receptor antagonist raclopride (0.03 mg/kg) were studied. The order of substitution and combination agonist/antagonist tests was presented non-systematically across rats.

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Drugs and injections [2]
SKF-82958, SKF-81297, SKF-38393, quinpirole, cocaine hydrochloride, and S(−) raclopride l-tartrate, were all purchased from RBI. All compounds were freshly prepared on the day of the experiment in 0.9% NaCl and administered in a volume of 1 ml/kg. To ensure solubility, SKF-82958, SKF-81297, and SKF-38393 were sonicated in a heated water bath for 30 min (1 mg/ml) before dilution. Pretreatment time for each drug was 15 min and drugs were administered via i.p. injection with the exception of SCH-39166 and raclopride. Both SCH-39166 and raclopride were administered subcutaneously with a pretreatment time of 30 min. All doses are expressed as free base.

[1]. Conformational analysis of D1 dopamine receptor agonists: pharmacophore assessment and receptor mapping. J Med Chem. 1996;39(1):285-296.

[2]. The dopamine D(1) receptor agonist SKF-82958 serves as a discriminative stimulus in the rat. Eur J Pharmacol. 2000;388(2):125-131.

[3]. Striatal and nigral D1 mechanisms involved in the antiparkinsonian effects of SKF-82958 (APB): studies of tremulous jaw movements in rats. Psychopharmacology (Berl). 1999;143(1):72-81.

9-chloro-5-phenyl-3-prop-2-enyl-1,2,4,5-tetrahydro-3-benzazepine-7,8-diol is a benzazepine.

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The present study demonstrates that the high-efficacy dopamine D1 receptor agonist SKF-82958 can serve as an effective discriminative stimulus in the rat. Pretreatment with SCH-39166, a selective dopamine D1/D5 receptor antagonist, attenuated the discriminative stimulus effects of SKF-82958. In contrast, pretreatment with the dopamine D2 receptor antagonist raclopride, did not result in appreciable antagonism. The antagonism of the SKF-82958 cue by a dopamine D1 receptor antagonist is consistent with previous studies where the discriminative stimulus effects of the dopamine D1 receptor agonists SKF-38393 and SKF-81297 were selectively attenuated by the dopamine D1 receptor antagonist SCH-23390 Cunningham et al., 1985, Kamien et al., 1987, Arnt, 1988, Reavill et al., 1993. Although not tested in this study, it would have been of interest to ascertain whether the antagonism of SKF-82958 by SCH-39166 was surmountable. Nonetheless, these results demonstrate clearly that the discriminative stimulus effects of SKF-82958 are primarily mediated by an interaction at dopamine D1-like receptors. [1]
Specificity of the SKF-82958 discriminative stimulus effects were further evident from substitution tests performed with the high- and low-efficacy dopamine D1 receptor agonists SKF-81297 and SKF-38393, and the dopamine D2 receptor agonist quinpirole, respectively. SKF-81297 fully substituted for the SKF-82958 cue at an intermediate dose, whereas the low-efficacy dopamine D1 receptor agonist SKF-38393 only partially substituted even up to doses that markedly reduced responding. Furthermore, quinpirole did not reproduce the discriminative stimulus effects of SKF-82958 at the doses tested. Together, this pattern of substitution further confirms the D1-like nature of the SKF-82958 discriminative stimulus. [1]
High-efficacy dopamine D1 receptor agonists are known to stimulate adenylate cyclase in in vitro rat brain preparations Stoof and Kebabian, 1981, Izenwasser and Katz, 1993. SKF-82958 stimulates adenylate cyclase with efficacy similar to that of dopamine O'Boyle et al., 1989, Anderson and Jansen, 1990, Izenwasser and Katz, 1993. In the same studies, SKF-81297 produced similar degrees of adenylate cyclase activation (81–100%) as dopamine and SKF-82958, whereas SKF-38393 stimulated adenylate cyclase to a lesser degree (45–60%). Together with the present findings, these data suggest that when high-efficacy dopamine receptor agonists are used as discriminative stimuli, only agonists with similar degrees of intrinsic efficacy are able to reproduce these effects, as evidenced from the full and partial substitution patterns observed with SKF-81297 and SKF-38393, respectively. In contrast, these results are not consistent with Reavill et al. (1993) who showed that SKF-38393 fully substituted for the high-efficacy dopamine D1 receptor agonist SKF-81297. More likely, this is due to differences in experimental testing procedures used between the studies and inherent differences in the stimulus properties of the two dopamine D1 receptor agonists (Rosenzweig-Lipson and Bergman, 1993). In this regard, perhaps further research (i.e., using different training doses) will help characterize these compounds as discriminative stimuli. [1]
The findings in the present study that the preferential dopamine D2 receptor agonist quinpirole did not substitute for the SKF-82958 cue, even when used at doses that decreased response rate, is consistent with other studies showing that dopamine D2-like receptor agonists do not substitute for dopamine D1-like receptor agonists and vice versa Cunningham et al., 1985, Kamien et al., 1987, Arnt, 1988, Reavill et al., 1993. Together, the findings that the discriminative stimulus effects of SKF-82958 are reproduced only by similar dopamine D1 receptor agonists and not by a dopamine D2 receptor agonist, and are attenuated by the selective dopamine D1/D5 receptor antagonist SCH-39166 but not by the dopamine D2 receptor antagonist raclopride, suggest that the SKF-82958 interoceptive stimulus is mediated via an interaction at dopamine D1 receptors. [1]
Also in the present study, substitution tests with various doses of indirect dopamine receptor agonist cocaine did not fully substitute for the SKF-82958 discriminative stimulus cue. It is possible that administration of higher doses of cocaine (highest dose tested 5.6 mg/kg) would have fully reproduced the discriminative stimulus effects of SKF-82958, but they were not tested during the course of the present experiments. However, when the similarly efficacious dopamine D1 receptor agonist SKF-81297 was used as the discriminative stimulus in squirrel monkeys, neither cocaine nor d-amphetamine fully substituted up to doses that disrupted responding (Rosenzweig-Lipson and Bergman, 1993). In addition, these data are consistent with others that show the converse to be true, where high- and low-efficacy dopamine D1 receptor agonists do not reliably substitute for psychostimulants. For example, in rats, SKF-38393 did not substitute for cocaine Barrett and Appel, 1989, Filip and Przegalinski, 1997 or d-amphetamine (Furmidge et al., 1991). Similarly, SKF-81297 did not fully reproduce the effects of d-amphetamine Furmidge et al., 1991, Reavill et al., 1993, and dihydrexadine did not substitute for cocaine (Witkin et al., 1991). Also, in primates, the dopamine D1 receptor agonists SKF-82958 and SKF-81297 only partially reproduced the effects of cocaine, even when used at doses that decreased response rates considerably (Spealman et al., 1991). Therefore, based on results from the present experiment and others, there does not appear to be full overlap between the mechanisms underlying the discriminative stimulus effects of dopamine D1 receptor agonists such as SKF-82958 and psychostimulants such as cocaine and vice versa. [1]
Recently, it has been suggested that dopamine D1-like receptor agonists may serve as a possible ‘replacement’ pharmacotherapy for cocaine addiction in part because the high-efficacy dopamine D1 receptor agonist SKF-82958, utilized in the present drug discrimination experiments, has been shown to block the reinstatement of cocaine self-administration in rats following a short extinction period (Self et al., 1996a). However, the doses of SKF-82958 utilized in the aforementioned studies produced marked rate disruption in our hands, suggesting that the blockade of cocaine reinstatement observed may have been due to behavioral disruption rather than a selective effect on cocaine reinforcement per se. Additionally, there is evidence that high-efficacy dopamine D1 receptor agonists are self-administered in both rats and primates Self and Stein, 1992, Self et al., 1996b, Weed and Woolverton, 1995, Weed et al., 1997, Grech et al., 1996 raising the possibility that they have inherent abuse liability. In contrast, dopamine D1 receptor agonists with lower intrinsic efficacy are not readily self-administered Woolverton et al., 1984, Katz and Witkin, 1992, Grech et al., 1996, Weed et al., 1997, and can serve as functional antagonists in vivo by blocking some of the behavioral effects of cocaine, suggesting a role for these drugs in the treatment of cocaine addiction Katz and Witkin, 1992, Spealman et al., 1997. However, it remains to be determined whether other behavioral effects associated with dopamine D1 receptor agonists preclude their use. Pre-clinical studies specifically designed to address the possibility of using partial dopamine D1 receptor agonists as pharmacotherapies for cocaine abuse are warranted. [1]
In conclusion, the present study demonstrates, for the first time, that the high-efficacy dopamine D1 receptor agonist SKF-82958 can serve as a reliable discriminative stimulus in the rat and is mediated by a dopamine D1-like receptor mechanism. Furthermore, although some overlap exists, the stimulus effects of the direct dopamine D1 receptor agonist SKF-82958, are distinct from that elicited by the indirect dopamine receptor agonist, cocaine. [2]

C19H20CLNO2

410.73254

329.118

C, 69.19; H, 6.11; Cl, 10.75; N, 4.25; O, 9.70

80751-65-1

SKF-82958 hydrobromide;74115-01-8

1225

Typically exists as solid at room temperature

1.234 g/cm3

473.7ºC at 760 mmHg

240.3ºC

1.616

4.823

2

3

3

23

399

0

OC1=C(O)C=C2C(C3=CC=CC=C3)CN(CC=C)CCC2=C1Cl

HJWHHQIVUHOBQN-UHFFFAOYSA-N

InChI=1S/C19H20ClNO2/c1-2-9-21-10-8-14-15(11-17(22)19(23)18(14)20)16(12-21)13-6-4-3-5-7-13/h2-7,11,16,22-23H,1,8-10,12H2

9-Chloro-5-phenyl-3-prop-2-enyl-1,2,4,5-tetrahydro-3-benzazepine-7,8-diol

(±)-SKF 82958; Chloro-APB; SKF-81297 HBr; SKF-81297; Skf-82958; 80751-65-1; Cl-Apb; SKF 82958; SK&F 82958; SK&F-82958; 6-Chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; 9-chloro-5-phenyl-3-prop-2-enyl-1,2,4,5-tetrahydro-3-benzazepine-7,8-diol; SKF 81297; SKF81297; (±)-SKF 82958 Chloro-AP

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)