D3 receptor ( EC50 = 0.64 nM )

In vitro activity: PD-128907 HCl is a potent and selective dopamine D3 receptor agonist with an EC50 of 0.64 nM with a 53-fold selectivity over the dopamine D2 receptor.[1] PD-128907 shows roughly a l000-fold selectivity for human D3 receptors (Ki, 1 nM) versus human D2 receptors (Ki, 1183 nM) and a l0000-fold selectivity versus human D4 receptors (Ki, 7000 nM) when using [³H]spiperone as the radioligand in CHOKl-cells.[2] PD 128907 is employed in the investigation of these receptors' functions in the brain, including their function as inhibitory autoreceptors, which control the release of additional dopamine.[3]

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The functional potency of a series of dopamine agonists for increasing mitogenesis, measured by incorporation of [3H]thymidine, was established in transfected cell lines expressing human D2 or D3 receptors. The functional selectivity of agonists markedly differs from their binding selectivity. (+)7-OH-DPAT, pramipexole, quinerolane and PD-128907, the most D3 receptor-selective compounds in binding studies, were 7, 15, 21 and 54 times more potent, respectively, at the D3 than at the D2 receptor in the functional test. Bromocriptine displayed a 10-fold functional selectivity toward the D2 receptor. The known behavioural actions of D3 selective agonists support a role for the D3 receptor in motor inhibitions, which should be taken into account for the treatment of motor dysfunctions by dopamine agonists. [1]

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PD-128907 [4a R, 10 b R-(+)-trans-3, 4, 4a, 10 b - tetrahydro - 4- n -propyl2 H,5H-[1]benzop-yrano[4,3-b]1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (Ki, 1 nM) versus human D2 receptors (Ki, 1183 nM) and a 10000-fold selectivity versus human D4 receptors (Ki, 7000 nM) using [3H]spiperone as the radioligand in CHO-K1-cells. Studies with [3H]PD 128907, showed saturable, high affinity binding to human D3 receptors expressed in CHO-K1 cells (CHO-K1-D3) with an equilibrium dissociation constant (Kd) of 0.99 nM and a binding density (Bmax) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D2 receptors (CHO-K1-D2). The rank order of potency for inhibition of [3H]PD 128907 binding with reference DA agents was consistent with reported values for D3 receptors. These results indicate that [3H]PD 128907 is a new, highly selective D3 receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DA D3 receptors. [2]

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The present study determined the biochemical and pharmacological effects of PD 128907 [R-(+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H- [1]benzopyrano[4,3-b]-1,4-oxazin-9-ol], a dopamine (DA) receptor agonist that shows a preference for the human D3 receptor. In transfected Chinese hamster ovary cells (CHO K1), PD-128907 displaced [3H]spiperone in a biphasic fashion which fit best to a two-site model, generating Ki values of 20 and 6964 nM for the high- and low-affinity sites for the D2L receptors and 1.43 and 413 nM for the corresponding sites for the D3 receptors. Addition of sodium and the GTP analog Gpp(NH)p to both the D2L and D3 caused a modest reduction in the affinity of the compound suggestive of an agonist type action. In agonist binding ([3H]N-0437), PD 128907 exhibited an 18-fold selectivity for D3 versus D2L, a selectivity similar to that found with antagonist binding to the high-affinity sites. PD 128907 exhibited only weak affinity for D4.2 receptors (Ki = 169 nM). No significant affinity for a variety of other receptors was observed. PD-128907 stimulated cell division (measured by [3H]thymidine uptake) in CHO p-5 cells transfected with either D2L or D3 receptors exhibiting about a 6.3-fold greater potency in activating D3 as compared to D2L receptors [4].

PD-128907 effectively lowers the synthesis of DA in rats treated with GBL and in rats receiving normal treatment.[4] PD-128907 (3 mg/kg) totally avoids the convulsive and deadly effects of cocaine, reducing the toxicity from overdose.[5] A D3-linked mechanism provides the protection, which also extends to seizure kindling.[6]

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Dopamine (DA) autoreceptors expressed along the somatodendritic extent of midbrain DA neurons modulate impulse activity, whereas those expressed at DA nerve terminals regulate both DA synthesis and release. Considerable evidence has indicated that these DA autoreceptors are of the D2 subtype of DA receptors. However, many pharmacological studies have suggested an autoreceptor role for the DA D3 receptor. This possibility was tested with mice lacking the D3 receptor as a result of gene targeting. The basal firing rates of DA neurons within both the substantia nigra and ventral tegmental area were not different in D3 receptor mutant and wild-type mice. The putative D3 receptor-selective agonist R(+)-trans-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-(1)benzopyrano(4,3-b)-1,4-oxazin+ ++-9-ol (PD-128907) was equipotent at inhibiting the activity of both populations of midbrain DA neurons in the two groups of mice. In the gamma-butyrolactone (GBL) model of DA autoreceptor function, mutant and wild-type mice were identical with respect to striatal DA synthesis and its suppression by PD-128907. In vivo microdialysis studies of DA release in ventral striatum revealed higher basal levels of extracellular DA in mutant mice but similar inhibitory effects of PD 128907 in mutant and wild-type mice. These results suggest that the effects of PD 128907 on dopamine cell function reflect stimulation of D2 as opposed to D3 receptors. Although D3 receptors do not seem to be significantly involved in DA autoreceptor function, they may participate in postsynaptically activated short-loop feedback modulation of DA release. [3]

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In vivo the compound was active in reducing DA synthesis both in normal and gamma-butyrolactone (GBL) treated rats; in the GBL model, the decrease was greater in the higher D3-expressing mesolimbic region as compared with striatum which has a lower expression of D3 receptors. PD-128907 decreased DA release (as measured by brain microdialysis) both in rat striatum, nucleus accumbens and medial frontal cortex, as well as in monkey putamen. Behaviorally PD 128907 decreased spontaneous locomotor activity (LMA) in rats at low doses, whereas at higher doses stimulatory effects were observed. PD-128907 at high doses reversed the reserpine-induced decrease in LMA and induced stereotypy in combination with the D1 agonist SKF 38393 indicating postsynaptic DA agonist actions. It is unclear which of the subtypes of DA receptors might be mediating the pharmacological effects of PD 128907. However, the present findings indicating that PD 128907 shows a preference for DA D3 over D2L and D4.2 receptors indicates that its action at low doses may be due to interaction with D3 receptors and at higher doses, with both D2 and D3 receptors. [4]

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Cocaine abuse is a public health concern with seizures and death being one consequence of overdose. In the present study, dopamine D(3/)D(2) receptor agonists dose dependently and completely prevented the convulsant and lethal effects of cocaine. The D(3)-preferring agonists R-(+)-trans-3,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol) [(+)-PD-128907], (+)-7-hydroxy-dipropylaminotetralin, and the mixed D(3/)D(2) agonists quinpirole and quinelorane were all effective against cocaine toxicity in mice. The anticonvulsant effects of these compounds occurred at doses below those that produced motor impairment as assessed in the inverted screen test. Protection against the convulsant effects of the selective dopamine uptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy] ethyl]-4-[3-phenyl-propyl]piperazine (GBR 12909) was also conferred by (+)-PD 128,907. The possible selectivity of the effects of (+)-PD 128,907 (3 mg/kg) for these dopaminergic compounds was demonstrated by its general lack of protective efficacy against a host of convulsants acting through other neural mechanisms [pentylenetetrazol, (+)-bicuculline, and picrotoxin, 4-aminopyridine, and t-butylbiclyclophosphoorothionate, N-methyl-d-aspartate, kainate, pilocarpine, nicotine, strychnine, aminophylline, threshold electric shock, and 6-Hz electrical stimulation]. Direct and correlational evidence suggests that these effects were mediated by D(3) receptors. Protection was stereospecific and reversible by an antagonist of D(3) receptors [3-[4[1-(4-[2[4-(3-diethyamino-propoxy)-phenyl]-benzoimidazol-l-yl]-butyl)-1H-benzoimidazol-2-yl]-phenoxy]-propyl)-diethyl-amine; PD 58491] but not D(2) receptors [3[[4-(4-chlorophenyl)-4 hydroxypipeidin-1-yl]methyl-1H-indole; L-741,626]. Anticonvulsant potencies were positively associated with potencies in a functional assay of D(3) but not D(2) receptor function. Together, these findings suggest that the prevention of cocaine convulsions and lethality by (+)-PD-128907 may be due to D(3) receptor-mediated events. [5]

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Previous findings have demonstrated a protective role for dopamine D(3)/D(2) receptor agonists in the convulsant and lethal effects of acutely administered cocaine. Data are provided here to establish that the protection occurs through a D(3)-linked mechanism and that protection is extended to seizure kindling. The D(3) antagonist SB-277011-A [4-quinolinecarboxamide,N-[trans-4-[2-(6-cyano-3,4-dihydro-2(1H)-isoquinolinyl)ethyl]-cyclohexyl]-(9CI)] prevented the anticonvulsant effect of the D(3)/D(2) receptor agonist (+)-PD-128907 [(R-(+)-trans-3,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol)] on cocaine-induced seizures. The protection afforded by the D(3)/D(2) agonist, (+)-PD-128,907, was eliminated in D(3) receptor-deficient mice. In D(2) receptor knockout mice, the anticonvulsant effects of (+)-PD-128,907 were preserved. (+)-PD-128907 also prevented the acquisition and expression of cocaine-kindled seizures engendered by repeated daily dosing with 60 mg/kg cocaine. (+)-PD-128,907 also blocked the seizures induced in mice fully seizure kindled to cocaine. Although repeated dosing with cocaine increased the potency of cocaine to produce seizures and lethality (decreased ED(50) values), daily coadministration of (+)-PD-128,907 significantly prevented this potency shift. In mice treated daily with cocaine and (+)-PD-128,907, the density, but not the affinity, of D(3) receptors was increased. The specificity with which (+)-PD-128907 acts upon this cocaine-driven process was demonstrated by the lack of a significant effect of (+)-PD-128,907 on seizure kindling to a GABA(A) receptor antagonist, pentylenetetrazol. Taken together and with literature findings, the data indicate that dopamine D(3) receptors function in the initiation of a dampening mechanism against the toxic effects of cocaine, a finding that might have relevance to psychiatric disorders of drug dependence, schizophrenia, and bipolar depression [6].

Lipand-binding assays. [2]
The assay conditions are described below. A series of equilibrium saturation studies were conducted using different Tris-HCl buffers. Briefly, 50 pl of [‘Hlligand (1 nM, final concentration in competition studies), 50 pl of either drug or buffer, and 400 pl of brain or CHO-Kl cell membranes in appropriate ice cold buffer were added to polypropylene micro tubes to give a total volume of 500 l_~l. Incubation proceeded for 60 min at 25-C and was terminated by rapid filtration followed by four washes with 1 ml buffer on a Brandel MR48 cell harvester through Whatman GF/B glass fiber filters (pre-soaked for about one hour in 0.5% PEI). Following the addition of 10 ml of liquid scintillation Ready Gel cocktail and an overnight extraction, the radioactivity remaining on the filters was counted with a Beckman LS 6800 liquid scintillation counter (50% efficiency). Specific binding was defined as total binding minus binding in presence of 1 pM haloperidol and this ranged from 90-95%. All assays were performed in triplicates. Crude membranes from CHOKl cells ranged from 40 pg-60 pg of protein per assay tube. Protein was determined by the Bradford assay using the microplate reader for analysis.

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Saturation studies. [2]
Saturation binding curves were determined with nine increasing concentrations of [w]PD-128907 (0.039-10.0 r&l). As described previously, incubations proceeded for 60 min at 25-C with the tissue homogenates. The effects of different ions on the binding of [3H]PD-128907 were examined by using different buffers (TE = 25 mM Tris HCl, 1 mM EDTA; TEM = 25 mM Tris HCl, 1 mM EDTA, 6 mM MgCl,, TEN = 2.5 mM Tris HCl, 1 mM EDTA, 12

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Kinetic experiments. [2]
Association and dissociation experiments were conducted with [‘H]PD-128907 (1 .O nM, final concentration) using TEM buffer. In association experiments, specific binding was defined in the presence of 1 pM haloperidol and samples were filtered at various times. In dissociation studies, samples were also filtered at various times (following equlibrium as determined above) by the addition of excess PD-128907 (20 pM final concentration). The ratio of the rate constants for the dissociation and the association (k-l/k+l) was used to calculate the &of r3H]PD 128907 (n = 3).

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Comoetition studies. [2]
Inhibition constants (IS,) were determined for various agents using 1 nM [SH]PD-128907 in CHO-Kl-D, cell membranes and assays were conducted with TEM buffer. Eight different concentrations of each ligand was prepared and studies were performed in triplicate. The effect of Gpp(NH)p, a non hydrolyzable GTP analog was studied on the inhibition of [“H]PD 128907 binding to CHO-Kl-D, membranes by DA.

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(+)-PD 128907 hydrochloride is a selective agonist of the D2/D3 dopamine receptor. Its Kis values for human and rat D3 receptors are 1.7, 0.84 nM, and 179, 770 n M, respectively.

Tissue culture. [2]
CHO cells and consequently transfected with human D,, and D, cDNA were maintained under an atmosphere of 95% air and 5% CO, at 37.C. CHO cells were grown in and subcultured in F-12 medium containing 100 U/ml penicillin-streptomycin and dialyzed fetal bovine serum. The media were changed every 2-3 days and at least 18 hr before harvesting of the cells. Confluent cultures were harvested by replacement of medium with cold phosphate-buffered saline (PBS) containing 0.05% EDTA followed by centrifugation at 1000 g for 2 min. The pellets obtained were suspended in appropriate vol of ice-cold buffer (25 mM Tris-HCl, 1 mM EDTA, pH 7.4, TE buffer) and centrifuged at 20000 g for 15 min at 4-C. The final pellets obtained were suspended in TE buffer and homogenized with a Polytron at setting 6 for 5 s for use in radioligand binding assays or stored at -8O*C. CHO-Kl cell membranes for use in the assay were homogenized with a Polytron at setting 6 for 5 s.

Extracellular single-unit recordings. [3]
All methods for extracellular single-cell recordings were similar to those previously reported (White et al., 1995), although modified somewhat for the mouse (Xu et al., 1994). Briefly, mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a standard stereotaxic apparatus with a specialized adapter for the mouse. Body temperature was maintained at 36–37.5°C with a thermostatically controlled heating pad. A 28 gauge (3/8 inch) hypodermic needle was placed in a lateral tail vein through which additional anesthetic (as required) and drugs of study were administered. A burr hole was drilled in the skull, and the dura was retracted from the area overlying the VTA and SN, 0.4–1.3 mm anterior to lambda and 0.2–1.0 mm lateral to the midline. Recording electrodes were made by pulling glass tubing [outer diameter (o.d.), 2.0 mm], which was prefilled with fiberglass, and by breaking the tip back to a diameter of 1–2 μm. Electrodes were filled with 2 m NaCl saturated with 1% (w/v) fast green dye and typically exhibitedin vitro impedances of 1–3 MΩ (at 135 Hz). Electrode potentials were passed through a high impedance amplifier/filter and displayed on an oscilloscope. Individual action potentials were discriminated electronically and monitored with an audio amplifier. Integrated rate histograms, generated by the output of the window discriminator, were plotted by a polygraph recorder, whereas digitized counts of cellular activity were obtained for off-line analysis. Electrodes were lowered to a point 0.5 mm above the VTA and SN and then slowly advanced with a hydraulic microdrive through the DA cell regions (3.2–5.0 mm ventral to the cortical surface). DA cells were identified by standard physiological criteria (Bunney et al., 1973; Wang, 1981;Sanghera et al., 1984) and were recorded for 3–6 min to establish a baseline firing rate. To determine the sensitivity of impulse-modulating somatodendritic autoreceptors, we administered PD-128907 to each mouse through the tail vein, using a cumulative dose regimen in which each dose doubled the previous dose, at 60–90 sec intervals. After agonist-induced inhibition, the D2-class receptor antagonist eticlopride was administered (0.1–0.2 mg/kg) to reverse the effect and confirm receptor mediation. At the end of each experiment, the cell location was marked by ejecting fast green dye, and the spot was verified by routine histological assessment (described below).

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γ-Butyrolactone experiments. [3]
The l-aromatic amino acid decarboxylase inhibitor NSD 1015 was administered 30 min before death (100 mg/kg, i.p.). γ-Butyrolactone (GBL) was administered (750 mg/kg, i.p.) 5 min before NSD 1015 to eliminate impulse flow in DA neurons (Walters and Roth, 1976). PD-128907 was injected 5 min before GBL. Mice were killed by decapitation, and the brains were quickly removed. Dorsal and ventral striatum were dissected on a chilled glass plate with the aid of a mouse brain matrix designed to allow coronal sections to be cut rapidly and reproducibly (Activational Systems, Warren, MI). Two slices (2 mm each) were taken, beginning at the rostral boundary of the olfactory tubercle. The most anterior slice was the source of ventral striatum, whereas both slices were used to obtain dorsal striatum. The ventral striatum was dissected with angular cuts originating at the lateral olfactory tracts and ending at the midline (average weight, 18.5 mg). The remainder of the striatal region from that slice as well as the similar region from the more caudal slice was considered the dorsal striatum (average weight, 35.8 mg). Tissues were kept at −80°C. To measure tissue catechols, we weighed frozen tissues and then sonically disrupted them in a homogenization solution consisting of 100 mmHClO4, 5 mmNa2S2O5, and α-methyl dopa, an internal standard. After centrifugation (25,000 ×g for 10 min), aliquots of the supernatant were processed by alumina extraction as described previously (Galloway et al., 1986). The 3,4-dihydroxyphenylalanine (DOPA) content of samples was determined using an HPLC system consisting of a Bioanalytical Systems (BAS) Phase II ODS 3 μm column (100 × 3.2 mm), a BAS LC4C electrochemical detector, and a Scientific Systems model 222C HPLC pump. The mobile phase consisted of 0.1 mNaH2PO4, 1 mm EDTA, 0.2 mm 1-octane-sulfonic acid, and 3% methanol, adjusted to pH 2.7 with phosphoric acid. DOPA content was quantified based on both internal and external standards.

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In vivo microdialysis experiments. [3]
Mice used for dialysis experiments weighed 28–35 gm. Concentric microdialysis probes were constructed as described previously, with fused-silica inlet and outlet lines (Wolf et al., 1994). Dialysis membrane (molecular weight cutoff, 6000; o.d., 250 μm) was obtained from Spectrum (Los Angeles, CA). Data were not corrected for in vitro probe recovery because probes may suffer differential alterations during insertion. Consistent with this assumption, data sets corrected for recovery often exhibit greater variability than do those that are uncorrected (Xue et al., 1996). Probes were stereotaxically implanted under sodium Brevital (8 mg/kg, i.p.). Stereotaxic coordinates were, relative to bregma, anterior, 1.5 mm; lateral, 1.5 mm; and ventral, 2.7–4.7 mm. Ventral coordinates indicate exposed regions of dialysis membrane (2 mm). After surgery, mice were placed in Plexiglas cages (23 × 46 mm) and allowed to recover overnight. Food and water were available ad libitum. Dialysis cages were equipped with balance arms (Instech, Plymouth Meeting, PA), and homemade liquid swivels and tethers were constructed from plastic syringes and tubing. These were used because commercially available swivels were too heavy for use with mice. Probes were perfused overnight at 0.3 μl/min with artificial CSF (aCSF) consisting of (in mm): 2.7 KCl, 140 NaCl, 1.2 CaCl2, 1 MgCl2, 0.3 NaH2PO4, and 1.7 Na2HPO4, pH 7.4. The next morning, the perfusion rate was increased to 2 μl/min for 2–3 hr before the experiment was begun. Experiments consisted of 1 hr of perfusion with control aCSF to determine basal DA efflux, 1 hr of perfusion with aCSF containing PD-128907, and a 1 hr recovery period during which control aCSF was perfused. Thus, administration of PD-128907 occurred ∼20 hr after probe implantation. Fractions were collected every 20 min. After each experiment, mice were anesthetized and perfused intracardially with normal saline followed by 10% formalin. Probe placement was examined in sections stained with cresyl violet. Only data from mice with verified probe placements were included in the analysis. Dialysates were analyzed for DA content using the HPLC system described for GBL experiments. Chromatographic conditions were optimized for early elution of DA to obtain maximum sensitivity. The mobile phase consisted of 0.1 m NaH2PO4, 0.5 mm EDTA, 2 mm 1-octane-sulfonic acid, and 16% methanol, adjusted to pH 4.9. Peaks were recorded using a dual-channel chart recorder and were quantified by comparison with the peak heights of external standards run with every experiment.

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Dissolved in water; 3 mg/kg; i.p. injection

Mouse

[1]. Neuroreport. 1995 Jan 26;6(2):329-32.

[2]. Life Sci. 1995;57(15):1401-10.

[3]. J Neurosci. 1998 Mar 15;18(6):2231-8.

[4]. J Pharmacol Exp Ther. 1995 Dec;275(3):1355-66.

[5]. J Pharmacol Exp Ther. 2004 Mar;308(3):957-64.

[6]. J Pharmacol Exp Ther. 2008 Sep;326(3):930-8.

As mentioned above, studies conductedwith [SH]PD-128907 in CHO-Kl-D, membranes in the presence of Na’ (120 mM) showed that the K, was slightly lower, although not statistically significant. These effects may indicate that Na’is not inducing a conformational change in the D, receptor as has previously been suggested for D, receptors. Besides Na’, guanyl nucleotides have also been shown to decrease the binding of agonists to G-protein linked receptors. Inclusion of 100 PM of the nonhydrolyzable GTP analog, Gpp(NH)p in the DA displacement curves did not alter the competition curves for DA, indicating a lack of any significant functional coupling of the human D, receptor to a G-protein regulated signal transduction system in CHO-Kl cells (Table II). These findings are in agreement with previously published findings in CHO and MN9D cells indicating a lack of or only weak coupling of D, receptors to G-proteins (see ref. 2.5 and 26 for opposite results). However, another study has shown that recombinant D,receptors heteregously expressed in CHO cells functionally interact with endogenous G proteins. The reasons for these differences not clear but may be due to different signalling pathways in certain clone of the cells used where G protein modulation have been described. In agreement with our present data, weak or no modulatory effects of guanyl nucleotide on DA binding at native D,receptors were also observed using [SH]7-OH-DPAT and [‘*‘1]7-OH-PIPAT, respectively. The absence of appropriate subtypes of G proteins in the various cell lines and/or the D, receptor are possible explanations for the phenomenon if the natural coupling mechanism/channel is not present or is not G-protein linked. [‘H]PD-128907 appears to be a very selective D,radioligand with high specific activity, good specific yield and low non-specific binding. [3H]PD 128907 shoud be appropriate for such studies as autoradiography to evaluate the presence and distribution of D, receptors in brain and possibly to characterize the function, if any, of this receptor subtype. [2]

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Our microdialysis studies indicated a significant increase in basal DA efflux in the ventral striatum of D3 receptor mutant mice compared with their wild-type littermates. Although “no-net flux” dialysis studies are often needed to quantify differences in basal transmitter efflux in vivo (for review, see Justice, 1993), the difference between D3 receptor mutant and wild-type mice was quite robust, with all but one mutant mouse showing higher basal DA efflux than any of the wild-type mice. Increased basal DA efflux might be expected if nerve terminal D3 autoreceptors normally exert a tonic inhibitory influence on DA release. However, the results obtained with PD 128907 are incompatible with this simple interpretation because this putative D3 receptor-selective agonist inhibited DA release by the same absolute amount in D3 receptor mutant and wild-type mice. Thus, no reduction of DA autoreceptor modulation occurred as a result of the D3 receptor mutation. Although the percent decrease caused by PD-128907 was smaller in the mutant mice, this effect was attributable to their higher basal DA levels, a factor known to reduce the ability of DA agonists to suppress
DA release (Cubeddu and Hoffman, 1982; Dwoskin and Zahniser, 1986; for review, see Wolf and Roth, 1987).
An alternative explanation for our findings is that DA release is normally modulated both by D2 release-modulating autoreceptors and by negative feedback pathways engaged by postsynaptic D3 (and other D2-class) receptors. Loss of D3 receptor-mediated inhibitory feedback would explain increased basal DA efflux, whereas activation of D2release-modulating autoreceptors by PD-128907 would explain the normal inhibitory effects on DA release. This model is consistent with the D3 mRNA findings indicating that D3 receptors are distributed primarily within target neurons of the ascending DA systems. In ventral striatal terminal fields where the D3receptor is most highly expressed (nucleus accumbens shell, olfactory tubercle, and islands of Calleja), there is good agreement between levels of D3 receptor mRNA and D3 receptor binding sites (Diaz et al., 1995), suggesting that most D3receptors exist on dendrites, soma, or local terminals of intrinsic neurons. If postsynaptic D3 receptors are coupled to feedback pathways that normally exert an inhibitory influence on DA release, the loss of such feedback might lead to increased basal DA efflux but leave D2 autoreceptor-mediated effects intact.[3]

C14H20CLNO3

285.77

285.113

C, 58.84; H, 7.05; Cl, 12.41; N, 4.90; O, 16.80

112960-16-4

112960-16-4; 23594-64-9; 300576-59-4 (HCl)

11957668

White to light yellow solid powder

145-153°C

2.676

2

4

2

19

286

2

Cl.CCCN1CCO[C@@H]2C3C=C(O)C=CC=3OC[C@@H]12

DCFXOTRONMKUJB-QMDUSEKHSA-N

InChI=1S/C14H19NO3.ClH/c1-2-5-15-6-7-17-14-11-8-10(16)3-4-13(11)18-9-12(14)15;/h3-4,8,12,14,16H,2,5-7,9H2,1H3;1H/t12-,14-;/m1./s1

(4aR,10bR)-4-propyl-3,4a,5,10b-tetrahydro-2H-chromeno[4,3-b][1,4]oxazin-9-ol;hydrochloride

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.)