D₃ Receptor ( Ki = 1.4 )

NGB 2904 induces mitotic activity and antagonizes quinpirole (100 nM) with an IC50 of 5.0 nM [1]. The receptor binding profiles of compounds 4 and 5/NGB-2904 as summarized in Table 1 were determined by in vitro binding studies using membranes from CHO cells transfected with individual human, primate or rat receptor subtype cDNAs. Both compounds displayed high affinity for the D3 receptor with greater than 150-fold selectivity over all other dopamine receptor subtypes. Similar selectivity was observed over ~l receptors. In the case of 4, moderate affinity (125 nM) and selectivity (100-fold) was found for the serotonin 5-HT2. A global receptor screen (Panlabs) for binding sites not listed in Table 1 indicated no inhibition of greater than 50% at 1 mM.
In order to determine the functional action of 4 and 5/NGB-2904 at the D 3 receptor, the effect on mitogenesis in D3-transfected CHO cells was measuredJ 2 Agonist activation of D 3 receptors stimulates [3H]thymidine uptake in CHO cells. The D 3 receptor agonists dopamine and quinpirole both stimulate [3H]thymidine incorporation into CHO.hD3 cells in a dose dependent manner, while the D 3 antagonist haloperidol, and both NGB 2849 and NGB 2904 are receptor antagonists. Haloperidol, NGB 2849 and NGB 2904 antagonize 100 nM quinpirole stimulated mitogenesis with an IC50 values of 8.8, 6.8 and 5.0 nM, respectively [1].

NGB 2904 (26 μg/kg; single subcutaneous injection) improves amphetamine (26 mg/kg)-stimulated locomotion in wild-type mice [3]. In wild type, NGB 2904 (0.026 μg-1 mg/kg; once daily for 7 days or as a single subcutaneous injection) stimulates spontaneous locomotion [3].

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Accumulating evidence indicates that dopamine (DA) D(3) receptor antagonists appear highly promising in attenuating cocaine reward and relapse in preclinical models of addiction. In the present study, we investigated the effects of the novel D(3)-selective antagonist NGB-2904 (N-(4-[4-{2,3-dichlorophenyl}-1-piperazinyl]butyl)-3-fluorenylcarboxamide) on cocaine self-administration, cocaine-enhanced brain stimulation reward (BSR), and cocaine-triggered reinstatement of drug-seeking behavior in male Long-Evans rats. We found that: (1) acute intraperitoneal (i.p.) administration of NGB-2904 (0.1-10 mg/kg) failed to alter cocaine self-administration (0.5 mg/kg/infusion) under fixed-ratio 2 (FR2) reinforcement, but 1 or 5 mg/kg NGB 2904 significantly lowered the break-point for cocaine self-administration under progressive-ratio (PR) reinforcement; (2) cocaine (1, 2, and 10 mg/kg) significantly enhanced electrical BSR (decreased brain reward thresholds), while NGB 2904 significantly inhibited the enhancement of BSR elicited by 2 mg/kg, but not 10 mg/kg of cocaine; (3) NGB-2904 alone neither maintained self-administration behavior nor altered brain reward thresholds; and (4) NGB-2904 significantly inhibited cocaine-triggered reinstatement of extinguished drug-seeking behavior, but not sucrose-plus-sucrose-cue-triggered reinstatement of sucrose-seeking behavior. Overall, these data show that the novel D(3)-selective antagonist NGB 2904 attenuates cocaine's rewarding effects as assessed by PR self-administration, BSR, and cocaine-triggered reinstatement of cocaine-seeking behavior. Owing to these properties and to its lack of rewarding effects (as assessed by BSR and by substitution during drug self-administration), NGB 2904 merits further investigation as a potential agent for treatment of cocaine addiction. [2]

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The dopamine D3 receptor is believed to play an important role in regulation of rodent locomotor behavior, and has been proposed as a therapeutic target for substance abuse, psychotic disorders, and Parkinson's disease. One model of dopamine D3 receptor function, based on studies utilizing D3 receptor knockout mice and D3 receptor-preferring agonists, proposes that D3 receptor stimulation is inhibitory to psychostimulant-induced locomotion, in opposition to the effects of concurrent dopamine D1 and D2 receptor stimulation. Recent progress in medicinal chemistry has led to the development of highly-selective dopamine D3 receptor antagonists. In order to extend our understanding of D3 dopamine receptor's behavioral functions, we determined the effects of the highly-selective dopamine D3 receptor antagonist NGB-2904 on amphetamine-stimulated and spontaneous locomotion in wild-type and dopamine D3 receptor knockout mice. NGB-2904 (26.0 microg/kg s.c.) enhanced amphetamine-stimulated locomotion in wild-type mice, but had no measurable effect in dopamine D3 receptor knockout mice. Of a range of doses (0.026 microg-1.0 mg/kg) given acutely or once daily for seven days, the highest dose of NGB 2904 (1.0 mg/kg) stimulated spontaneous locomotion in wild-type mice, but was without measurable effect in dopamine D3 receptor knockout mice. These behavioral effects of NGB 2904 contrast with those described for other highly D3 receptor-selective antagonists, which have not previously demonstrated an effect on spontaneous locomotor activity. In combination, these data add to the behavioral profile of this novel D3 receptor ligand and provide further support for a role for dopamine D3 receptor inhibitory function in the modulation of rodent locomotion [3].

General procedure [2]
After recovery from surgery, each rat was placed into a test chamber and allowed to lever-press for i.v. cocaine (1 mg/kg/injection) delivered in 0.08 ml over 4.6 s, on an FR1 reinforcement schedule. During the 4.6 s injection time, additional responses on the active lever were recorded but did not lead to additional infusions. Each session lasted 3 h. The FR1 reinforcement schedule was used for 3–5 days until stable cocaine self-administration was established. The initial cocaine dose of 1 mg/kg/infusion was chosen on the basis of our previous experience that this dose produces the most rapid and facile acquisition of cocaine self-administration behavior. Subsequently, subjects were randomly assigned to one of the following four experiments: (1) cocaine self-administration under an FR2 reinforcement schedule, (2) cocaine self-administration under a PR reinforcement schedule, (3) NGB-2904 or saline replacement testing in experienced cocaine self-administering rats, or (4) cocaine-triggered reinstatement of drug-seeking behavior. In all experiments, NGB-2904 was given 30 min prior to testing because preliminary data showed that NGB-2904's effects occurred approximately 30 min after systemic administration.

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Cocaine self-administration under FR2 reinforcement [2]
After transition from FR1 reinforcement, subjects (n=10) were allowed to continue cocaine (0.5 mg/kg/infusion) self-administration under FR2 reinforcement until the following criteria for stable cocaine-maintained responding were met: <10% variability in inter-response interval and <10% variability in the number of presses on the active lever for at least 3 consecutive days. The dose of cocaine was chosen on the basis of previous findings that rats self-administering cocaine at 0.5 mg/kg/infusion display highly stable self-administration behavior. In addition, previous studies have shown that 0.5 or 1 mg/kg/infusion cocaine lies within the range of the descending limb of the cocaine dose–response self-administration curve, where stable and reliable dose-dependent effects have been observed (Weissenborn et al, 1998; Parsons et al, 1998; Xi et al, 2005). Furthermore, we chose 0.5 mg/kg rather than 1 mg/kg of cocaine in order to increase the work demand (ie lever presses) of the animals for the same amount of drug intake. In our previous experience, this approach increases the sensitivity of measuring changes in drug-taking or drug-seeking behavior. To avoid cocaine overdose during the self-administration period, each animal was limited to a maximum of 50 cocaine injections per session. After stable rates of responding were established, each subject randomly received one of four doses of NGB-2904 (0.1, 1, 5, and 10 mg/kg i.p.) or vehicle (1 ml of 25% 2-hydroxypropyl-β-cyclodextrin solution) 30 min prior to the test session. Animals then received an additional 5–7 days of self-administration of cocaine alone until the baseline response rate was re-established prior to testing the next dose of NGB-2904. The order of testing for the various doses of NGB-2904 was counterbalanced according to a Latin square design.

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Cocaine self-administration under PR reinforcement [2]
Initial cocaine self-administration under FR1 and FR2 reinforcement was identical to that outlined above. After stable cocaine self-administration under FR2 reinforcement was established, the subjects were switched to cocaine self-administration (0.5 mg/kg/injection) under a PR schedule, during which the work requirement of lever presses needed to receive a single i.v. cocaine infusion was progressively raised within each test session (see details in Richardson and Roberts, 1996) according to the following PR series: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328, 402, 492, and 603 until the break-point was reached. The break-point was defined as the maximal workload (ie number of lever presses) completed for the last cocaine infusion prior to a 1-h period during which no infusions were obtained by the animal. Animals were allowed to continue daily sessions of cocaine self-administration under PR reinforcement conditions until day-to-day variability in break-point fell within 1–2 ratio increments for 3 consecutive days. Once a stable break-point was established, subjects were assigned to four subgroups to determine the effects of three different doses of NGB-2904 (0.1, 1, and 5 mg/kg i.p.) or vehicle (1 ml 25% 2-hydroxypropyl-β-cyclodextrin solution) on PR break-point for cocaine self-administration. Since it is relatively difficult to reachieve basal break-point levels after each drug test, we chose to use a between-subjects design rather than a within-subjects design for this experiment.

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NGB-2904 or saline self-administration in rats formerly self-administering cocaine [2]
After a stable pattern of daily cocaine self-administration (0.5 mg/kg/infusion) under FR2 reinforcement was established for at least 3 consecutive days, the animals were divided into five groups (n=8 each): (1) cocaine (0.5 mg/kg/infusion) was available for self-administration on the following days in the usual 3-h test sessions; (2) cocaine was replaced by NGB-2904 (0.1 mg/kg/infusion); (3) cocaine was replaced by NGB-2904 (0.5 mg/kg/infusion); (4) cocaine was replaced by heroin (0.05 mg/kg/infusion); and (5) cocaine was replaced by saline (0.08 ml/infusion). Since animals might take several days to support self-administration for a novel reinforcer, each replacement test was repeated for 3–5 days. The doses of NGB-2904 were chosen on two grounds. First, NGB-2904's maximum solubility in the 5% 2-hydroxypropyl-β-cyclodextrin solution used as vehicle in this experiment is approximately 3 mg/ml, making 0.5 mg/kg/infusion the maximum feasible unit dose. Second, the cumulative i.v. NGB-2904 dose within the initial 30 min (5–8 infusions × 0.1 or 0.5 mg/kg/infusion, see Figure 2) was approximately 0.5–4 mg/kg, which is higher than the i.p. doses (0.1–5 mg/kg) of NGB-2904 found to be effective in the self-administration, BSR, and reinstatement of drug-seeking experiments detailed below, making failure to self-administer an a fortiori finding. Heroin and saline were chosen as positive and neutral reinforcer controls.

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Testing the effects of cocaine and/or NGB-2904 on BSR [2]
Once a baseline θ0 value was achieved (<15% variation in θ0 over 5 continuous days), the effects of cocaine and/or NGB-2904 on BSR were assessed. On test days, animals randomly received one of three different doses of NGB-2904 (0.1, 1, and 5 mg/kg i.p.) or vehicle (1 ml 25% 2-hydroxypropyl-β-cyclodextrin) 30 min prior to a cocaine injection (1, 2, or 10 mg/kg i.p.). After each test, animals received an additional 5–7 days of BSR restabilization until a new baseline θ0 was established. The order of testing for various doses of NGB-2904 was counterbalanced according to a Latin square design. The effect of NGB-2904 on cocaine-enhanced BSR was evaluated by comparing cocaine-induced alterations in θ0 value in the presence or absence of each dose of NGB-2904 pretreatment.

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Extinction and testing for reinstatement [2]
After stable cocaine self-administration was established, animals were exposed to extinction conditions, during which cocaine was replaced by saline, and the cocaine-associated cue-light and tone were turned off. Active lever-pressing led only to saline infusion. Daily 3 h extinction sessions for each rat continued until that rat lever-pressed <10 times per 3 h session for at least 3 consecutive days. After successful achievement of extinction, animals were divided into four groups for reinstatement testing. On the reinstatement test day, each group of rats received either the vehicle (25% 2-hydroxypropyl-β-cyclodextrin) or one dose of NGB-2904 (0.1, 1, and 5 mg/kg i.p.). At 30 min after vehicle or NGB-2904 administration, all rats were given a priming injection of cocaine (10 mg/kg i.p.) immediately before the reinstatement testing began. During the reinstatement test, the conditions were identical to those in extinction sessions. Cocaine-induced active lever-pressing responses (reinstatement) were recorded, although these lever-pressing responses did not lead to either cocaine infusions or presentation of the conditioned cues. Reinstatement test sessions lasted 3 h.

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Drugs [2]
To decrease injection resistance and solution osmotic pressure, 5% 2-hydroxypropyl-β-cyclodextrin was used as vehicle for i.v. NGB-2904 self-administration. We have previously observed no differences in behavioral effects between 5 and 25% 2-hydroxypropyl-β-cyclodextrin.

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Experiment 1: acute and sub-chronic effects of NGB-2904 [3]
Mice were removed from the vivarium and transported to the testing facility (located in an adjacent building) the day before testing (at ∼ 1500). Subjects were placed in the Residential Activity Chambers and allowed to acclimate for 16–18 h before drug administration, during which time, lab chow and water were available. At ∼ 1000 the following day, food and water were removed from the chambers prior to drug administration, so that feeding behaviors would not compete with locomotor behavior. Mice were injected with one of the following: vehicle (50% PEG 400), 0.026, 2.6, 260.0, 333.0 or 1000 μg/kg NGB-2904. Subjects were then placed back in the Residential Activity Chambers, and locomotor activity was recorded for 2 h. After the two-hour testing period, mice were returned to their home cages overnight. Injections were repeated once daily for 7 days, and the distance traveled during the first 2 h on each day was used to calculate the average distance traveled for the seven-day period. Each animal received only one dose of NGB-2904 on day 1 and continued to receive the same dose on all seven test days.

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Experiment 2: effect of NGB-2904 on amphetamine-stimulated locomotion [3]
In a series of pilot experiments, it was determined that NGB-2904 concentrations in the low microgram range increased amphetamine-stimulated locomotion (data not shown). The dose tested in this experiment was derived from these pilot experiments. Mice were acclimated to the Residential Activity Chambers for 1 h, then injected with vehicle (50% PEG 400) or NGB-2904 (26.0 μg/kg). Thirty minutes later, mice were injected with amphetamine (2.5 mg/kg) or 0.9% saline. Locomotor activity was recorded for 3 h after the second injection. We have previously determined that the amphetamine 2.5 mg/kg dose induces primarily elevated locomotor behavior in both wild-type and D3 receptor knockout mice, with negligible competing stereotyped behaviors observed (McNamara et al., 2006). All injections were subcutaneous in a volume of 1.0 ml/kg. Each animal was used for a single behavioral observation, to avoid alterations in locomotor response through the effects of behavioral sensitization. The number of D3 receptor mutant mice available for study limited the experiment to a single NGB-2904 dose.
NGB-2904 was dissolved by heating overnight to 60 °C in 50% polyethylene glycol 400.[3]

[1]. NGB 2904 and NGB 2849: two highly selective dopamine D3 receptor antagonists. Bioorg Med Chem Lett. 1998 Oct 6;8(19):2715-8.

[2]. The novel dopamine D3 receptor antagonist NGB 2904 inhibits cocaine's rewarding effects and cocaine-induced reinstatement of drug-seeking behavior in rats. Neuropsychopharmacology. 2006 Jul;31(7):1393-405.

[3]. The dopamine D3 receptor antagonist NGB 2904 increases spontaneous and amphetamine-stimulated locomotion. Pharmacol Biochem Behav. 2007 Apr;86(4):718-26.

N-[4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyl]-9H-fluorene-2-carboxamide is a member of fluorenes.

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N-(4-[4-¿2, 3-dichlorophenyl¿-1-piperazinyl]butyl)-3-fluorenylcarboxamide and N-(4-[4-¿2, 3-dichlorophenyl¿-1-piperazinyl]butyl)-2-biphenylenylcarboxamide were prepared in several steps from 2,3-dichloroaniline. These compounds were identified as highly selective dopamine D3 receptor antagonists. [1]

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In conclusion, NGB 2904 attenuates cocaine's rewarding effects and selectively inhibits relapse to drug-seeking behavior, and has no apparent rewarding, aversive, or locomotor effects. Therefore, NGB 2904 or other highly selective D3 antagonists may be promising as pharmacotherapeutic agents to treat cocaine abuse and may provide in vivo tools with which to further characterize the role of DA D3 receptors in drug addiction.[2]

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Taken together, our findings contribute to a growing body of data suggesting that NGB 2904 acts as a selective D3 receptor antagonist in vivo (Gilbert et al., 2005, Xi et al., 2006) and provides a new and useful tool in elucidating the functional role of the D3 receptor. Given the high degree of in vitro selectivity of NGB 2904 and the lack of measurable effect in D3 receptor mutant mice, our findings provide further support for the hypothesis that the D3 receptor plays an inhibitory role in the modulation of certain domains of rodent locomotor behavior. As with any study using knockout mice, our results must be interpreted with caution, as it is possible that differences in drug response between wild-type and knockout mice could have resulted from compensatory adaptations, rather than from deletion of the D3 receptor gene. Based on its demonstrated behavioral effects and in vitro selectivity, NGB 2904 may prove to be a useful instrument for clarifying the role of the dopamine D3 receptor in substance abuse, psychosis, and Parkinson's disease.[3]

C28H29CL2N3O

494.46

493.17

C, 68.02; H, 5.91; Cl, 14.34; N, 8.50; O, 3.24

189060-98-8

189061-11-8 (HCl); 189060-98-8

9891901

White to off-white solid powder

708.8ºC at 760 mmHg

382.4ºC

2.41E-20mmHg at 25°C

7.092

1

3

7

34

670

0

C1CN(CCN1CCCCNC(=O)C2=CC3=C(C=C2)C4=CC=CC=C4C3)C5=C(C(=CC=C5)Cl)Cl

PFIWYJNBKGCVFM-UHFFFAOYSA-N

InChI=1S/C28H29Cl2N3O.ClH/c29-25-8-5-9-26(27(25)30)33-16-14-32(15-17-33)13-4-3-12-31-28(34)21-10-11-24-22(19-21)18-20-6-1-2-7-23(20)24;/h1-2,5-11,19H,3-4,12-18H2,(H,31,34);1H

N-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl]-9H-fluorene-2-carboxamide hydrochloride

NGB 2904; NGB2904; 189060-98-8; 9H-Fluorene-2-carboxamide, N-[4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyl]-; CHEMBL300780; N-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl]-9H-fluorene-2-carboxamide; N-(4-(4-(2,3-Dichlorophenyl)-1-piperazinyl)butyl)-3-fluorenylcarboxamide; N-[4-[4-(2,3-Dichlorophenyl)-1-piperazinyl]butyl]-9H-fluorene-2-carboxamide; NGB-2904

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)

Ethanol : ~4.55 mg/mL (~9.20 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.)