5-HT4 receptors

1. The interaction of the novel antagonist, SDZ 205-557 (2-methoxy-4-amino-5-chloro benzoic acid 2-(diethylamino) ethyl ester), at 5-HT3 and 5-HT4 receptors has been assessed in vitro and in vivo. 2. In guinea-pig hippocampus and in the presence of 0.4 microM 5-carboxamidotryptamine, 5-HT4-mediated stimulation of adenylyl cyclase was competitively antagonized by SDZ 205,557, with a pA2 value of 7.5, and a Schild slope of 0.81. In rat carbachol-contracted oesophagus, 5-HT4-receptor mediated relaxations were surmountably antagonized by SDZ 205,557 with a similar pA2 value (7.3). This value was agonist-independent with the exception of (R)-zacopride, against which a significantly lower value (6.4) was observed. 3. In functional studies of 5-HT3 receptors, SDZ 205,557 exhibited an affinity of 6.2 in guinea-pig ileum compared with 6.9 at binding sites labelled by [3H]-quipazine in NG108-15 cells [2].

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Guinea-pig hippocampal adenylyl cyclase [2]
Adenylyl cyclase activity (basal activity = 1.8 nmol 30 min-' mg-' protein) in membranes isolated from the guinea-pig hippocampus was stimulated by 5-HT ( 2 fold) with a -log EC50 value of 6.4 (Figure 1). The sigmoidal concentration-response curve to 5-HT appeared to be monophasic. By contrast, 5-carboxamidotryptamine (5-CT) produced a biphasic response curve. Low concentrations (0.4pM) of 5-CT were sufficient to stimulate maximally the initial phase (Figure 1). Previous studies have demonstrated that the increase in adenylyl cyclase activity elicited by 5-HT in the presence of 0.4 IM 5-CT is due to the stimulation of 5-HT4 receptors (Shenker et al., 1987). In the absence of 5-CT, maximally effective concentrations (100 gM) of SDZ 205-557 and spiperone reduced the stimulation in response to 10 LM 5-HT by 44% and 64%, respectively. Stimulation of adenylyl cyclase by 5-HT was abolished when the membrane preparation was incubated in the presence of both antagonists (Figure 2). In the presence of 5-CT, responses to 5-HT were antagonized, in a concentration-dependent manner, by both tropisetron and SDZ 205,557, with - log ICm values of 6.4 and 5.1 (mean, s.e.mean <10%), respectively. In contrast, responses to 5-HT were unaffected by propranolol (0.1 mM), ketanserin (1 LM) or methysergide (1 gLM; data not shown). The apparent affinity of SDZ 205,557 at the 5-HT4 receptor mediating the stimulation of adenylyl cyclase was derived by Schild regression analysis. The pA2 value was 7.5 with a Schild slope of 0.81 (Figure 3).

In the anaesthetized, vagotomized micropig, SDZ 205-557 produced only a transient blockade of 5-HT4-mediated tachycardia. This contrasted with tropisetron, which was active for over 60 min after administration. The half-lives for the inhibitory responses of SDZ 205,557 and tropisetron were 23 and 116 min, respectively. 4. In conclusion, SDZ 205,557 has similar affinity for 5-HT3 and 5-HT4 receptors. The apparent selectivity observed in guinea-pig is due to the atypical nature of the 5-HT3 receptor in this species. The short duration of action of this novel antagonist may complicate its use in vivo. SDZ 205,557 should, therefore, be used with appropriate caution in studies defining the 5-HT4 receptor [2].

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Rat oesophageal muscularis mucosae [2]
In preparations pre-contracted with 10 jM carbachol, all agonists induced a concentration-dependent relaxation. The potencies and intrinsic activities to agonists studied are shown in Table 1. The rank order was 5-HT = 5-MeOT> BIMU-l ) SC-53116 > BIMU-8>(S)-zacopride>(R)-zacopride. SDZ 205-557 (1 nM-10pM) lacked intrinsic activity at the oesophageal 5-HT4 receptor in that no relaxations were seen. In the presence of SDZ 205,557, concentration-response curves to all agonists were shifted to the right in a competitive fashion. Schild regression analysis performed with SDZ 205,557 (1-10 tM) and using 5-HT as the agonist, resulted in a Schild slope not significantly different from unity (1.24, 95% confidence limits 0.98-1.50) (Figure 4). When the unity constraint was imposed, a pA2 value of 7.3 (95% confidence limits: 7.2-7.4) was determined. The apparent affinities (- log KB) obtained with a single concentration of SDZ 205,557 against the other agonists were similar to this value (Table 1). The exception to this was the affinity when (R)-zacopride was used as the agonist. In this case the - log KB value was significantly lower. Tropisetron (3 1M) antagonized the relaxations induced by 5-HT with a concentration-ratio of 35 (95% confidence limits, 27-45) and an apparent affinity (- log KB) of 7.0 ± 0.04. SDZ 205,557 (3 gM) antagonized responses to 5-HT with a concentration-ratio of 98 (41-237). In the presence of both SDZ 205,557 (3 1M) and tropisetron (3 1M), responses to 5-HT were shifted to the right with a combined concentration-ratio of 153 (99.8-234; mean, 95% confidence limits). This was not significantly different from that predicted by the combination of two competitive antagonists interacting at the same site (i.e. 133 fold).

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Interactions at S-HT3 receptors [2]
Experiments in guinea-pig isolated ileum were conducted in the presence of methysergide (1 1M) and 5-methoxytryptamine (10 M) to desensitize selectively 5-HTI, 5-HT2 and 5-HT4 receptors. At ileal 5-HT3 receptors SSDZ 205-557 (10 ILM) antagonized responses to 5-HT, with an apparent affinity (- log KB) of 6.2 ± 0.08. Affinities for other 5-HT3 antagonists reported by Eglen et al. (1990), under similar conditions are included for comparison (Table 2). The rank order of antagonist affinity at ileal 5-HT3 receptors was therefore: (S)-zacopride > tropisetron >ondansetron > SDZ 205,557. In binding studies, at 5-HT3 receptors identified by [H]-quipazine in NG108-15 cell membranes (Table 3), SDZ 205,557 displacement isotherms yielded an apparent affinity (- log Kj) of 6.9 ± 0.2. The Hill coefficient (1.3 ± 0.1) was not significantly different from unity, suggesting an interaction of SDZ 205,557 at a homogeneous population of sites. The rank order of apparent affinities was (S)-zacopride> ondansetron > tropisetron > MDL 72222 >SDZ 205,557 > metoclopramide. The values estimated in binding sites were greater then those estimated in functional studies (Table 2), in agreement with previous reports by Butler et al. (1990).

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Tachycardia in anaesthetized pig [2]
5-HT, renzapride and (R,S)-zacopride elicited a dosedependent tachycardia in the anaesthetized pig with 5-HT being significantly more potent than either benzamide (Figure 5). Preliminary studies showed that consecutive, reproducible dose-response curves to 5-HT in the pig can be constructed at intervals of 90-120 min. Heart rate responses returned to baseline within 10-15min after each dose of 5-HT, with responses treated cumulatively after successive doses of the benzamides. 5-HT and (R,S)-zacopride acted as full agonists zacopride acted as full agonists and renzapride as a partial agonist (Table 3). The responses to all these agonists were antagonized by tropisetron (data not shown). To assess the inhibitory activity of tropisetron and SDZ 205-557, single doses of the antagonists were administered against the ED54 dose for 5-HT (3-10tgg kg-', i.v.), previously obtained by individual titration. The variances of the control and treatment groups did not differ sufficiently to utilize ranked changes from baseline; therefore a parametric repeated measures ANOVA was used to analyze this data. In these overall ANOVAs, the effects of treatment, time and treatment x time interaction were all highly significant (P<0.0001). Subsequent pairwise contrasts (one-way ANOVAs) showed that SDZ 205,557, administered at 6.0 mg kg-', i.v., significantly (P< 0.05) inhibited the tachycardic response to 5-HT for only 3 min following administration whereas no significant effect was seen for the remainder of the experiment. In contrast, tropisetron (5 mg kg-', i.v.) administered against similar doses of 5-HT, significantly (P< 0.05) inhibited responses for 120 min following administration (Figure 6). The half-lives for the inhibitory responses were 23 (17-35) and 116 (85-175) min for SDZ 205,557 and tropisetron, respectively (values are mean with 95% confidence intervals).

Competition radioligand binding studies [2]
Membranes were prepared from NG108-15 cells cultured under conditions described previously (Sharif et al., 1991). In competition binding studies, 5-HT3 receptors in NG108-15 cell membranes were labelled with 0.5 nM [3H]-quipazine and non-specific binding was defined by 1 JAM (S)-zacopride. The membrane homogenates were incubated in 25 mM Tris-Krebs (pH 7.4 at 250C) with radioligand and varying concentrations of SDZ 205-557, or other standard 5-HT3 receptor antagonists, in a final assay volume of 0.5 ml. The incubations were carried out for 45 min at 250C and were terminated by rapid vacuum filtration over Whatman GF/B filters using a Brandell 48 cell harvester. This was immediately followed by 8 s of washing with ice-cold 0.1 M NaCl. The filters were pretreated with 0.3% polyethyleneimine in order to reduce filter binding of the radioligand and the radioactivity retained on the filters was determined by liquid scintillation spectrometry. All competition data were analyzed by iterative curve fitting procedure as described previously (Michel & Whiting, 1984). The apparent dissociation constant (K.) of competing ligands was calculated from ICso values by the Cheng-Prusoff equation (Cheng & Prusoff, 1973). [2]

Guinea-pig hippocampal adenylyl cyclase [2]
Male guinea-pigs (Hartley, 250-500 g) were killed by CO2 asphyxiation. The hippocampi were then rapidly dissected, and homogenized (20 strokes) in a glass homogenizer in 9 ml of ice-cold buffer, of the following composition (mM): sucrose 300, Tris-HCl 20, EGTA 1, Na2EDTA 2.5 and dithiothreitol 1 (pH 7.4, 23°C). This homogenate was then diluted, 1:8 (v/v) with buffer and centrifuged (10 min, 39,000 g, 4C). The pellet was resuspended in 9 ml of buffer and the membrane suspension used directly in the adenylyl cyclase assay. Antagonists were added 30 min prior to addition of agonist. Measurements of adenylyl cyclase activity were performed, three times, according to the method of Alvarez & Daniels (1990) with each sample run in triplicate. The final composition of the incubation medium was (mM): [at32P]-adenosine 5'-triphosphate ([&32P]-ATP, 0.25 pCi) 0.5, MgSO4 5, TrisHCl 44 (pH 7.4), 1-methyl-3-isobutylxanthine 1.0, sucrose 50, EDTA 1.0, EGTA 0.2, dithiothreitol 0.2, adenosine 3':5'- cyclic monophosphate (cyclic AMP) 2, guanosine 5'-triphosphate (GTP) 0.1, phosphoenol pyruvate 20 and pyruvate kinase 6 units ml-'. The reaction was initiated by the sequential addition of the membrane suspension (40 #Al) to the incubation medium in a total reaction volume of 200 Jl. Incubations were performed for 30 min at 370C and terminated by the addition of 20 ftl of a stock solution of [3H]-cyclic AMP (0.005 JCi) in 2.2 N HCl. Labelled cyclic AMP was added to estimate and correct for recovery of the nucleotide following column chromatography. The tube contents were heated at 95C for 4 min and then cooled in an ice-water bath. Neutral alumina (1.3 g) was dispensed into disposable polypropylene columns with a Uniflow adjustable powder measure. The columns were placed on a plexiglas rack designed to hold the columns and to fit over a box of 100 scintillation vials. An aliquot (200 pl) of the solution contained in each tube was pipetted onto the columns and allowed to flow into the dry alumina. Cyclic AMP was then eluted with 4 ml of 0.1 M ammonium acetate, pH 7. The effluent (3.2 ml) was collected into scintillation vials, mixed with 15 ml scintillation fluid and counted for 3H and 32p in a liquid scintillation spectrometer. [2]

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Rat isolated oesophageal muscularis mucosae [2]
The method used was that previously described by Baxter et al. (1991). Male rats (Sprague-Dawley, 200-250 g) from Charles River were killed by CO2 asphyxiation, the thoracic oesophagus removed and placed in Tyrode solution (composition, mM: NaCl 139.0, KCl 2.7, MgCl2 6H2O 1.1, NaH2PO4 0.4, glucose 5.6, NaHCO3 11.8 and CaCl2-6H2O 1.8). The outer proprial muscle coat was cut longitudinally and gently peeled away, leaving the inner muscularis mucosae. Silk threads were then tied through the lumen on both ends of the tissues, which were then mounted vertically in 10 ml tissue baths containing Tyrode solution with 1 JAM methysergide, 30 gM cocaine and 30 JM corticosterone. The baths were maintained at 37C and constantly aerated with a mixture of 95% 02 and 5% CO2. An initial resting tension of 1 g was applied to the tissues, then adjusted to 0.5 g tension at 15 min intervals thereafter. After 1 h of equilibration, 100 JM pargyline was added to the baths for 30 min, followed by a 15 min washout. At this point, the tissues were exposed to 50 mM KCl for 5 min, washed four times, followed by an additional 30 min equilibration period with a 15 min wash cycle. Carbachol (3 ,.M) was added to the baths to contract the preparations. Once a stable contraction had been established (usually 30 min) the agonist was then added cumulatively to the bath to induce relaxation. After establishing the control concentration-response curve for the agonist, the preparations were then washed. SDZ 205-557 (1-10IOM) was applied to the baths and allowed to equilibrate with the tissues for 60 min before the second agonist concentration-response curve. Since all 5-HT4 agonists at high concentrations have the propensity to antagonize muscarinic receptors (Baxter et al., 1991), a final concentration-response curve was constructed in the presence of both 10 JM 5-methoxytryptamine and SDZ 205,557 after a further 60 min. This procedure established the agonist concentration-range attributable to 5-HT4 agonism alone as distinct from additional relaxation due to muscarinic receptor antagomsm. Agonist potencies were determined by nonlinear iterative curve fitting procedures (Michelson et al., 1992), using the relationship described by Parker & Waud (1971). Apparent antagonist affinities (- log KB) were estimated by the relationship - log KB = - log [Antagonist]/(concentration ratio - 1) (Furchgott, 1972). Concentration ratios were measured at the agonist concentration which elicited 30% of the maximal relaxation, since under some conditions the effects observed at higher concentrations may have reflected muscarinic receptor antagonism. Against 5-HT itself, the apparent affinity was determined by the method of Arunlakshana & Schild (1959), wherein three concentrations of SDZ 205-557 were used, and the slope of the Schild plot determined by regression analysis. [2]

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Anaesthetized micropig studies [2]
The method used was modified from that described by Villalon et al. (1990). Yucatan micropigs (male and female; 14.9-20.8 kg) were pretreated with ketamine HCl (approx. 30 mg kg-', i.m.), anaesthetized with pentobarbitone sodium (20 mg kg-') via a marginal ear vein, intubated, and mechanically ventilated with room air by an animal respirator. A femoral artery was cannulated for the measurement of arterial blood pressure via a Gould/Statham pressure transducer (P231D). Dual cannulae were inserted into the ipsilateral femoral vein, one cannula for continuous infusion of supplemental anaesthetic (pentobarbitone sodium 8- 15 mg kg-' h-') and the second cannula for compound administration. A limblead II ECG was monitored via subcutaneously placed electrodes and heart rate (HR) was determined by a cardiotachometer triggered by the aortic pressure signal form. Following a midline cervical incision, the vagus nerves were bilaterally transected. Blood gas parameters were periodically monitored via a blood gas analyzer and blood gas values were stabilized within a normal (pH 7.2; Pco2, 33 mmHg; Po2, 96 mmHg) physiological range by adjustments of ventilatory rate, tidal volume, and positive endexpiratory pressure prior to continuing an experiment. 5-HT, tropisetron and SDZ 205-557 were administered in base equivalent doses. 5-HT, dissolved in 0.154 M NaCI, was administered at bolus i.v. doses of 1, 3, 10, 30 and 1I00 g kg-' (0.05 ml kg-' doses) in each animal and a doseresponse curve constructed. An ED50 dose for 5-HT was determined visually from each dose-response curve. SDZ 205,557 and tropisetron were dissolved in a 3:7 propylene glycol:normal saline (v/v) mixture. Animals were assigned randomly to 3 treatment groups: vehicle (3:7 propylene glycol: normal saline), SDZ 205,557 (6 mg kg- , i.v.), or tropisetron (5 mg kg-', i.v.). Doses for SDZ 205-557 and tropisetron were determined in preliminary dose-finding experiments (data not shown). These doses represented those that maximally antagonized 5-HT-induced tachycardia and/ or were the maximum feasible dose based on compound supply and micropig weight. The ED50 dose of 5-HT (determined from a previous experiment) was administered 3 times at 10-15 min intervals to determine a control response (mean of 3 responses). Following administration of vehicle, SDZ 205,557, or tropisetron in a volume of 0.1 ml kg-l, the ED50 dose of 5-HT was administered in a volume of 0.03 ml kg-' at 3, 10, 20, 30, 45, 60, 75, 90, 105, and 120 min thereafter. [2]

[1]. SDZ 205-557, a selective antagonist at 5-HT4 receptors in the isolated guinea pig ileum. Eur J Pharmacol. 1991 Aug 6;200(2-3):373-4.

[2]. The action of SDZ 205,557 at 5-hydroxytryptamine (5-HT3 and 5-HT4) receptors. Br J Pharmacol. 1993 Feb;108(2):376-82.

In the anaesthetized, vagotomized micropig, 5-HT elicited a tachycardic response in the presence of 5-HT1, 5-HT2, 5-HT3, M2-muscarinic and P-adrenoceptor blockade. These responses were antagonized by high doses of tropisetron and mimicked by (R,S)-zacopride and renzapride. Similar observations have been reported by Villalon et al. (1990) in the Yorkshire pig and concur with biochemical data showing that porcine myocardial 5-HT4 receptors stimulate adenylyl cyclase (Kaumann, 1990). The tachycardia may be due to subsequent activation of a kinase (Kaumann et al., 1991) and closure of potassium channels (Bockaert et al., 1992). 5-HTinduced tachycardia in the micropig, thus, provides a suitable in vivo assay to study 5-HT4 compounds. The administration of SDZ 205-557 failed to antagonize, except briefly (3 min) after injection, the tachycardic responses to 5-HT. The doses of 5-HT were submaximal and adequately blocked by tropisetron. The short-lived antagonism by SDZ 205,557 in vivo contrasts with the sustained antagonism seen in vitro (at least 60 min; see Methods). The compound, at least at the dose tested, appears to be subject to rapid metabolism, probably due to hydrolysis at the ester moiety in the SDZ 205-557 molecule. It should be noted, however, that such rapid degradation may not be so marked at higher doses and additional experiments are required to study this. In conclusion, SDZ 205,557 acted as a surmountable 5- HT4 receptor antagonist in guinea-pig hippocampus and rat oesophagus, although it had no selectivity between mouse neuroblastoma 5-HT3 and 5-HT4 receptors. In guinea-pig, a 5-HT4/5-HT3 selectivity was evident due to the atypical nature of the guinea-pig 5-HT3 receptor. The short duration of action in vivo together with this limited selectivity, suggests that caution should be exercised in its use to define the 5-HT4 receptor. [2]

C14H21CLN2O3

300.78

137196-67-9

Typically exists as solids at room temperature

CCN(CCOC(C1=CC(Cl)=C(N)C=C1OC)=O)CC

2-(Diethylamino)ethyl 4-amino-5-chloro-2-methoxybenzoate; SDZ 205-557; SDZ 205557; SDZ-205-557; 2-Methoxy-4-amino-5-chlorobenzoic acid 2-(diethylamino)ethyl ester; Benzoic acid, 4-amino-5-chloro-2-methoxy-, 2-(diethylamino)ethyl ester; DTXSID90160102; ...; 137196-67-9;

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.)