5-HT6 Receptor (Ki = 2.04 nM)

To identify any other off-target activities, compound SUVN-502 (5al) was evaluated at Novascreen in their commercial selectivity panel of over 100 target sites including receptors (64), enzymes (5), peptides (18), growth factors (5), ion channels (6), steroids, immunological factors, second messengers, and prostaglandins (Supporting Information). Compound SUVN-502 (5al) at 1 μM showed <50% inhibition at all the tested receptors, barring dopamine D3, and adrenergic alpha 2A and 2C. In an independent experiment, 5al showed a Ki value of 616 nM at dopamine D3 and 2570 nM adrenergic alpha 2A receptor. At alpha 2C receptor, the functional affinity (Kb) value was 619 nM. The lead candidate, 5al, has shown no significant binding toward the hERG potassium channel when tested in an astemizole binding assay, and no changes in the QTc intervals were noticed in human Phase-1 clinical trials.
The permeability of SUVN-502 (5al) was evaluated in the Caco-2 intestinal epithelial cell line, where it showed high permeability (Papp = 19.6 × 10–6 cm/s). The efflux ratio [Papp (B - A)/Papp (A - B)] was less than 2, indicating minimal interaction with efflux transporters such as P-gp. The extent of protein binding for 5al was determined in rat, dog, and human plasma using equilibrium dialysis. The plasma protein binding of 5al was found to be 99.0, 98.6, and 98.0% in rat, dog, and human, respectively [1].

Having seen the increase in acetylcholine levels upon standalone treatment, the effects of therapeutic (3 mg/kg p.o.) and subtherapeutic (1 mg/kg p.o.) doses of Masupirdine/SUVN-502 (5al) were evaluated in combination with donepezil (1 mg/kg s.c.) and memantine (1 mg/kg s.c.) for modulation of acetylcholine in the ventral hippocampus of male Wistar rats (Figure 5). This study design is based on the premise that this combination may result in improved therapeutic efficacy and also offer several therapeutic benefits, for instance, reduction in the doses of therapeutic agents, which may evade the side effects associated with dosing of higher amounts. If the hypothesis is correct, these scenarios may offer a novel mechanism with improved drug efficacy and tolerability compared to the existing approved therapeutic options.

Treatment with donepezil (1 mg/kg s.c.) and memantine (1 mg/kg s.c.) combination produced an increase in hippocampal acetylcholine levels, which reached to a maximum of 1203 ± 106% of basal levels. Compound Masupirdine/SUVN-502 (5al) , in combination with donepezil and memantine, produced a mean maximum increase of 1383 ± 194% and 2136 ± 288% after 1 and 3 mg/kg p.o., respectively. An increase in acetylcholine produced by the combination of Masupirdine/SUVN-502 (5al) (1 and 3 mg/kg p.o.), donepezil, and memantine was significantly higher than the increase produced by a donepezil and memantine combination. This increase of acetylcholine in the cotreatment group was devoid of any cholinergic side effects. In a separate study involving pharmacokinetic evaluation, a nonsignificant difference in the plasma exposures of donepezil, memantine, or Masupirdine/SUVN-502 (5al) was observed when administered either alone or in triple combination, thus ruling out the possibility of a pharmacokinetic interaction mediated effect (data on file). The aforementioned results from the preclinical study provide the support for the potential therapeutic utility of 5-HT6R antagonist Masupirdine/SUVN-502 (5al) in combination with donepezil and memantine for the treatment of cognitive disorders.

In Phase-1 human clinical trial studies, Masupirdine/SUVN-502 (5al) showed a favorable safety and pharmacokinetic profile in a single ascending dose in healthy male and female subjects and multiple ascending doses for 14 days in healthy elderly male subjects. Gender and food did not have a significant effect on the pharmacokinetics of Masupirdine/SUVN-502 (5al) . It was well tolerated in humans with adequate plasma exposures for efficacy and favorable half-life suitable for once a day treatment. A Phase-2 proof of concept study (ClinicalTrials.gov Identifier: NCT02580305) is ongoing for evaluating the safety and efficacy of 5al in subjects with moderate AD, receiving stable doses of donepezil and memantine. To the best of our knowledge, this is the first clinical trial wherein a triple combination [5-HT6R antagonist (5al) + Donepezil + Memantine] is being used for evaluating the efficacy of a 5-HT6R antagonist [1].

Determination of 5-HT6 receptor Ki by Radioligand Binding Assay: [1]
The procedure used was previously described. In brief, receptor source and radioligand used were human recombinant receptor expressed in HEK-293 cells and [3H] Lysergic Acid Diethylamide (LSD), respectively. The final ligand concentration was 1.5 nM and non-specific determinant was Methiothepin mesylate – [0.1 µM]. Methiothepin mesylate was used as a positive control. Reactions were carried out in 50 mM Tris-HCl (pH 7.4) containing 10 mM MgCl2, 0.5 mM EDTA for 60 min at 37 °C. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined and compared to control values in order to ascertain any interactions of test compound(s) with the cloned serotonin - 5-HT6 binding site and reported as Ki values. These studies were conducted and the data were analyzed using standard radioligand binding techniques as described above. Under these tested assay conditions the Ki value obtained for methiothepin mesylate (reference compound) is 0.5 ± 0.04 nM.

---

Determination of Kb for 5-HT6 Receptor: [1]
The procedure used was previously described. A stable CHO cell line expressing recombinant human 5-HT6 receptor and pCRE-Luc reporter system was used for cell-based assay. The assay offers a non-radioactive based approach to determine binding of a compound to GPCRs. In this specific assay, the level of intracellular cyclic AMP which is modulated by activation or inhibition of the receptor is measured. The recombinant cells harbor luciferase reporter gene under the control of cAMP response element. The above cells were plated in 96 well clear bottom white plates at a density of 5 x 104 cells/well using Hams F12 medium containing 10% fetal bovine serum (FBS) and incubated S4 overnight at 37 oC and 5% CO2 followed by serum starvation for 18-20 h. Increasing concentrations of test compounds were added along with 10 µM serotonin in OptiMEM to the cells. The incubation was continued at 37 oC in CO2 incubator for 4 h. After 4 h cells were lysed using lysis buffer and luciferase assay buffer was added to each well and counts per second were recorded using luminescence counter. From counts per second (CPS) obtained, percent binding was calculated for each well by taking 10 µM 5-HT as 100% bound and vehicle as 0% bound. The percent bound values were plotted against compound concentrations and data were analyzed using a nonlinear, iterative curve-fitting computer program of Graph pad Prism 4 software. The Kb and IC50 values were calculated using concentration of the agonist used in the assay and its EC50 values in the same software. Under these tested assay conditions the Kb value obtained for methiothepin mesylate (reference compound) is 0.7 ± 0.05 nM.

---

5-HT1A Binding Experimental Procedures: [1]
The procedure used was previously described. Membrane preparation from recombinant human 5-HT1A cell line and radioligand 8-Hydroxy-DPAT, [Propyl2,3-ring-1,2,3- 3H] were purchased commercially. All other reagents used in buffer preparation were purchased commercially. The final ligand concentration was 1.75 nM; non-specific determinant was 5-HT [10 µM] and 5-HT1A membrane protein (16 µg/ well). Serotonin was used as a positive control. Reactions were carried out in 50 mM Tris pH 7.4 containing 0.5 mM EDTA, 10 mM MgSO4 and 0.1% ascorbic acid buffer for 120 min at 25 ºC. Reaction was stopped by rapid filtration followed by six washes of the binding mixture using 96 well harvest plate pre coated with 0.33% polyethyleneimine. The plate was dried and the bound radioactivity collected on the filters was determined by scintillation counting using MicroBeta TriLux. Radioligand binding in the S5 presence of non labeled compound was expressed as a percent of the total binding and plotted against the log concentration of the compound. Ki values were determined using a nonlinear, iterative curve-fitting computer program of Graph pad Prism 4 software (Table 1). Under these tested assay conditions the Ki value obtained for serotonin (reference compound) is 0.2 ± 0.03 nM.

---

5-HT2A Binding Experimental Procedures: [1]
The procedure used was previously described Membrane preparation from recombinant human 5-HT2A cell line and radioligand Ketanserin hydrochloride, [Ethylene-3H]-(R-41468) were purchased from Perkin Elmer. All other reagents used in buffer preparation were purchased from Sigma. The final ligand concentration was 1.75 nM; non-specific determinant was 1-NP [10 µM] and 5-HT2A membrane protein (5µg/ well). 1-NP was used as a positive control. Reactions were carried out in 67 mM Tris pH 7.6 containing 0.5 mM EDTA buffer for 60 min at 25 ºC. Reaction was stopped by rapid filtration followed by six washes of the binding mixture using 96 well harvest plate pre coated with 0.33% polyethyleneimine. The plate was dried and the bound radioactivity collected on the filters was determined by scintillation counting using MicroBeta TriLux. Radioligand binding in the presence of non labeled compound was expressed as a percent of the total binding and plotted against the log concentration of the compound. Ki values were determined using a nonlinear, iterative curve-fitting computer program of Graph pad Prism 4 software (Table 1). Under these tested assay conditions the Ki value obtained for 1-(1- naphthyl)piperazine hydrochloride (reference compound) is 15.4 ± 0.7 nM.

---

5-HT2C Binding Experimental Procedures: [1]
S6 The procedure used was previously described. In brief, membrane preparation from recombinant human 5-HT2C cell line, radioligand Mesulergine, [NMethyl3H] were purchased from Perkin Elmer. All other reagents used in buffer preparation were purchased from Sigma. The final ligand concentration was 1.25 nM; non-specific determinant was Mianserine [10 µM], 5-HT2C membrane protein (30 µg/well) and Ysi Polylysine SPA Beads, 1.0 mg/ well. Mianserine was used as a positive control. Reactions were carried out in 54 mM Tris (pH 7.4) containing 10.8 mM MgCl 2, 0.54 mM EDTA, 10.8 µM Pargyline, 0.108% Ascorbic acid, pH 7.4 buffer for 180 min at 25 °C. The plate was read in a MicroBeta TriLux. Radioligand binding in the presence of non labeled compound was expressed as a percent of the total binding and plotted against the log concentration of the compound. Ki values were determined using a nonlinear, iterative curvefitting computer program of Graph pad Prism 4 software (Table 1). Under these tested assay conditions the Ki value obtained for mianserine (reference compound) is 2.8 ± 0.3 nM.

---

5-HT4B Binding Experimental Procedures: [1]
The procedure used was previously described.7 In brief, membrane preparation from recombinant human 5-HT4B cell line was purchased commercially, Radioligand GR113808, [N-methyl3H] was purchased commercially. All other reagents used in buffer preparation were purchased from Sigma. The final ligand concentration was 0.5 nM; non-specific determinant was GR113808 [10 µM] and 5- HT4B membrane protein (5 µg/well). GR113808 was used as a positive control. Reactions were carried out in 25 mM Tris-HCl (pH 7.4) buffer for 120 min at 25 °C. Reaction was stopped by rapid filtration followed by six washes of the binding mixture using 96 well harvest plate pre coated with 0.33% polyethyleneimine. The plate was dried S7 and the bound radioactivity collected on the filters was determined by scintillation counting using MicroBeta TriLux. Radioligand binding in the presence of non labeled compound was expressed as a percent of the total binding and plotted against the log concentration of the compound. Ki values were determined using a nonlinear, iterative curve-fitting computer program of Graph pad Prism 4 software (Table 1). Under these tested assay conditions the Ki value obtained for GR113808 (reference compound) is 16.2 ± 0.8 nM.

---

Determination of IC50 Values for Rat 5-HT7 Receptor: [1]
The procedure used was previously described. 8 A stable CHO cell line expressing recombinant rat 5-HT7 receptor and pCRE-Luc reporter system was used for cell-based assay. The assay offers a non-radioactive based approach to determine binding of a compound to GPCRs. In this specific assay, the level of intracellular cyclic AMP which is modulated by activation or inhibition of the receptor is measured. The recombinant cells harbor luciferase reporter gene under the control of cAMP response element. The above cells were plated in 96 well clear bottom white plates at a density of 5 x 104 cells/well using Hams F12 medium containing 10% fetal bovine serum (FBS) and incubated overnight at 37 oC and 5% CO2 followed by serum starvation for 18-20 h. Increasing concentrations of test compounds were added along with 10 µM serotonin in OptiMEM to the cells. The incubation was continued at 37 oC in CO2 incubator for 4 h. After 4 h cells were lysed using lysis buffer and luciferase assay buffer was added to each well and counts per second were recorded using luminescence counter. From CPS obtained, percent binding was calculated for each well by taking 10 µM serotonin as 100% bound and vehicle as 0% bound. The percent bound values were plotted against compound concentrations and data were analyzed using a nonlinear, iterative curve-fitting computer program of Graph pad Prism 4 software. The IC50 S8 value was calculated using the concentration of the agonist used in the assay and its EC50 values in the same software (Table 1). Under these tested assay conditions the Kb value obtained for methiothepin mesylate (reference compound) is 0.6 ± 0.04 nM (IC50 = 117 ± 6.6 nM).

---

Protocol for Protein Binding: [1]
Unbound fractions of Masupirdine/SUVN-502 (5al) in plasma were determined using Rapid Equilibrium dialysis. The dialysate chambers were loaded with 750 µL of 100 mM phosphate buffer (pH 7.4) in triplicates. The matrix chambers were loaded with 500 µL of the plasma spiked with Masupirdine/SUVN-502 (5al) at a final concentration of 1 µM. 50 µL of the sample was removed from both the chambers at 0 h. The S19 plate was sealed and incubated at 37 °C for 6 h at 100 rpm. After 6 h, 50 µL of the sample was removed from both the chambers. Equal volumes of buffer or plasma were added to the plasma / microsomal and buffer samples respectively to create identical sample matrices for analysis. The samples were precipitated with 150 µL of acetonitrile containing internal standard. All the samples were centrifuged at 10000 rpm for 10 min at 4 °C. The supernatants were transferred to vials and were analyzed by LC-MS/MS.

---

CYP Inhibition: [1]
The inhibition profiles of test compounds were determined using the marker probe substrate reactions of CYP enzymes in human liver microsomes as described elsewhere. 17 The final incubation mixture is comprised of phosphate buffer (100 mM, pH 7.4), marker probe substrate specific to each enzyme, and human liver microsomes. Reactions were initiated by adding NADPH (1 mM) to a final volume of 200 µL. After specified incubation times for each CYP isoform, 120 µL of the incubation mixture was terminated with 240 µL of acetonitrile containing internal standard. Samples were centrifuged at 2500xg for 10 min at 4 °C.

---

CYP Induction: [1]
Human cryopreserved hepatocytes from three donors were thawed and recovered in universal cryopreserved recovery medium and centrifuged at 100 g for 10 min. Viability was assessed via trypan blue dye exclusion. The hepatocytes were plated in collagen coated 96-well plates with approximately 65,000 cells per well and were cultures in an incubator kept at 37 °C in a humidified atmosphere of 5% carbon dioxide and 95% air. The hepatocytes were cultured for 1 S21 day before they were treated with test compounds and positive control inducers (1A2: 50 μM omeprazole; 2B6: 1000 μM phenobarbital; 3A4: 20 μM rifampin). Human hepatocytes from 3 different donors were used in the study. The treatment was performed for an additional 3 days. After the 3 day treatment period, medium was changed to HIM containing a specified P450 substrate and the cells were incubated for 30 min. At the end of the incubation period, the medium was harvested in to 96 well plates and were stored at -80 °C until analysis. Cell viability of the hepatocytes was determined with the WST cell viability assay using the tetrazolim salt, WST-1. The hepatocytes were incubated with the HIM containing WST-1 reagent at 1: 10 dilution for 2 h, followed by quantification of absorbance at 450 nm using a UV plate reader. Total RNA was isolated from hepatocytes using the RNeasy 96 kit according to instructions provided by the manufacturer for isolation. Reverse transcription was performed with 100 ng of isolated RNA using the High capacity cDNA reverse transcription kit. Gene expression was measured using 7500 Fast Real-Time PCR. RT Reactions were quantitated using Taqman universal mix and specific primer sets. The relative quantity of the target gene was compared with that of the endogenous control housekeeping gene expression (GAPDH) as determined by the ΔΔCT method.

Permeability study: [1]
Trans epithelial transport in Caco-2 cells was conducted at RCC Gen biotec GmbH (RCCGBT) Switzerland as per their standard operating procedure. The cells were maintained in DMEM medium supplemented with 10% fetal calf serum, 1% nonessential amino acids and 1% Gentarnicin. The cells were seeded in 12 well Polyester (PET) transwell dishes at a density of about 300,000 cells/ cm 2 using Caco-2 medium. The cells were cultivated at 37 °C/ 5% CO2 in a humidified incubator. The medium was renewed every 2-3 days. After cultivation of about 10 days the TEER was determined every three days until it reached a plateau. Transwell cavities with a TEER of >280Ω cm 2 were used in the assay. For transepithelial transport study TEER was S18 measured prior to the start of the assay, medium was removed and cells were washed twice with pre-warmed (37 ºC) Hank's buffer to remove traces of medium. The assay was started by adding test compound dissolved in to either the apical or basolateral cavity to a final concentration of 10 µM. Samples of 100 µl were taken at different time points (20, 40, 60, 80, 100 and 120 min). The removed volume was replaced by adding 100 µL Hank's buffer and the reduction in concentration was taken into account in the evaluation of the results. The samples were analyzed using LCMS/MS analysis. [1]

---

Determination of Adrenergic Alpha 2A Receptor Ki by Radioligand Binding Assay: [1]
In brief; receptor source and radioligand used were HT29 cells and [3H] MK-912, respectively. The final ligand concentration was 0.75 nM and non-specific determinant was L (-)-Norepinephrine - [100 µM]. Oxymetazoline HCl was used as a positive control. Reactions were carried out in 33 mM Tris-HCl (pH 7.5) containing 1 mM EDTA, 140 mM NaCl and 100 µM Gpp(NH)p (guanyl-5'-yl-imidodiphosphate) for 60 min at 25 °C. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined and compared to control values in order to ascertain any interactions of test compound(s) with the human adrenergic alpha 2A binding site and reported as Ki values. These studies were conducted and the data were analyzed using standard radioligand binding techniques as described above. Under these tested assay conditions the Ki value obtained for oxymetazoline HCl (reference compound) is 1.3 ± 0.06 nM.

---

Determination of Kb Values for Adrenergic Alpha 2C Receptor: [1]
A stable CHO cell line expressing recombinant human adrenergic alpha 2C receptor and pCRE-Luc reporter system was used for cell-based assay. The assay offers a non-radioactive based approach to determine binding of a compound to GPCRs. In this specific assay, the level of intracellular cyclic AMP which is modulated by activation or inhibition of the receptor is measured. The recombinant cells harbor luciferase reporter gene under the control of cAMP response element. The above cells were plated in 96 well clear bottom white plates at a density of 5 x 104 cells/well using Hams F12 medium containing 10% fetal bovine serum (FBS) and incubated overnight at 37 oC and 5% CO2 followed by serum starvation for 18-20 h. Increasing concentrations of test compounds were added along with 1 µM epinephrine and 1 µM forskolin in OptiMEM to the cells. The incubation was continued at 37 oC in CO2 incubator for 4 h. After 4 h cells were lysed using lysis buffer and luciferase assay buffer was added to each well and counts per second were recorded using luminescence counter. From CPS obtained, percent binding was calculated for each well by taking 10 µM epinephrine as 100% bound and vehicle as 0% bound. The percent bound values were plotted against compound concentrations and data were analyzed using a nonlinear, iterative curve-fitting computer program of Graph pad Prism 4 software. The Kb and IC50 values were calculated using concentration of the agonist used in the assay and its EC50 values in the same software. Under these tested assay conditions the Kb value obtained for spiroxatrine (reference compound) is 3.6 ± 0.3 nM.

---

Determination of Dopamine D3 receptor Ki by Radioligand Binding Assay: [1]
In brief; receptor source and radioligand used were rat recombinant receptor expressed in CHO cells and [3H] 7-OH DPAT, respectively. The final ligand concentration was 0.8 nM and non-specific determinant was Dopamine – [1 µM]. (±)-7-OH-DPAT HBr was used as a positive control. Reactions were carried out in 50 mM Tris-HCl (pH 7.4) containing 120 mM NaCl for 60 min at 25 °C. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined and compared to control values in order to ascertain any interactions of test compound(s) with the cloned Dopamine D3 binding site and reported as Ki values. These studies were conducted and the data were analyzed using standard radioligand binding techniques as described above. Under these tested assay conditions the Ki value obtained for (±)-7-OH-DPAT HBr (reference compound) is 0.4 ± 0.02 nM.

In Vivo Brain Microdialysis [1]
Male Wistar rats (240–300 g body weight) were stereotaxically implanted with a microdialysis guide cannula in ventral hippocampus (AP: −5.2 mm, ML: +5.0 mm, DV: −3.8 mm) under isoflurane anesthesia. Coordinates were taken according to the atlas for rat brain with reference points taken from bregma and vertical from the skull. The rats were allowed to recover individually for 4 days in a round-bottom Plexiglas bowl with free access to feed and water. After surgical recovery of 4–5 days, male Wistar rats were connected to a dual quartz lined two-channel liquid swivel on a counter balance lever arm, which allowed unrestricted movements of the animal. Sixteen hours before the study, a pre-equilibrated microdialysis probe (4 mm dialysis membrane) was inserted into the ventral hippocampus through the guide cannula. On the day of study, a probe was perfused with artificial cerebrospinal fluid (aCSF; NaCl 147 mM, KCl 3 mM, MgCl2 1 mM, CaCl2·2H2O 1.3 mM, NaH2PO4·2H2O 0.2 mM and Na2HPO4·7H2O 1 mM, pH 7.2) at a flow rate of 1.5 μL/min, and a stabilization period of 2 h was maintained. Five basal samples were collected at 20 min intervals prior to the treatment of compound Masupirdine/SUVN-502 (5al) (1 or 3 mg/kg p.o.) or vehicle. For studies involving combination, donepezil (1 mg/kg s.c.) and memantine (1 mg/kg s.c.) were administered 30 min after administration of Masupirdine/SUVN-502 (5al) . Dialysates were collected for an additional period of 6 h post-treatment (4 h post-treatment for studies involving combination) of Masupirdine/SUVN-502 (5al) , and dialysates were stored below −50 °C until quantification of acetylcholine by LC-MS/MS. Concentrations of acetylcholine were converted as percent change from mean basal acetylcholine concentrations with 100% defined as the average of five predose values. The percent changes in acetylcholine levels were compared with vehicle. In studies involving combination, percent changes in acetylcholine levels after combination of Masupirdine/SUVN-502 (5al) , donepezil, and memantine were compared with the donepezil and memantine combination using two-way analysis of variance (time and treatment), followed by Bonferroni’s post-test. Statistical significance was considered at a p value less than 0.05. Incorrect probe placement was considered as a criterion to reject the data from animal.

---

Pharmacokinetic Study in Rats: [1]
Male Wistar rats (225 ± 25 g) were used as experimental animals. Three animals were housed in each cage. Two days prior to dosing day, male Wistar rats (225 - 250 g) were anesthetized with isoflurane for surgical placement of jugular vein catheter. Animals were fasted overnight before oral dosing (p.o.) and food pellets were allowed 2 h post dosing, whereas during intravenous dosing food and water were provided ad libitum. Three rats each were dosed with test compound orally (10 mg/kg) and intravenously (10 mg/kg). 10 mL/kg for oral and 2 mL/kg for intravenous was used as dosing volume and water as a vehicle for preparation of dose formulation. At each time point blood was collected through jugular vein and immediately replenished with an equivalent volume of normal saline into freely moving rats. Collected blood was transferred into a labeled vial containing 10 µL of heparin as anticoagulant. Blood samples were collected at following time points: pre dose, 0.08 (only i.v.), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post dose (n=3). Blood was centrifuged at 4000 rpm for 10 min. Plasma was prepared and stored at - 70 °C until analysis. The concentrations of the compounds were quantified in plasma by qualified LC-MS/MS method using suitable extraction technique. The compounds were quantified in the calibration range around 2-2000 ng/mL in plasma. Study samples were analyzed using calibration samples in the batch and quality control samples spread across the batch. Pharmacokinetic parameters Cmax, AUC0-t, t1/2 and bioavailability were calculated by non compartmental model using standard non-compartmental model by using WinNonLin 5.0.1 version Software package. We have evaluated the pharmacokinetic profiling of a reported reference 5-HT6 antagonist (SAM-531) in rats under similar experimental conditions to that of lead compounds (Table 2). The reported values are in agreement with the published report.

---

Rodent Brain Penetration Study: [1]
Male Wistar rats (225 ± 25 g) were used as experimental animals. Three animals were housed in each cage. Animals were given water and food ad libitum throughout the experiment and maintained on a 12 h light/dark cycle. Male Wistar rats were fasted overnight and compound was administered per orally (p.o.; dosing volume 2.5 mL/kg) at 10 mg/kg (n = 3). Blood was collected by cardiac puncture at pre dose 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24 and 32 h after dosing (n=3/ time point), immediately brain was isolated and carcasses were discarded. The brain was homogenized with 4 volumes of ice cooled water. Plasma and brain homogenates (20%) were stored frozen at -20 °C until analysis. The plasma and brain levels of compound was determined by validated LC-MS/MS method using solid phase extraction technique, quantified in the calibration range of 2-2000 ng/mL in plasma and brain homogenates. Pharmacokinetic evaluation was performed using the validated software WinNonlin version 4.1. Extent of brain-plasma ratio was calculated (Cb/Cp) at Tmax.

---

Protocol for Object Recognition Test: [1]
For object recognition test, Male Wistar rats 10-12 weeks old were used. Arena was 50 x 50 x 50 cm. open field was made up of acrylic. On day 1, rats were habituated to individual test arenas for 20 min in the absence of any objects. On day 2 (24 h after the habituation), rats were subjected to familiarization phase (T1). The rats were placed individually in the open field containing two identical objects (a1 and a2) for 3 min. The recognition trial (T2) trial was carried out after 24 h after the T1 trial. Rats were allowed to explore the open field in presence of one familiar object (a3) and one novel object (b) for 3 min. The exploratory behavior towards the objects during the familiarization and recognition phases were recorded. We have evaluated a reported reference 5-HT6 antagonist (SAM-531) in object recognition task model under similar experimental conditions, except the route of administration. SAM-531 showed efficacy at doses of 1 and 3 mg/kg as shown below (Figure 1).

---

Pharmacokinetic Study in Dogs: [1]
Masupirdine/SUVN-502 (5al) was administered to fasted Beagle dogs (n = 3/dose/ group) at 10 mg/kg, orally on Day 8 and non-fasted dogs were treated with intravenous route at a dose of at 3.0 mg/kg on Day 1. For oral administration, compound was weighed directly into a gelatin capsule. Water for injection was used as a vehicle for intravenous administration at a dosing volume of 1 mL/kg. On Day 1 and Day 8 of the study, blood samples were obtained for plasma drug concentrations following oral (capsule) and intravenous (bolus) administration. Samples were collected at 15 min, 30 min, 45 min and 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 48 and 72 h after the oral dose. Whereas, after the intravenous dose, blood samples were collected at 5 min, 10 min, 20 min, 30 min and 1, 2, 4, 6, 8, 12, 24, 48 and 72 h. On each occasion, approximately 3 ml of blood was drawn from the jugular vein and collected into lithium heparin blood collection tubes. Blood samples were centrifuged at 4000 rpm for 10 min at 4 °C. Plasma was transferred to plastic (polypropylene) tubes and placed on dry ice. The samples were stored at -80 ± 10 °C until analysis.

---

Protocol for Fear Conditioning Model: [1]
Experiment was carried out over a period of two days. On day 1, rats were placed in the operant behavior chamber and allowed to acclimatize for 2 min. Rats received a conditioned stimulus (CS) (tone for 10 sec) followed by an unavoidable foot shock (unconditioned stimulus (US): electric shock of 0.5 - 0.7 mA for 3 sec). Following a 1 min interval between each administration, tone and shock were repeated to deliver a total of three CS-US pairings. Rats were administered Masupirdine/SUVN-502 (5al) (1 h). Scopolamine (0.3 mg/kg, s.c.) was administered 120 S22 min after training. On day 2, rats were placed in the operant behavior chamber and total freezing time scored for a period of 5 min.

The pharmacokinetic study of SUVN-502 (5al)was carried out in nonrodent species, Beagle dogs (at 10 mg/kg p.o. and 3 mg/kg i.v. dose). It was well absorbed into systemic circulation with high oral exposures (AUC0-t 1356 ± 1067 ng.h/mL), favorable terminal half-life 3.5 h, and high intravenous clearance (46.4 ± 10.9 mL/min/kg). The volume of distribution was 7.5 L/kg, indicating that 5al was widely distributed in tissues. Absolute oral bioavailability was found to be 35%. The CYP3A4 inhibition for compound 5al was assessed with testosterone, another probe substrate. It inhibited the testosterone hydroxylation with an IC50 value of 2.64 μM. Compound 5al was also evaluated for the inhibitory potential against other CYP isoforms, CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, and 2E1, in pooled human liver microsomes using specific CYP isoform marker activities. It had moderately inhibited mephenytoin hydroxylation (2C19) with IC50 values of 5.7 μM and did not inhibit the other CYP450 enzymes significantly (IC50 > 10 μM). Also the CYP induction potential of 5al was evaluated in human plateable hepatocytes using both mRNA expression and enzyme activities as end points. The results indicated that 5al did not induce CYP1A2, CYP2B6, and CYP3A4 at tested concentrations of 0.01, 0.1, and 1.0 μM. Compound 5al was evaluated in additional animal models of cognition and neurochemistry. It reversed the scopolamine induced emotional memory deficits in a contextual fear conditioning task at 1, 3, and 10 mg/kg p.o. dose (Figure 3).[1]

[1]. Discovery and Development of 1-[(2-Bromophenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole Dimesylate Monohydrate (SUVN-502): A Novel, Potent, Selective and Orally Active Serotonin 6 (5-HT6) Receptor Antagonist for Potential Treatment of Alzheimer's Disease. J Med Chem.2017 Mar 9;60(5):1843-1859.

Optimization of a novel series of 3-(piperazinylmethyl) indole derivatives as 5-hydroxytryptamine-6 receptor (5-HT6R) antagonists resulted in identification of 1-[(2-bromophenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole dimesylate monohydrate (Masupirdine/SUVN-502 (5al)) as a clinical candidate for potential treatment of cognitive disorders. It has high affinity at human 5-HT6R (Ki = 2.04 nM) and selectivity over 100 target sites which include receptors, enzymes, peptides, growth factors, ion channels, steroids, immunological factors, second messengers, and prostaglandins. It has high selectivity over 5-HT2A receptor. It is orally bioavailable and brain penetrant with robust preclinical efficacy. The combination of 5al, donepezil, and memantine (triple combination) produces synergistic effects in extracellular levels of acetylcholine in the ventral hippocampus. Preclinical efficacy in triple combination and high selectivity over 5-HT2A receptors are the differentiating features which culminated in selection of 5al for further development. The Phase-1 evaluation of safety and pharmacokinetics has been completed, allowing for the initiation of a Phase-2 proof of concept study. [1] In summary, the focused SAR around the 3-(piperazinylmethyl) indole scaffold led to the finding that the central indole core is optimal for affinity at 5-HT6R. Replacing it with 4-azaindole, 7-azaindole, or indazole resulted in moderately potent compounds. Smaller halo and alkoxy substitutions were tolerated, preferably at the C5 position of indole. 3-(Piperazinylmethyl) indole is optimal for affinity. Further chain elongation, i.e. 3-(piperazinylethyl)indole, is not tolerated. N-Aryl sulfonamide is critical for affinity; replacing it with benzyl and benzoyl gave less potent compounds. Halo and lower alkoxy are preferred substitutions on the N-aryl sulfonamide ring. The in vitro affinity, selectivity, physicochemical properties, in vivo efficacy, and safety evaluations resulted in the identification of Masupirdine/SUVN-502 (5al), as a developmental candidate. 5al is a crystalline dimesylate monohydrate salt with very high water solubility and permeability. It weakly inhibited recombinant cytochrome P450 3A4 while it does not inhibit CYP 2D6. Compound 5al is an orally bioavailable and brain penetrant 5-HT6R antagonist with robust efficacy in cognition models such as ORT and the contextual fear conditioning model. The triple combination of 5al, donepezil, and memantine produced additive augmentation in extracellular levels of acetylcholine in the ventral hippocampus without any cholinergic side effects, thus providing a neurochemical basis for the pro-cognitive activity observed in various behavioral models. The preclinical efficacy in triple combination coupled with selectivity over the 5-HT2A receptor is the differentiating feature of this compound. Compound 5al is nonmutagenic and nonclastogenic. The clinical portions of the single and multiple ascending dose studies assessing safety and pharmacokinetics have been completed, allowing for the initiation of the Phase-2 proof of concept study. The complete biological characterization of 5al covering the detailed rationale for the use of a triple combination in a Phase-2 POC trial will be reported in a forthcoming biology publication.[1]

C22H28BRN3O6S2

574.508222579956

669.048

C, 41.19; H, 4.81; Br, 11.92; N, 6.27; O, 21.47; S, 14.34

1791396-46-7

Masupirdine free base;701205-60-9

118142730

Pale purple to purple solid powder

2

11

5

39

743

0

CN1CCN(CC1)CC2=CN(C3=C2C=C(C=C3)OC)S(=O)(=O)C4=CC=CC=C4Br.CS(=O)(=O)O.CS(=O)(=O)O

LOZVXBFXRPRECW-UHFFFAOYSA-N

InChI=1S/C21H24BrN3O3S.2CH4O3S/c1-23-9-11-24(12-10-23)14-16-15-25(20-8-7-17(28-2)13-18(16)20)29(26,27)21-6-4-3-5-19(21)22;2*1-5(2,3)4/h3-8,13,15H,9-12,14H2,1-2H3;2*1H3,(H,2,3,4)

1-(2-bromophenyl)sulfonyl-5-methoxy-3-[(4-methylpiperazin-1-yl)methyl]indole;methanesulfonic acid

SUVN-502 dimesylate; 1791396-46-7; Masupirdine mesylate; Masupirdine (mesylate); Masupirdine dimesylate; BFE1VOB95D; 1-(2-bromophenyl)sulfonyl-5-methoxy-3-[(4-methylpiperazin-1-yl)methyl]indole;methanesulfonic acid; SUVN502 mesylate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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

DMSO : ~25 mg/mL (~37.28 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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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