Motilin Receptor ( pEC50 = 7.9 )
Motilin receptor (GPR38), a G protein-coupled receptor found on smooth muscle cells and enteric neurons in the stomach and duodenum. In vitro studies have demonstrated that camicinal selectively activates the human motilin receptor with a pEC50 of 7.9, which is comparable to erythromycin's pEC50 of 7.3. The compound shows no significant activity at a wide range of other receptors, including the structurally related ghrelin receptor (pEC50 < 6.0), demonstrating excellent selectivity for the motilin receptor over closely related GPCR targets.

GSK962040 hydrochloride, also known as camecicinal hydrochloride, exhibits negligible effects on ghrelin and other receptors, ion channels, and enzymes. When Camicinal hydrochloride (GSK962040 hydrochloride) was administered at 300 nmol L 1-10 μmol L 1, the cholinergic-mediated contraction amplitude in the rabbit stomach antrum was prolonged and reached a maximum of 248 ± 47% at 3 μmol L 1. The following were found in the samples: 10.4±0.01 (n=770), 7.3±0.29 (n=4), and 7.9±0.09 (n=17) for motilin, erythromycin, and Camicinal hydrochloride (GSK962040 hydrochloride) [1]. Dog motilin receptor activation is caused by camicinal hydrochloride (GSK962040 hydrochloride) (pEC50 5.79; intrinsic activity 0.72 relative to [Nle13]-motilin) [2]. Because its initial IC50 value on CYP3A4 was much greater than our recommended threshold of 10 μM [3], camicinal hydrochloride (GSK962040 hydrochloride) was preferred.
Camicinal demonstrates potent agonistic activity at the human motilin receptor in vitro with a pEC50 of 7.9, comparable to the natural ligand motilin and the macrolide erythromycin (pEC50 7.3). Activation of the motilin receptor on gastrointestinal smooth muscle cells triggers a cascade of intracellular signaling events leading to muscle contraction, mediated primarily through the Gαq subunit of the associated G protein, which stimulates phospholipase C (PLC) to hydrolyze PIP2 into IP3 and DAG, ultimately resulting in smooth muscle contraction. The compound activates long-lasting cholinergic contractility at low doses in the antrum with greater efficacy than current therapeutic options.

Camicinal (GSK962040) (5 mg free base·kg 1) also raises the overall weight of the feces after two hours after administration (21.2 ± 4.5 g; P < 0.05). [1]. Camicinal (GSK962040) causes mean plasma concentrations >1.14 μmol L 1 to cause dose-related phasic contractions that last 48 and 173 minutes for 3 and 6 mg kg 1, respectively. Migratory motor complex (MMC) activity returns when the effects of Camicinal (GSK962040) wear off. The 3 mg kg 1 Camicinal (GSK962040) had no effect on the migration of motor complex recovery; however, at the 6 mg kg 1 dose, MMC recovered 253 minutes after delivery as opposed to 101 minutes after saline (n = 5 each).[2]. The oral bioavailability (Fpo) of camicinal (GSK962040) is 48 (13%). In contrast to short-term effects, camicinal (GSK962040) demonstrated long-term effects (T1/2) 46.9 (5.0 min at 3 μM) [Nle13].Motilin (T1/2) 11.4 (at 0.3 μM) for 1.5 minutes [3]. The compound camicinal (GSK962040) is less active in the fundus and small intestine but significantly increases cholinergic activity in the gastric antrum [4].
In vivo, camicinal activates motilin receptors in the stomach and upper intestine, stimulating coordinated gastric contractions and accelerating gastric emptying. In a randomized, double-blind, placebo-controlled study in mechanically ventilated feed-intolerant critically ill patients, a single enteral dose of 50 mg camicinal showed a trend toward accelerated gastric emptying (adjusted geometric means: pre-treatment BTt1/2 117 minutes vs. post-treatment 76 minutes; 95% CI 0.39-1.08) and increased glucose absorption (AUC240min pre-treatment: 28.63 mmol.min/L vs. post-treatment: 71.63 mmol.min/L; 95% CI 1.68-3.72). When patients who did not have detectable plasma concentrations of camicinal were excluded, camicinal significantly accelerated gastric emptying (pre-treatment BTt1/2 121 minutes vs. post-treatment 65 minutes; 95% CI 0.32-0.91). No tolerance effect was observed in patients with gastroparesis and type 1 diabetes after repeat dosing over 14 days.

Motilin Receptor Agonist FLIPR Assay[3]
The potency and efficacy of target compounds at the human motilin receptor were studied using a fluorometric imaging plate reader (FLIPR) and Chinese hamster ovary (CHO-K1) cells which stably express the human motilin receptor. Briefly, the human motilin receptor was cloned (PCR from human genomic DNA) into pCDNA3.1 in the vector, subcloned into pENTR/D-TOPO (using the pENTR D-TOPO directional cloning kit) and then recombined into pCIN1GW vector (LR clonase gateway reaction kit) to give the pCIN1 motilin receptor. CHO-K1 cells (ATCC No. CCl-61) were then stably transfected with the pCIN1 motilin receptor plasmid. These cells were grown as a monolayer in DMEM/HamF-12 supplemented with 10% v/v of FBS, 2 mM GlutaMAX, and 1 mg/mL Geneticin. Stimulation of this cell line with motilin causes intracellular signaling leading to an increase in intracellular calcium which was measured using calcium sensitive fluorescent dyes and quantified using a FLIPR. Briefly, cells were seeded onto 384-well black-walled, clear-bottom microtiter plates (10 000 cells/well) and incubated for 24 h. On the day of assay, media were aspirated from cell plates using a cell washer (leaving 10 μL of media). Cells were immediately loaded with loading buffer (Tyrodes (Elga water + 145 mM NaCl + 5 mM KCl + 20 mM HEPES + 10 mM glucose + 1 mM MgCl2) + 1.5 mM CaCl2 + 0.714 mg/mL Probenicid (predissolved in 1 M NaOH) + 0.5 mM brilliant black + 2.5 μM Fluo 4 dye) and incubated at 37.5 °C for 1 h. Master compound plates were prepared in 100% DMSO. A top concentration of 3 mM was used (giving 12 μM final concentration in assay), and this was serially diluted 1 in 4. Then 1 μL from the master plate was transferred to a daughter plate, to which 50 μL of compound dilution buffer (Tyrodes + 1 mg/mL BSA + 1.5 mM CaCl2) was added. An amount of 10 μL from the compound plates was then added immediately to cell plates using a FLIPR 3 calcium imaging instrument, and changes in fluorescence were measured over a 1 min time frame. Maximum change in fluorescence over baseline was used to determine agonist response, and concentration response curves were constructed using a four-parameter logistic equation. The intrinsic activity of target compounds was calculated by using the maximum asymptote of its concentration−response curve relative to the maximum asymptote of the motilin concentration−response curve.

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CYP 3A4 Time Dependent Inhibition (TDI) Assay[3]
Inhibition was determined by quantifying the production of fluorescent metabolite following incubation of CYP3A4 specific profluorescent probe substrate diethoxyfluorescein (DEF), with heterologously expressed CYP3A4 in E. coli (Cypex) and the test compound or positive control (troleandomycin). A NADPH-regenerating system (cofactor) was prepared as follows: 7.8 mg/mL glucose 6-phosphate (27.65 mM), 1.7 mg/mL NADP (2.22 mM), and glucose 6-phosphate dehydrogenase at 6 enzyme units/mL were made up in 2% w/v sodium bicarbonate solution. Incubation mixtures containing enzyme, probe substrate, and 50 mM potassium phosphate buffer (at pH 7.4) were prepared, and 220 μL was added to each well of a 96-well plate. An amount of 5 μL of the serially diluted test compounds was added, and the plate was incubated at 37 °C for 10 min. To start the reaction, 25 μL of cofactor was added. The production of fluorescence was then measured every minute over a 30 min time frame at 37 °C. A summary of the assay conditions is given in Table 5.
In vitro enzyme/receptor binding assays for camicinal typically involve measuring the compound's ability to displace a radiolabeled motilin ligand from membranes expressing the human motilin receptor (GPR38). Competition binding experiments are performed using membrane preparations from cells transfected with the human motilin receptor, incubated with increasing concentrations of camicinal and a fixed concentration of radiolabeled motilin ligand. After incubation at room temperature for a specified period, bound and free ligand are separated by rapid filtration through glass fiber filters, and radioactivity is measured by scintillation counting. pEC50 values are determined from competition binding curves using nonlinear regression analysis.

There is an urgent clinical need for a safe, efficacious stimulant of gastric emptying; current therapies include erythromycin (an antibiotic with additional properties which preclude chronic use) and metoclopramide (a 5-hydroxytryptamine type 4 receptor agonist and an antagonist at brain D2 receptors, associated with movement disorders). To move away from the complex motilide structure of erythromycin, a small molecule motilin receptor agonist, GSK962040, was identified and characterized. The compound was evaluated using recombinant human receptors, rabbit and human isolated stomach preparations known to respond to motilin and in vivo, by measuring its ability to increase defecation in conscious rabbits. At the human motilin receptor, the pEC50 (the negative logarithm to base 10 of the EC50 value, the concentration of agonist that produces 50% of the maximal response) values for GSK962040 and erythromycin as agonists were, respectively, 7.9 and 7.3; GSK962040 had no significant activity at a range of other receptors (including ghrelin), ion channels and enzymes. In rabbit gastric antrum, GSK962040 300 nmol L(-1)-10 micromol L(-1) caused a prolonged facilitation of the amplitude of cholinergically mediated contractions, to a maximum of 248 +/- 47% at 3 micromol L(-1). In human-isolated stomach, GSK962040 10 micromol L(-1), erythromycin 10 micromol L(-1) and [Nle13]-motilin 100 nmol L(-1), each caused muscle contraction of similar amplitude. In conscious rabbits, intravenous doses of 5 mg kg(-1) GSK962040 or 10 mg kg(-1) erythromycin significantly increased faecal output over a 2-h period. Together, these data show that GSK962040, a non-motilide structure, selectively activates the motilin receptor. Simplification of the structural requirements to activate this receptor greatly facilitates the design of potentially new medicines for gastroparesis[1].
Cellular assays for camicinal typically utilize cell lines (such as CHO or HEK293 cells) stably or transiently transfected with the human motilin receptor (GPR38) to measure agonist-induced calcium mobilization or other downstream signaling events. Cells are loaded with a calcium-sensitive fluorescent dye (such as Fluo-4 or Fura-2) and fluorescence changes are monitored upon addition of increasing concentrations of camicinal. The potency of camicinal is expressed as pEC50, derived from concentration-response curves. These assays confirm that camicinal acts as a full agonist at the motilin receptor with comparable efficacy to the natural ligand motilin.

Rat Pharmacokinetic Studies[3]
The pharmacokinetics and oral bioavailability of the HCl salt of compound 12/Camicinal (GSK962040) were investigated in the male Sprague−Dawley rat (n = 3). The study was carried out on 2 study days with a period of 2 days between each study day. On study day 1, compound 12 was dissolved in 0.9% (w/v) saline at a target concentration of 0.2 mg of free base/mL. Compound 12 was administered as a 1 h intravenous infusion at 5 (mL/kg)/h to three rats to achieve a target dose of 1 mg of free base/kg. Serial blood samples were taken from each rat up to 12 h after the start of the infusion. On study day 2, compound 12 was suspended in 1% (w/v) methylcellulose at a target concentration of 1 mg of free base/mL. Three rats received an oral gavage dose of compound 12 administered at 5 mL/kg to achieve a target dose of 5 mg of free base/kg. Serial blood samples were taken from each rat up to 12 h after dosing. Diluted blood samples were analyzed for compound 12 by LC/MS/MS (LLQ was 5 ng/mL, 0.012 μM).
The systemic exposure of the HCl salt of compound 16 following oral suspension administration was investigated the male Sprague−Dawley rat. Compound 16 was dosed to three rats orally by gavage at a target dose of 5 mg of free base/kg. Compound 16 was prepared on the day of dosing in 1% (w/v) aqueous methylcellulose at a concentration of 1 mg of free base/mL and administered at 5 mL/kg. Serial blood samples were taken from each rat up to 8 h after dose administration. Diluted blood samples were analyzed for parent compound by LC/MS/MS.

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Dog Pharmacokinetic Studies[3]
The pharmacokinetics and oral bioavailability of the HCl salt of compound 12/Camicinal (GSK962040) were investigated in the male beagle dog (n = 3). The study was carried out on 2 study days with a period of 7 days between each study day. On study day 1, compound 12 was dissolved in 0.9% (w/v) saline at a concentration of 0.4 mg of free base/mL. Compound 12 was administered as a 1 h intravenous infusion at 5 (mL/kg)/h to three dogs to achieve a target dose of 2 mg of free base/kg. Serial blood samples were taken from each dog up to 30 h after the start of the infusion. On study day 2, compound 12 was suspended in 1% (w/v) methylcellulose at a concentration of 1 mg of free base/mL. The same three dogs each received an oral gavage dose of compound 12 administered at 5 mL/kg to achieve a target dose of 5 mg of free base/kg. Serial blood samples were taken from each dog up to 30 h after dosing. Diluted blood samples were analyzed for compound 12 by LC/MS/MS (LLQ was 5 ng/mL, 0.012 μM).

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Background: GSK962040, a small molecule motilin receptor agonist, was identified to address the need for a safe, efficacious gastric prokinetic agent. However, as laboratory rodents lack a functional motilin system, studies in vivo have been limited to a single dose, which increased defecation in rabbits. Motilin agonists do not usually increase human colonic motility, so gastric prokinetic activity needs to be demonstrated.
Methods: The effect of intravenous GSK962040 on gastro-duodenal motility was assessed in fasted dogs implanted with strain gauges. Activity was correlated with blood plasma concentrations of GSK962040 (measured by HPLC-MS/MS) and potency of GSK962040 at the dog recombinant receptor [using a Fluorometric Imaging Plate Reader (Molecular Devices, Wokingham, UK) after expression in HEK293 cells].
Key results: GSK962040 activated the dog motilin receptor (pEC(50) 5.79; intrinsic activity 0.72, compared with [Nle(13) ]-motilin). In vivo, GSK962040 induced phasic contractions, the duration of which was dose-related (48 and 173 min for 3 and 6 mg kg(-1) ), driven by mean plasma concentrations >1.14 μmol L(-1) . After the effects of GSK962040 faded, migrating motor complex (MMC) activity returned. Migrating motor complex restoration was unaffected by 3 mg kg(-1) GSK962040 but at 6 mg kg(-1) , MMCs returned 253 min after dosing, compared with 101 min after saline (n=5 each).
Conclusions & inferences: The results are consistent with lower potency for agonists at the dog motilin receptor, compared with humans. They also define the doses of GSK962040 which stimulate gastric motility. Correlation of in vivo and in vitro data in the same species, together with plasma concentrations, guides further studies and translation to other species.[2]
In vivo animal studies for camicinal typically involve administration to rodent or dog models to assess effects on gastrointestinal motility, gastric emptying rate, and pharmacokinetic parameters. In dogs, oral or intravenous administration of camicinal is followed by monitoring of gastric antral contractions using manometry or scintigraphy. Gastric emptying of solid or liquid meals is assessed using radiolabeled test meals or the 13C-octanoic acid breath test. Pharmacokinetic parameters including Cmax, Tmax, AUC, and bioavailability are determined from serial blood sampling. In critically ill patients, a single enteral dose of 50 mg camicinal demonstrated efficacy in accelerating gastric emptying.

The study found that the presence of a more polar cyano group was unfavorable to the oral pharmacokinetics of compound 16. In male Sprague-Dawley rats, the oral exposure of compound 16 was low and unstable, while the exposure of the fluorinated analog 12 was higher and more stable. After determining its intravenous pharmacokinetics, the oral bioavailability (Fpo) of compound 12 was found to be 48 ± 13%. This promising result prompted us to determine the pharmacokinetics of compound 12 in male beagle dogs, and we were pleased to find that its oral bioavailability was 51 ± 16%. [1] Compound 12 was also evaluated for its inhibitory effects on other major human CYP isoenzymes, and it was found to have good inhibitory properties (1A2, 2C19, 2C9 IC50 > 100 μM, 2D6 IC50 = 34 μM). In addition, no TDI interaction was observed on these isoenzymes on CYP3A4 with 7BQ as a substrate. It exhibits high selectivity for the closely associated human ghrelin receptor (pEC50 < 6.0) and no interaction was observed on hERG channels (pIC50 = 4.8 in binding assay). In vitro plasma protein binding is acceptable (83% in humans, 63% in rats), and it is highly soluble in water and a range of simulated gastrointestinal fluids (hydrochloride, >1 mg/mL). [1] Furthermore, the duration of action of compound 12 in native tissue assays of the rabbit antrum has been determined. Full results will be reported elsewhere, but in brief, compound 12 showed a sustained effect (T1/2 = 46.9 ± 5.0 min at 3 μM) compared to the short-acting effect of [Nle13]motilin (0.3 μM, T1/2 = 11.4 ± 1.5 min). Its duration of action is also longer than that of erythromycin 1 (T1/2 = 24.0 ± 5.6 min at 3 μM), which has been successfully used clinically for low-dose repeated administration to improve gastric emptying. Therefore, these data may suggest that compound 12 is less likely to develop rapid tolerance when administered appropriately in vivo. The overall selectivity of compound 12, its efficacy in human native gastric tissue, and its prokinetic activity in a rabbit whole intestinal transport model have also been determined, and these data will be reported in full elsewhere. [1]
Camicinal demonstrates favorable pharmacokinetic profiles with good oral bioavailability and systemic exposure. Following oral administration, the compound is absorbed and reaches peak plasma concentrations within a predictable timeframe. The compound shows linear pharmacokinetics across the studied dose range. Camicinal is well tolerated in multiple trials when given as a single oral dose of up to 125 mg. The compound is metabolized primarily in the liver, and its pharmacokinetic profile supports once-daily or twice-daily dosing. In the NUTRIATE study, some patients did not have detectable plasma concentrations of camicinal, suggesting variable absorption in the critically ill population.

Preclinical toxicity studies have demonstrated a favorable safety profile for camicinal with no liabilities at the hERG channel, supporting its progression into clinical development. In multiple clinical trials, camicinal was well tolerated with no serious drug-related adverse events or changes in ECG chemistry when given as a single oral dose of up to 125 mg. Adverse events occurred evenly between the placebo and treatment groups and were generally mild. No tolerance effect was observed after repeat dosing over 14 days. The compound meets the criteria of an ideal motilin agonist with low potential for off-target toxicity.

[1]. GSK962040: a small molecule, selective motilin receptor agonist, effective as a stimulant of human and rabbit gastrointestinal motility. Neurogastroenterol Motil, 2009. 21(6): p. 657-64, e30-1.

[2]. GSK962040: a small molecule motilin receptor agonist which increases gastrointestinal motility in conscious dogs. Neurogastroenterol Motil, 2011. 23(10): p. 958-e410.

[3]. Discovery of N-(3-fluorophenyl)-1-[(4-([(3S)-3-methyl-1-piperazinyl]methyl)phenyl)acetyl]-4-pi peridinamine (GSK962040), the first small molecule motilin receptor agonist clinical candidate. J Med Chem, 2009. 52(4): p. 1180-9.

[4]. Regional- and agonist-dependent facilitation of human neurogastrointestinal functions by motilin receptor agonists. Br J Pharmacol, 2012. 167(4): p. 763-74.

Camicinal belongs to the acetamide class of compounds. Camicinal has been used in clinical trials for the treatment of gastroparesis. N-(3-fluorophenyl)-1-[(4-([(3S)-3-methyl-1-piperazinyl]methyl)phenyl)acetyl]-4-piperidinamine 12 (GSK962040) is a novel small molecule motilin receptor agonist. It has excellent activity against both recombinant human motilin receptor and native rabbit motilin receptor, and its agonist activity can enhance the amplitude of nerve-mediated contractions in isolated gastric antrum tissue. Compound 12 has shown promising pharmacokinetic characteristics in rats and dogs, and these results, combined with further analysis in native human tissue and rabbit gastrointestinal transport models, make it a candidate for further development. [1] In summary, compound 12 exhibits very desirable overall properties and has been selected as a candidate for further development. Therefore, compound 12 is a novel selective small molecule motilin receptor agonist (molecular weight 424), which was developed through a broad structure-activity relationship study based on the activity of recombinant human motilin receptor and is the first compound of its kind to enter Phase I clinical trials, and is expected to be used to treat diseases associated with delayed gastric emptying. Therefore, compound 12 is a novel selective small molecule motilin receptor agonist (molecular weight 424), which was developed through a broad SAR program based on the activity of recombinant human motilin receptor and is the first compound of its kind to enter Phase I clinical trials, and is expected to be used as a drug to treat diseases associated with delayed gastric emptying. [1]
Camicinal (GSK962040) has been investigated in multiple Phase II clinical trials for the treatment of gastroparesis and other gastrointestinal motility disorders. The NUTRIATE study (NCT01039805) was a randomized, double-blind, placebo-controlled trial assessing the pharmacodynamic effects and pharmacokinetics of single doses of camicinal in critically ill feed-intolerant patients. Results showed that when absorbed, a single enteral dose of 50 mg camicinal accelerates gastric emptying and increases glucose absorption. The compound is a first-in-class, non-macrolide motilin receptor agonist designed to avoid the antibiotic resistance and off-target effects associated with macrolide prokinetics like erythromycin. Development status may vary, and further clinical studies may be needed to establish efficacy in larger patient populations.

C25H33N4OF.HCL

461.01506

460.24

C, 65.13; H, 7.43; Cl, 7.69; F, 4.12; N, 12.15; O, 3.47

923565-22-4

Camicinal;923565-21-3

25222897

Typically exists as solid at room temperature

3.542

3

5

6

32

560

1

O=C(N1CCC(NC2=CC=CC(F)=C2)CC1)CC3=CC=C(CN4C[C@H](C)NCC4)C=C3.Cl

MZEVMNJFEUATKJ-FYZYNONXSA-N

InChI=1S/C25H33FN4O.ClH/c1-19-17-29(14-11-27-19)18-21-7-5-20(6-8-21)15-25(31)30-12-9-23(10-13-30)28-24-4-2-3-22(26)16-24;/h2-8,16,19,23,27-28H,9-15,17-18H2,1H3;1H/t19-;/m0./s1

1-[4-(3-fluoroanilino)piperidin-1-yl]-2-[4-[[(3S)-3-methylpiperazin-1-yl]methyl]phenyl]ethanone;hydrochloride

Camicinal hydrochloride; 923565-22-4; GSK962040 (HCl salt); GSK962040 (hydrochloride); GSK962040 hydrochloride; 1-[4-(3-fluoroanilino)piperidin-1-yl]-2-[4-[[(3S)-3-methylpiperazin-1-yl]methyl]phenyl]ethanone;hydrochloride; CHEMBL489679;

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 (e.g. under nitrogen), 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)

H2O : ~100 mg/mL (~216.91 mM)
DMSO : ~100 mg/mL (~216.91 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.42 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.42 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (216.91 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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