Neuropeptide Y (NPY) Y1 receptor
BIBO-3304 TFA: Y1 receptor (neuropeptide Y receptor subtype Y1)【2†L46】
IC50 values:
- Human Y1 receptor (SK-N-MC cells): 0.38 ± 0.06 nM【2†L66-L67】
- Human Y1 receptor (BHK cells): 0.69 ± 0.16 nM【2†L62-L63】
- Rat Y1 receptor (HEK 293 cells): 0.72 ± 0.42 nM【2†L67】
- Rat Y1 receptor (high affinity site in hypothalamus): 1.4 ± 0.9 nM (32 ± 6% of total receptors)【2†L77-L78】
Low affinity (IC50 >1000 nM) for human Y2, human/rat Y4, and human/rat Y5 receptors【2†L48-L49】【2†L70-L76】.

1. The novel Y1-selective argininamide derivative BIBO 3304 ((R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphen ylacetyl)-argininamide trifluoroacetate) has been synthesized and was examined for its subtype selectivity, its in vitro antagonistic properties and its food intake inhibitory properties. 2. BIBO 3304 displayed subnanomolar affinity for both the human and the rat Y1 receptor (IC50 values 0.38+/-0.06 nM and 0.72+/-0.42 nM, respectively). The inactive enantiomer of BIBO 3304 (BIBO 3457) had low affinity for both the human and rat Y1 receptor subtype (IC50> 1000 nM). BIBO 3304 showed low affinity for the human Y2 receptor, human and rat Y4 receptor as well as for the human and rat Y5 receptor (IC50 values > 1000 nM). [1]

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In order to test the physiological relevance of Y1 receptor signaling in human islets, human islets were cultured and stimulated with glucose in the presence or absence of the Y1 receptor antagonist BIBO3304. Similar to rodent islets, human islets treated with BIBO3304 exhibited enhanced insulin secretion in response to a glucose challenge [2].
As shown in Fig. 4f, cAMP was significantly upregulated in the presence of 100 nM Exenatide and 20 mM glucose compared to glucose alone. The addition of 50 nM PYY was able to significantly reduce the Exenatide-induced increase in cAMP and this inhibitory effect was abolished in the presence of the Y1 receptor specific antagonist BIBO3304 [2].

BIBO3304 TFA (30 μg; bilateral paraventricular nucleus injection) reduces postfasting hyperphagia [1]. BIBO3304 TFA (15-60 μg) dose-dependently inhibits the feeding response mediated by 1 μg NPY [1]. BIBO3304 TFA (0.5 μM; orally) significantly elevates blood insulin levels [2].

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3. 30 microg BIBO 3304 administered into the paraventricular nucleus inhibited the feeding response induced by 1 microg NPY as well as the hyperphagia induced by a 24 h fast implying a role for Y1 receptors in NPY mediated feeding. The inactive enantiomer had no effect. 4. BIBO 3304 inhibits neither the galanin nor the noradrenaline induced orexigenic response. but it blocked feeding behaviour elicited by both [Leu31, Pro24]NPY and NPY (3 36) suggesting an interplay between different NPY receptor subtypes in feeding behavior. 5. The present study reveals that BIBO 3304 is a subtype selective nonpeptide antagonist with subnanomolar affinity for the Y1 receptor subtype that significantly inhibits food intake induced by application of NPY or by fasting [1].

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Mice that received BIBO3304 exhibited significantly increased serum insulin levels (Fig. 1l, m) confirming that blocking Y1 receptor signaling is able to enhance physiologically triggered insulin secretion. To further test whether this pharmacological intervention could improve the outcome of islet transplantation, we repeated the transplantation experiments using a minimal number of WT islets, and then treated half of the mouse cohort orally with BIBO3304 and the other half with placebo. As a control for islet quality, a group of mice were transplanted with an optimal islet mass. Recipient mice that were treated with placebo failed to achieve normoglycemia and remained diabetic throughout the course of the experiment (Fig. 2a, b). In contrast, mice transplanted with the minimal amount of WT islets and treated with BIBO3304 rapidly achieved normoglycemia (Fig. 2a). Strikingly, when antagonist treatment was stopped at day 10 post-transplantation these mice were able to maintain normoglycemia till the end of the experiment at day 60 (Fig. 2a). This suggests that transient inhibition of Y1 receptor signaling in the early period after islet transplantation is sufficient to normalize glucose homeostasis. Consistent with the improved glycemic control, glucose tolerance at day 5 post-transplantation was also significantly improved in the BIBO3304-treated group compared to the placebo group (Fig. 2c, d). Consistently, BIBO3304-treated mice also showed a superior DIo being 12.28-fold higher than placebo-treated mice (DIo = 0.066 ± 0.0082 vs. 0.819 ± 0.324 for placebo vs. BIBO3304, respectively; n = 4 mice per group). Analysis of BIBO3304-treated compared to untreated islet grafts did not reveal any obvious differences in islet morphology, apoptosis, graft vascularization, or endoplasmic reticulum (ER) stress response (Supplementary Figs. 3, 4a), however, the number of Ki67-positive β-cells in the BIBO3304-treated grafts was significantly increased (Fig. 2e, f). Together, these data demonstrate that the improved glycemic control in BIBO3304-treated mice was a consequence of increased insulin secretion and islet proliferative capacity in response to changes in physiological glucose levels (Fig. 2e–h)[2].
BIBO-3304 TFA: In Vivo: BIBO-3304 TFA (30 μg, unilateral PVN injection) inhibited the feeding response induced by 1 μg NPY in rats by approximately 50% (food intake: 1.87 ± 0.3 g vs. 3.58 ± 0.33 g for NPY alone, n=18)【2†L151-L155】. The inactive enantiomer BIBO 3457 had no effect【2†L155-L157】. BIBO-3304 TFA (30 μg) did not inhibit noradrenaline (30 μg) or galanin (4 μg) induced feeding【2†L157-L162】. BIBO-3304 TFA blocked feeding elicited by NPY (2-36) (1 μg), NPY (3-36) (1 μg), and [Leu31, Pro34]NPY (2 μg)【2†L163-L168】. Bilateral PVN injection of 15 μg BIBO-3304 TFA per side (total 30 μg) attenuated hyperphagia in 24 h fasted rats, particularly during the first 2 h of refeeding【2†L170-L172】. Oral administration of 0.5 μM BIBO-3304 TFA 2 h prior to a glucose load significantly increased serum insulin levels in C57BL/6 mice. In alloxan-induced diabetic mice transplanted with a minimal mass (60 islets) of wild-type islets, daily oral treatment with 0.5 μM BIBO-3304 TFA for 9 days rapidly achieved normoglycemia, which was maintained even after treatment cessation (up to day 60 and >260 days). The treatment also significantly improved glucose tolerance and increased the insulin disposition index. In human islet transplant recipients (NOD RAG1-/- mice), oral BIBO-3304 TFA (0.5 μM) accelerated recovery to normoglycemia, improved glucose tolerance, and increased circulating human insulin levels. In a full MHC mismatch allogeneic islet transplant model, BIBO-3304 TFA restored metabolic function of a minimal islet mass. In NOD mice, daily treatment with 0.5 μM BIBO-3304 TFA from 6 weeks of age delayed the onset of hyperglycemia by 2-4 weeks. Increasing the dose 10-fold (to 5 μM) after diagnosis of hyperglycemia (>12 mM) reversed the trend and further delayed diabetes onset. BIBO-3304 TFA treatment also significantly improved glucose tolerance in NOD mice at 15 and 19 weeks of age. [1][2]

BIBO-3304 TFA: Receptor binding assays were performed using membrane homogenates from various cell lines expressing different NPY receptor subtypes or from rat hypothalamus【2†L54-L87】. For the rat hypothalamus, the tissue was homogenized in sucrose buffer and centrifuged, and the resulting pellet was resuspended in a Krebs-Ringer buffer【2†L54-L57】. The membrane preparation was incubated with 30 pM [125I]NPY and increasing concentrations of BIBO-3304 TFA (10⁻¹³ - 10⁻⁴ M) for 45 min at 37°C【2†L57-L59】. Non-specific binding was determined in the presence of 100 nM NPY【2†L60】. For cell-based assays, SK-N-MC (human neuroblastoma, endogenous Y1), SMS-KAN (human neuroblastoma, Y2), BHK cells (stably expressing human Y1, Y2, or rat Y4), HEK 293 cells (stably expressing rat Y1 or human Y5), and CHO cells (transiently transfected with human Y4 or rat Y5) were used【2†L62-L87】. Cells were incubated with 30 pM [125I]NPY and varying concentrations of BIBO-3304 TFA for 2-3 hours at room temperature【2†L62-L87】. Bound radioactivity was measured after centrifugation and washing【2†L59-L60】. The ability of BIBO-3304 TFA to antagonize NPY-mediated inhibition of cAMP synthesis was assessed in SK-N-MC cells【2†L91-L99】. Cells were preincubated with papaverine and the antagonist, then stimulated with NPY and forskolin【2†L94-L96】. After incubation, cAMP levels were measured using a commercial kit【2†L97-L99】. The pKb value was determined from dose-ratios of NPY dose-response curves in the presence and absence of the antagonist【2†L106-L107】. For cAMP measurement in islets, islets were cultured with glucose, Exenatide (GLP-1 receptor agonist), and/or PYY in the presence of IBMX. Intracellular cAMP was measured using an ELISA kit and normalized to total protein content. [1][2]

Human Y1 receptor stably expressed in baby hamster kidney (BHK) cells [1]
Cells were grown in DMEM with 4.5 g/l glucose, 10% fetal calf serum, 1% PENStrep, 1 mg ml71 G-418, 1 mg ml71 hygromycin B. 96 h before receptor binding assay 1 mM isopropylthiogalactoside (IPTG) was added in order to induce expression (Lac Switch Expression System from Stratagene). Con¯uent cells were removed with 0.06% EDTA/PBS (1 min incubation) and resuspended in 15 ml incubation bu€er (MEM/25 mM HEPES+1% bovine serum albumine, 50 mM PMSF, 0.1% bacitracin, 3.75 mM CaCl2). After 10 min centrifugation at RT (150 6 g), the pellet was resuspended in 50 ml incubation bu€er, respun and resuspended in 30 ml incubation bu€er. After counting, the cells were diluted to a ®nal concentration of 2.5 6 105 cells ml71 . Two hundred microliters of this cell suspension was incubated 3 h at RT with 30 pM [ 125I]NPY and increasing concentrations of test compounds (10713 ± 1074 M) in a total volume of 250 ml. The incubation was stopped by 10 min centrifugation, 3000 6 g at 48C. The pellet was resuspended with 0.25 ml PBS recentrifuged and the pellet measured in a g-counter.

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Rat Y1 receptor expressing human embryonic kidney (HEK) 293 cells [1]
Confluent cells were removed with 0.02% EDTA/PBS and resuspended in 10 ml incubation bu€er (MEM/25 mM HEPES+0.5% BSA, 50 mM PMSF, 0.1% bacitracin, 3.75 mM CaCl2). After 5 min centrifugation (150 6 g) the pellet was resuspended in equal volume and after further centrifugation in 10 ml incubation bu€er. The cells were diluted to a concentration of one mio cells ml71 . 100 ml of this cell suspension was incubated 3 h at RT with 30 pM [ 125I]NPY solutions and increasing concentrations of test compounds in a total volume of 250 ml. The incubation was stopped as described for rat hypothalamus.

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Human Y5 receptor stably transfected in HEK 293 cells [1]
Centrifuged cells were cultivated as described for BHK/Y1 cells except that a concentration of 0.7 mg ml71 G-418 was used, no hygromycin added and IPTG induction was not necessary. The incubation bu€er cell cultivation and receptor binding was performed as described. Final concentration was 550 H.A. Wieland et al Novel Y1 receptor antagonist BIBO 3304 and its effect on feeding 1.5 6 106 cells ml71 and centrifugation stopped as described for Y1/BHK cells.

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BIBO-3304 TFA: For the ex vivo glucose-stimulated insulin secretion (GSIS) assay, islets were isolated from mice and cultured for 48 hours. Batches of five islets were pre-incubated in KRB buffer with 2 mM glucose for 1 hour, then incubated for 1 hour in KRB buffer with 0.1% BSA and various glucose concentrations (2, 11, or 20 mM) with or without 100 nM Exenatide, 50 nM PYY, and/or 0.5 μM BIBO-3304 TFA. Insulin release was measured by radioimmunoassay. For islet perfusion studies, islets were perfused with 11 mM glucose and insulin release was measured over time. For the [Ca²⁺]i measurement, single β-cells were dispersed from islets and loaded with fura-2. Cytosolic Ca²⁺ concentrations were measured by dual-wavelength microfluorometry. For immunofluorescence, pancreatic or graft sections were stained with antibodies against insulin, Ki67, p-CREB, and CD31. For western blotting, islet lysates were probed with phospho-specific CREB antibody. For quantitative RT-PCR, RNA was extracted from islets and gene expression was analyzed for Gk, Pc, Mdh2, Hadh, and other genes. [1][2]

Animal/Disease Models: Adult male Chbb:Thom rat, body weight 300 to 340 g [1]
Doses: 30 μg
Route of Administration: Bilateral paraventricular nucleus injection
Experimental Results: Hyperphagia after fasting was attenuated, especially before refeeding Within 2 hrs (hrs (hours)).

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Animal/Disease Models: 7weeks old C57BL/6JAusb mice[2]
Doses: 0.5 μM
Route of Administration: Oral
Experimental Results:Serum insulin levels were Dramatically increased.

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Food-intake studies Adult male Chbb:Thom rats weighing between 300 and 340 g were individually housed and maintained on a 12 : 12 h lightdark cycle beginning at 06.00 h. Tap water and standard laboratory chow were available throughout except in the experiments where the animals were fasted for 24 h. After 1 week of habituation to their new housing conditions, the animals were anaesthetized with sodium pentobarbital (60 mg kg71 , i.p) for the placement of stainless steel guide cannulae. Bilateral guide cannulae (26 gauge) were placed 1 mm above the paraventricular nucleus according to the stereotaxic coordinates (Paxinos & Watson, 1986): AP:71.8, L:0.5, V:7.0. Guide cannulae were maintained in place on the skull with small metal screws and dental acrylic cement. Cannulae were closed with a stainless steel xstylet when not in use. Rats were allowed to recover for at least 1 week and were adapted to the injection procedure. On the day of the experiments drugs were injected between 08.00 and 09.00 h. Injection cannulae (33 gauge) were inserted 1 mm beyond the tips of the guide cannulae. The injection cannulae were attached by polyethylene tubing to a Hamilton microsyringe mounted in an infusion pump. Injection volume was 0.5 ml infused with a rate of 0.0125 ml s71 . In the ®rst set of experiments groups of 6 ± 12 rats received increasing doses (0.5 ± 32 mg, unilateral) of NPY receptor agonists into the PVN and food intake was monitored for at least 2 h. On the ®rst treatment day the groups were randomly assigned to the various doses. Rats had a wash-out period of at least 3 days between injections, after which the groups were randomized again to test the next agonist. Not more than 5 ± 6 injections were given in total. In the second set of experiments BIBO 3304 or its inactive enantiomer were given 10 min before the injection of di€erent NPY receptor agonists, galanin or noradrenaline. All compounds were applied into the PVN and for each experiment 8 ± 22 rats were used and for each dose a di€erent group of rats were used. In the last series of experiments BIBO 3304 was given to animals which were fasted for 24 h. Five minutes after bilateral PVN injection of BIBO 3304 the rats (n=12) were given free access to food and food intake was monitored for another 24 h.[1]

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Y1lox/lox mice were generated as previously described and crossed with mice expressing the Cre recombinase gene under the control of the rat insulin-2 promoter Tg(Ins2-cre)25Mgn/J(INS2cre/+) to generate Y1lox/lox/INS2cre/+ mice. INS2cre/+ mice were also crossed onto Gt(ROSA)26Sor tm9(EGFP/Rpl10a)Amc/J mice in order to produce β-cell specific expression of the EGFP–L10a fusion protein for mRNA isolation and qPCR analysis. For clarity, islet tissue from these mice is referred to as WT and Y1−/− respectively, throughout the text. Age-matched and sex-matched mice on a C57BL/6Ausb background were used for all experiments except stated otherwise. Female NOD mice were housed under a controlled temperature of 22 °C and a 12-hour light cycle (lights on from 0700 to 1900 hours) with ad libitum access to water and a standard chow diet (6% calories from fat, 21% calories from protein, 71% calories from carbohydrate, 14.0 MJ/kg). To avoid the stress caused by gavage, specific Y1 receptor antagonist BIBO 3304 was dissolved in distilled water and administrated daily in a form of jelly for the duration stated in the text. This method of drug delivery was developed in our lab and described previously31. In brief, we trained mice to voluntarily eat a vehicle jelly before the start of an experiment. After 2–5 days training, over 95% of mice consumed the entire portion of jelly (195 μl for a 25 g mouse) within 1 min of being placed in the cage and maintained a high avidity for jelly throughout the study period. At the commencement of an experiment, mice received BIBO 3304 containing jelly once per day for the time period indicated in each study, while control mice received vehicle jelly. [2]

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BIBO-3304 TFA: For the food intake studies, adult male Chbb:THOM rats (300-340 g) were housed individually and implanted with bilateral guide cannulae targeting the PVN【2†L114-L120】. BIBO-3304 TFA or its inactive enantiomer was injected into the PVN (0.5 μL volume at 0.0125 μL/s) 10 minutes before the injection of NPY receptor agonists, galanin, or noradrenaline【2†L122-L132】. Food intake was monitored for 2 hours【2†L131】. In another experiment, BIBO-3304 TFA was administered bilaterally into the PVN of rats fasted for 24 hours, and food intake was monitored for 24 hours【2†L132-L134】. For the islet transplantation studies, diabetes was induced in recipient mice by intravenous injection of alloxan (110 mg/kg). Islets were isolated from donor mice and transplanted under the kidney capsule. BIBO-3304 TFA was dissolved in distilled water and administered daily in a jelly form. Mice were trained to consume the jelly voluntarily. The dose used was 0.5 μM BIBO3304 in the jelly. In some experiments, the dose was increased to 5 μM. Treatment was given for various durations (e.g., 9 days, 10 days) and then discontinued. Blood glucose levels were monitored regularly. Glucose tolerance tests were performed after a 6-hour fast by intraperitoneal or intravenous administration of glucose (1 g/kg). Serum insulin levels were measured by ELISA or RIA. Nephrectomy was performed to confirm graft dependence. For human islet transplantation, immunodeficient NOD RAG1-/- mice were used. Human islets were transplanted under the kidney capsule, and recipient mice were treated daily with placebo or BIBO-3304 TFA (0.5 μM). [1][2]

[1]. Subtype selectivity of the novel nonpeptide neuropeptide Y Y1 receptor antagonist BIBO 3304 and its effect on feeding in rodents. Br J Pharmacol. 1998 Oct;125(3):549-55.

[2]. Inhibition of Y1 receptor signaling improves islet transplant outcome. Nat Commun. 2017 Sep 8;8(1):490.

Insufficient insulin secretion is a pathological feature of both type 1 and type 2 diabetes and reduces the success rate of islet cell transplantation. This article demonstrates that the Y1 receptor signaling pathway inhibits insulin release from β cells and shows that insulin secretion can be enhanced through pharmacological means. Transplantation of Y1 receptor-deficient islets can accelerate the recovery of hyperglycemia to normal in chemically induced diabetic recipient mice, and short-term drug blocking of the Y1 receptor in transplanted mouse and human islets can achieve the same effect. In addition, treatment of non-obese diabetic mice with Y1 receptor antagonists can delay the onset of diabetes. Mechanistically, the Y1 receptor signaling pathway inhibits cAMP production in islets, thereby downregulating the activity of several key enzymes in glycolysis and ATP production through the CREB-mediated pathway. Therefore, regulating the Y1 receptor signaling pathway in β cells provides a unique therapeutic opportunity to correct the pathological state of type 1 diabetes and the insulin deficiency that occurs during islet transplantation. Islet transplantation is considered one of the potential treatments for type 1 diabetes, but low islet survival and impaired function limit its application. Loh et al. found that the Y1 receptor is expressed in β cells, and inhibiting its signaling pathway (including genetic and pharmacological methods) can improve islet function in mice and humans. [2] Since Y1 receptors may be involved in anxiolytic effects (Wahlestedt et al., 1993; Kask et al., 1996), antagonizing Y1 receptors may produce anxiety-like side effects. To assess whether the possible anxiety effect would interfere with BIBO 3304-mediated inhibition of the feeding response, we examined the interaction between BIBO 3304 and feeding responses mediated by other stimulants, such as norepinephrine and galanine. If anxiety plays a role in BIBO 3304-mediated inhibition of food intake, then norepinephrine- and galanine-induced food intake should also be inhibited. However, since neither galanine nor norepinephrine-induced food intake was inhibited, a significant anxiety component in the food intake inhibition properties of BIBO 3304 can be ruled out. Previous studies have shown that the feeding antagonism mediated by BIBP 3226 is not caused by general side effects under experimental conditions (O'Shea et al., 1997; Kask et al., personal communication). However, the data obtained using BIBP 3226 were not conclusive (Doods et al., 1996). Therefore, we tested the inactive enantiomer of BIBO 3304 to demonstrate that the inhibition of food intake was not due to the general toxicity of the structural components of BIBO 3304. In fact, the enantiomer did not affect the feeding response. This study identified BIBO 3304, a non-peptide compound that is active in the sub-nanomolar concentration range and selective for Y1 receptor subtypes. We hypothesize that Y1 receptors play an important role in NPY-induced feeding, and that BIBO 3304 is a novel tool for studying food intake and other central effects mediated by Y1 receptors. Furthermore, data using BIBO 3304 and agonists (e.g., [Leu31, Pro34]NPY) suggest that there are complex interactions among different NPY receptors in feeding behavior. [1]

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BIBO-3304 TFA: BIBO-3304 TFA is a non-peptide Y1 receptor antagonist with subnanomolar affinity and high selectivity for the Y1 receptor subtype【2†L46】. It is a valuable tool for studying the role of Y1 receptors in food intake and other central effects【2†L181-L183】. The compound acts by inhibiting Y1 receptor signaling, which reduces cAMP production and, via CREB-mediated pathways, downregulates key enzymes in glycolysis and ATP production, thereby enhancing insulin secretion. In the context of islet transplantation, short-term treatment with BIBO-3304 TFA can provide stable, long-term improvement in islet graft function. The compound has also been shown to delay the onset of diabetes in NOD mice, suggesting a potential clinical application in prolonging the honeymoon period in new-onset type 1 diabetes patients. [1][2]

C29H35N7O3

529.63330578804

643.273

C, 52.31; H, 4.92; F, 15.04; N, 12.94; O, 14.78

191868-14-1

191868-13-0;191868-14-1 (TFA);

5311021

White to off-white solid powder

5.748

7

9

13

46

862

1

O=C(C(C1C=CC=CC=1)C1C=CC=CC=1)N[C@@H](C(NCC1C=CC(CNC(N)=O)=CC=1)=O)CCC/N=C(\N)/N

FBMCYYWIBYEOST-GJFSDDNBSA-N

InChI=1S/C29H35N7O3.C2HF3O2/c30-28(31)33-17-7-12-24(26(37)34-18-20-13-15-21(16-14-20)19-35-29(32)39)36-27(38)25(22-8-3-1-4-9-22)23-10-5-2-6-11-23;3-2(4,5)1(6)7/h1-6,8-11,13-16,24-25H,7,12,17-19H2,(H,34,37)(H,36,38)(H4,30,31,33)(H3,32,35,39);(H,6,7)/t24-;/m1./s1

(2R)-N-[[4-[(carbamoylamino)methyl]phenyl]methyl]-5-(diaminomethylideneamino)-2-[(2,2-diphenylacetyl)amino]pentanamide;2,2,2-trifluoroacetic acid

191868-14-1; BIBO 3304 TRIFLUOROACETATE; BIBO3304; BIBO3304 (TFA); BIBO3304 TFA; BIBO-3304; BIBO 3457; BIBO-3457;

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 : ~100 mg/mL (~155.36 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.88 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 (3.88 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 (3.88 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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 (Please use freshly prepared in vivo formulations for optimal results.)