HIF-PHD (hypoxia-inducible factor prolyl hydroxylases) (Ki = 5.3 nM)

Hypoxia-inducible factor prolyl hydroxylases (PHDs) inhibitor stabilizes hypoxia inducible factor alpha, which increases erythropoietin (EPO) expression via the hypoxia response element. Therefore, PHDs inhibitors have been developed as novel therapeutic agents for anemia. Here, we characterize the in vitro and in vivo pharmacological profiles of TP0463518, 2-[[1-[[6-(4-chlorophenoxy)pyridin-3-yl]methyl]-4-hydroxy-6-oxo-2,3-dihydropyridine-5-carbonyl]amino]acetic acid, a novel potent PHDs inhibitor. TP0463518 competitively inhibited human PHD2 with a Ki value of 5.3 nM. TP0463518 also inhibited human PHD1/3 with IC50 values of 18 and 63 nM as well as monkey PHD2 with an IC50 value of 22 nM.

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Inhibition of PHDs activity [1]
As previously reported, the IC50 values of TP0463518 for human and rat PHD2 were 13 and 18 nM when HIF1α peptide was used as a substrate (Hamada et al., 2018). To elucidate the inhibitory profiles of TP0463518, we evaluated the IC50 values for human PHD1 and PHD3 using HIF1α peptide. TP0463518 inhibited PHD1 with an IC50 value of 18 nM (Table 1). Although TP0463518 also inhibited PHD3, the IC50 values were 3.5 and 4.8 times higher than those of PHD1/2. When using HIF2α peptide as a substrate, TP0463518 inhibited all the PHDs at potencies comparable to those obtained using HIF1α peptide as a substrate. The inhibitory profiles for other species were also investigated. TP0463518 inhibited monkey PHD2 with an IC50 value of 22 nM. To elucidate the mode of inhibition, the maximum activity and Km values were measured at each of the TP0463518 concentrations. The maximum activities (mP values) were 46, 47 and 47 for 0, 20 and 40 nM of TP0463518, respectively. The Km values were 0.10 μM without TP0463518, whereas they were 0.32 and 0.61 μM for 20 and 40 nM of TP0463518, indicating competitive inhibition. Competitive inhibition was confirmed using a double-reciprocal plot (Fig. 2). Based on the competitive inhibition, the Ki value of TP0463518 was calculated as 5.3 nM.

In normal mice and rats, TP0463518 significantly increased the serum EPO levels at doses of 5 and 20 mg/kg, respectively. The correlation factors for serum EPO and the serum TP0463518 levels were 0.95 in mice and 0.92 in rats. TP0463518 also increased the serum EPO level in 5/6 nephrectomized chronic kidney disease model rats at a dose of 10 mg/kg, with a correlation factor for serum EPO and the serum TP0463518 levels of 0.82. Finally, the effect of TP0463518 in monkeys was investigated. TP0463518 was promptly removed with a half-life of 5.2 h and increased the area under the curve (AUC) of EPO at a dose of 5 mg/kg. The EPO and TP0463518 levels were also correlated. These results suggest that TP0463518 induces endogenous EPO with a strong pharmacokinetic-pharmacodynamic correlation and may contribute to desirable hemoglobin control in patients with renal anemia. [1]

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Effect of TP0463518 on serum EPO levels in healthy rodents [1]
Next, to elucidate the EPO-producing effect of TP0463518 in rodents, single doses of TP0463518 were administered orally to healthy mice and rats. The serum EPO concentrations in the Balb/c mice at 6 h after administration are shown in Fig. 3A. The serum EPO concentrations increased in a dose-dependent manner, and a significant EPO-producing effect was observed at a dose of 5 mg/kg or more. A pharmacokinetic-pharmacodynamic (PK/PD) analysis showed an excellent correlation between the plasma TP0463518 concentrations and the serum EPO levels with a correlation factor of 0.95 (Fig. 3B).

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Effect of TP0463518 on serum EPO levels in CKD model rats [1]
To study the EPO-producing effect of TP0463518 in a CKD model, TP0463518 was administered to 5/6 Nx rats. As with the SD rats, the serum EPO levels were evaluated at the time when the maximum serum EPO concentration was obtained. The serum EPO levels increased significantly in a dose-dependent manner at a dose of 10 mg/kg or more (Fig. 5A). The serum TP0463518 concentration was also strongly correlated with the serum EPO concentration (Fig. 5B). The serum EPO concentration in the 5/6 Nx rats was comparable to that in the SD rats when the serum TP0463518 concentration was the same (Fig. 4B and Fig. 5B).

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Effect of TP0463518 on serum EPO levels in monkeys [1]
Finally, the effect of TP0463518 was studied in monkeys (Macaca fascicularis). The plasma TP0463518 concentrations peaked at 1.6 h after the administration of 20 mg/kg of TP0463518 and then decreased promptly during the distribution phase (Fig. 6A). The T1/2 during the elimination phase was 5.2 h. The serum EPO concentration peaked at 8 h post-administration in all the dosing groups and then decreased at 24 h (Fig. 6B). The serum EPO AUC increased significantly at a dose of 5 mg/kg or more (Fig. 6C). The serum EPO AUC was correlated with the plasma TP0463518 AUC (Fig. 6D).

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Prolyl hydroxylase (PHD) 1/2/3 pan inhibitors are known to potentially induce erythropoietin (EPO) production in both the kidney and liver. The 2-[[1-[[6-(4-chlorophenoxy)pyridin-3-yl]methyl]-4-hydroxy-6-oxo-2,3-dihydropyridine-5-carbonyl]amino]acetic acid (TP0463518) is a novel PHD 1/2/3 pan inhibitor; however, the main source of EPO production after TP0463518 administration remained to be investigated. We examined the effect of TP0463518 in inducing EPO production in the kidney and liver by measuring the hypoxia-inducible factor 2α (HIF-2α), EPO mRNA, and serum EPO levels in normal and bilaterally nephrectomized rats. Furthermore, we examined whether liver-derived EPO improved anemia in 5/6 nephrectomized (5/6 Nx) rats. TP0463518 scarcely increased the HIF-2α and EPO mRNA expression levels in the kidney cortex, whereas oral administration of TP0463518 at 40 mg/kg dramatically increased the HIF-2α level from 0.27 to 1.53 fmol/mg and the EPO mRNA expression level by 1300-fold in the livers of healthy rats. After administration of TP0463518 at 20 mg/kg, the total EPO mRNA expression level in the whole liver was 22-fold that in the whole kidney. In bilaterally nephrectomized rats, TP0463518 raised the serum EPO concentration from 0 to 180 pg/ml at 20 mg/kg. Furthermore, repeated administration of TP0463518 at 10 mg/kg increased the reticulocyte count in 5/6 Nx rats on day 7 and raised the hemoglobin level on day 14. The present study revealed that TP0463518 specifically induced EPO production in the liver and improved anemia. The characteristic feature of TP0463518 would lead to not only a more detailed understanding of the PHD-HIF2α-EPO pathway in erythropoiesis, but a new therapeutic alternative for renal anemia. SIGNIFICANCE STATEMENT: Prolyl hydroxylase (PHD) 1/2/3 pan inhibitors are known to potentially induce erythropoietin (EPO) production in both the kidney and liver; however, their effects on renal EPO production have been shown to vary depending on the experimental conditions. The authors found that 2-[[1-[[6-(4-chlorophenoxy)pyridin-3-yl]methyl]-4-hydroxy-6-oxo-2,3-dihydropyridine-5-carbonyl]amino]acetic acid (TP0463518), a PHD 1/2/3 pan inhibitor, specifically induced EPO production in the liver and that the liver-derived EPO was pharmacologically effective. Investigation of the effects of TP0463518 may pave the way for the development of a new therapeutic alternative for renal anemia patients [2].

Enzymatic assay [1]
The PHDs inhibition studies were performed using fluorescence polarization. FITC-HIF and 2-oxoglutarate were mixed with enzyme solution in a reaction buffer (20 mM Tris-HCl [pH 7.5], 5 mM KCl, 1.5 mM MgCl2, 10 μM FeSO4, 2 mM ascorbic acid, 1 mM DTT) with or without various concentrations of TP0463518. The concentrations of FITC-HIF and 2-oxoglutarate were twice the Km values of each enzyme. The reaction temperature was 30 °C, and the reaction time was optimized to each PHD enzyme to obtain the initial velocity (9–20 min). At the end of the reaction, a stop solution containing 20 mM of EDTA and anti-hydroxylated HIF antibody (Cell Signaling Technology, Inc.) was added to the reaction buffer. Then, the fluorescence (ex: 480 nm, em: 535 nm) was measured using EnVision (PerkinElmer Japan Co., Ltd.) to calculate the millipolarization (mP) value. The mP values and the corresponding hydroxylated HIF concentration were proportional, so we used the mP values as the activities. The IC50 values were calculated using SAS version 9.2 (SAS Institute, Tokyo, Japan) using a nonlinear least squares method. To determine the mode of inhibition, the activity of PHD2 was measured with various concentrations of 2-oxoglutarate (0.025–8 μM) and TP0463518 (0–40 μM). Then the apparent Vmax and Km corresponding to each TP0463518 concentration were compared. The mode of inhibition was confirmed using a double reciprocal plot. The Ki value was calculated according to the mode of inhibition (SAS 9.2).

Determination of serum EPO [1]
The serum EPO levels in mice, 5/6 Nx rats, and monkeys were measured using a commercially available EPO ELISA kit according to the manufacturer's manual with slight modifications. The serum EPO levels in healthy rats were measured using a sandwich immunoassay system. The rat EPO concentration from BioLegend and Meso Scale Diagnostics were confirmed to be comparable. EPO levels below the detection limits were calculated as being zero.

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Determination of plasma/serum TP0463518 concentration [1]
The plasma/serum concentrations of TP0463518 were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) consisting of an LC‐30AD HPLC syste and a Triple Quad 5500 Mass Spectrometer.

Nine-week-old Balb/c mice were randomly assigned to a vehicle or a 5–40 mg/kg dose of TP0463518 group. The mice were orally treated with 0.5% methyl cellulose or a TP0463518 dosing suspension. Blood was collected at 6 h after administration from the orbital plexus under deep anesthesia, and euthanasia was performed without awakening. An aliquot of blood was mixed with EDTA, and the remaining blood sample was left to stand at room temperature for 15 min. The samples were then centrifuged (2130×g for 10 min at 4 °C) to prepare the plasma and serum.
For the healthy rats study, 7-week-old SD rats (Japan SLC, Inc.) were randomly assigned to a vehicle or 1.25–160 mg/kg dose of TP0463518 group. For the chronic kidney disease (CKD) model study, 5/6 nephrectomized SD (5/6 Nx) rats were prepared at Japan SLC, Inc., as follows. Two-thirds of the left kidney were resected at 4 weeks of age and the right kidney was removed at 5 weeks of age. The rats were then transferred to our facility and kept until 10 weeks of age, at which time they had developed anemia. The rats were assigned to a vehicle or a 2.5–80 mg/kg dose of TP0463518 group, while ensuring that there was no imbalance in the variance and mean of their whole-blood hemoglobin levels. SD rats and 5/6 Nx rats were orally treated with 0.5% methyl cellulose or a TP0463518 dosing suspension. Approximately 0.6 mL of blood was collected from the tail vein at 8 h (SD rats) or 4 h (5/6 Nx rats) after administration. The serum samples were prepared using the same method as that used for mice.
Eight monkeys (9–12-year-old Macaca fascicularis.) were subjected to a fast for 16 h before administration and were re-fed at 8 h post-administration. Blood was collected from the cephalic vein or the femoral vein before (0) and 0.5, 1, 2, 4, 8, 12 and 24 h after administration. Plasma samples were prepared at all the time points, and serum samples were prepared at 0, 4, 8, 12 and 24 h after administration. The experiments were repeated weekly with increasing doses of TP0463518 from 0 (vehicle) to 20 mg/kg.

The half-life (T1/2) of TP0463518 in monkeys is 5.2 hours. This value is very close to the human T1/2 value of 1.3–5.6 hours predicted based on pharmacokinetic parameters in rats and dogs (Hamada et al., 2018). A T1/2 of 5 hours may be sufficient, as 2.5 mg/kg (one-eighth of the effective dose of 20 mg/kg) was ineffective in monkey studies. Based on these results, clinical trials of TP0463518 are currently underway, administered once daily. Since PHD inhibitors can modulate the expression of multiple genes, we believe it is necessary to pay attention to side effects related to their mechanism of action, especially VEGF induction. In daprodustat, the inhibitory activity is similar to that of TP0463518, and the formulation is administered once daily, but the trend in VEGF changes is not significant (Holdstock et al., 2016; Akizawa et al., 2017). To ensure the safety of conducting clinical trials, we performed dose titration and VEGF monitoring on healthy volunteers in our first human trial. [1]

[1]. TP0463518, a novel inhibitor for hypoxia-inducible factor prolyl hydroxylases, increases erythropoietin in rodents and monkeys with a good pharmacokinetics-pharmacodynamics correlation. Eur J Pharmacol. 2018 Nov 5;838:138-144.

[2]. TP0463518, a Novel Prolyl Hydroxylase Inhibitor, Specifically Induces Erythropoietin Production in the Liver. J Pharmacol Exp Ther. 2019 Dec;371(3):675-683.

PHD inhibitors protect HIFα from proteasome degradation by inhibiting HIFα hydroxylation (Schmid and Jelkmann, 2016). Subsequently, Epo expression, located downstream of the HIF response element, is upregulated, inducing hematopoiesis (Haase, 2006; Percy et al., 2008). In recent years, PHD inhibitors have been used in clinical studies to improve renal anemia, and several companies have reported a series of clinical proof-of-concept results (Akizawa et al., 2017; Martin et al., 2017; Provenzano et al., 2016). TP0463518 is a glycine-based PHD inhibitor (Hamada et al., 2018) currently undergoing clinical trials. This report summarizes the properties of TP0463518 in in vitro and in vivo studies. TP0463518 inhibits all human PHD1/2/3 on HIF1α and also inhibits PHD2 in rats and monkeys. TP0463518 is a competitive inhibitor of 2-ketoglutarate with a Ki value of 5.3 nM for human PHD2. These results indicate that TP0463518 has similar potency to daprodustat, which is currently undergoing phase III clinical trials (Ariazi et al., 2017). The IC50 values of TP0463518 for PHD3 are 3.5-fold and 4.8-fold higher than those for PHD1/2, indicating higher inhibitory activity against PHD1/2. Although TP0463518 is selective for PHD1/2, its Cmax in monkeys is significantly higher than its IC50 value (e.g., the IC50 for human PHD3 is 63 nM, compared to 27 ng/mL), therefore TP0463518 is considered capable of inhibiting all PHDs. TP0463518 also inhibits PHD2 when the substrate is HIF2α. Since HIF2α plays an important role in EPO production (Appelhoff et al., 2004; Kapitsinou et al., 2010), researchers subsequently conducted in vivo studies to investigate the effect of TP0463518 on EPO production. [1]
TP0463518 showed significant EPO induction in healthy mice and rats at doses of 5 mg/kg and 20 mg/kg, respectively, with good pharmacokinetic/pharmacodynamic correlations. In renal anemia, the production of erythropoietin (EPO) is impaired under hypoxic stimulation. To investigate the EPO induction effect of TP0463518 in a model animal of renal anemia, we administered TP0463518 to 5/6 nephrectomy (5/6 Nx) rats. The results showed that TP0463518 could induce EPO production with significant pharmacokinetic/pharmacodynamic (PK/PD) correlations. At the same dosage, serum EPO concentrations in 5/6 Nx rats were comparable to those in healthy SD rats. The number of EPO-producing cells (REP) in the kidneys of 5/6 Nx rats was estimated to be about one-sixth that of healthy SD rats. Therefore, we hypothesize that there may be a mechanism by which serum EPO levels rise to a certain level after TP0463518 administration, regardless of the amount of residual kidney. The following three possibilities can explain these mechanisms. [1] First, TP0463518 increased EPO production in the residual damaged kidneys of 5/6 nephrectomy (Nx) rats by about 6-fold. It has been reported that in gene knockout mice lacking PHD1/2/3 in EPO-producing cells, EPO mRNA levels in damaged kidneys were higher than in healthy kidneys when unilateral ureteral obstruction was established (Souma et al., 2016). This paper points out that myofibroblasts that are REP cells before transformation have the ability to express EPO in response to PHD deficiency. Therefore, in our experiments, damaged REP cells from 5/6 Nx rats may produce more EPO than normal REP cells. [1]
A second possibility is that TP0463518 induced more EPO under hypoxic conditions in 5/6 Nx rats. One paper reported that hypoxia and the iron chelator cyclopyrrolidone (CPX) synergistically enhanced reporter gene expression via EPO HRE (Wanner et al., 2000). The iron chelator removes iron from the enzyme and appears to inhibit the first step of the reaction (Hoffart et al., 2006). TP0463518 competes with 2-ketoglutarate and also appears to inhibit the first step of the reaction. Therefore, similar to the case with CPX, we expect TP0463518 and hypoxia to also produce a synergistic effect in our experiments. [1]
Finally, TP0463518 may increase extrarenal EPO production without increasing renal EPO production. It is known that liver-specific PHD1/2/3 triple knockout mice have increased hepatic EPO production (Minamishima and Kaelin, 2010). TP0463518 is a pan-inhibitor of PHD1/2/3, but with slightly weaker inhibitory efficacy against PHD3. In this case, TP0463518 cannot reach REP cells because EPO production in the kidneys is upregulated by inhibiting PHD2 alone (Takeda et al., 2008). We are currently investigating which organ is the main source of EPO, and all these possibilities will be explored in future studies. [1]
Next, we investigated the EPO-promoting effect of TP0463518 in cynomolgus monkeys (Macaca fascicularis). The AUC of serum EPO was positively correlated with the AUC of plasma TP0463518 and increased significantly at doses of 5 mg/kg or higher. The AUC of EPO, rather than its Cmax, is a key factor in increasing hemoglobin levels (Masunaga et al., 1989). Since high hemoglobin levels increase the risk of cardiovascular disease and stroke (Pfeffer et al., 2009; Singh et al., 2006), controlling the AUC of EPO is crucial for maintaining adequate hemoglobin levels. Unlike exogenous erythropoietin, PHD inhibitors modulate endogenous EPO levels; therefore, a stronger pharmacokinetic/pharmacodynamic (PK/PD) correlation is more beneficial for hemoglobin level control. Previous studies have shown that high doses of recombinant EPO (far exceeding the normal physiological range) used to treat anemia may increase the risk of cardiovascular events, and this risk is independent of elevated blood pressure (Szczech et al., 2008; Inrig et al., 2012). In our monkey experiments, serum EPO levels increased to 60 mU/mL after administration of 20 mg/kg of recombinant EPO. This increase is comparable to the physiological increase in endogenous EPO observed at high altitudes (Klausen et al., 1996) and is sufficient to improve anemia in monkeys and humans after once-daily administration (Akizawa et al., 2017; Flamme et al., 2014; Holdstock et al., 2016). As discussed by Flamme et al., erythropoietin therapy leads to serum EPO concentrations exceeding the normal physiological range, posing long-term safety concerns, while treatment with PHD inhibitors may not require such high EPO exposure. Therefore, TP0463518 can induce effective levels of EPO without exceeding the normal physiological range, thus improving anemia while having a lower risk of cardiovascular events than recombinant EPO. [1] Systemic conditional knockout of PHD2 increases serum VEGF concentrations (Takeda et al., 2007). The time of ineffective action of PHD inhibitors is considered crucial to reduce mechanism-related adverse reactions. The half-life (T1/2) of TP0463518 in monkeys is 5.2 hours. This value is very close to the human T1/2 value of 1.3–5.6 hours predicted based on pharmacokinetic parameters in rats and dogs (Hamada et al., 2018). A T1/2 of 5 hours may be sufficient, as 2.5 mg/kg (one-eighth of the effective dose of 20 mg/kg) was ineffective in monkey studies. Based on these results, clinical trials of TP0463518, which is administered once daily, are currently underway. Since PHD inhibitors regulate the expression of multiple genes, we believe it is necessary to pay attention to side effects related to their mechanism of action, especially VEGF induction. In daprodustat, the inhibitory activity is similar to that of TP0463518 and only requires once daily administration, but the trend of VEGF changes is not significant (Holdstock et al., 2016; Akizawa et al., 2017). To conduct clinical trials safely, we performed dose titration and VEGF monitoring in healthy volunteers in our first human trial. [1] Hypertension is a common side effect of erythropoiesis-promoting therapy. Since the EPO level induced by TP0463518 did not exceed the normal physiological range, we believe the risk of hypertension is low. In fact, no trend in blood pressure change was observed in the phase II clinical study of vadadustat. vadadustat can induce EPO levels not exceeding the normal physiological range (Martin et al., 2017; Pergola et al., 2016). Daprodustat has similar potency to TP0463518, and hypertension was only observed in a small number of patients (Akizawa et al., 2017). It is forthcoming to report that a single dose of TP0463518 does not affect vital signs, including blood pressure (Shinfuku et al., 2018). Based on this information, we believe the risk of hypertension caused by TP0463518 is low. Nevertheless, we plan to closely monitor blood pressure in future clinical trials. [1] In summary, TP0463518 competitively inhibits human PHD and inhibits PHD2 in rats and monkeys. TP0463518 not only increased serum EPO levels in healthy rodents but also in anemic rats and monkeys. In all tested animals, serum EPO concentration was significantly positively correlated with TP0463518 exposure. Currently, a once-daily dosing clinical trial of TP0463518 is underway, and proof-of-concept results will be published in the future. TP0463518 holds promise as a novel treatment option for easily controlling hemoglobin levels in patients with renal anemia.

C20H18CLN3O6

431.826424121857

431.088

C, 55.63; H, 4.20; Cl, 8.21; N, 9.73; O, 22.23

1558021-37-6

1558021-37-0

73052863

Light yellow to yellow solid powder

2.8

3

7

7

30

692

0

ClC1C=CC(=CC=1)OC1=CC=C(C=N1)CN1C(C(C(NCC(=O)O)=O)=C(CC1)O)=O

HMMHKGLPKAQOOH-UHFFFAOYSA-N

InChI=1S/C20H18ClN3O6/c21-13-2-4-14(5-3-13)30-16-6-1-12(9-22-16)11-24-8-7-15(25)18(20(24)29)19(28)23-10-17(26)27/h1-6,9,25H,7-8,10-11H2,(H,23,28)(H,26,27)

2-[[1-[[6-(4-chlorophenoxy)pyridin-3-yl]methyl]-4-hydroxy-6-oxo-2,3-dihydropyridine-5-carbonyl]amino]acetic acid

TP0463518; 1558021-37-6; 2-(1-((6-(4-Chlorophenoxy)pyridin-3-yl)methyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carboxamido)acetic acid; V65GPE6NTB; 2-[[1-[[6-(4-chlorophenoxy)pyridin-3-yl]methyl]-4-hydroxy-6-oxo-2,3-dihydropyridine-5-carbonyl]amino]acetic acid; TP-0463518; (1-((6-(4-Chlorophenoxy)pyridin-3-yl)methyl)-2,4-dioxopiperidine-3-carbonyl)glycine; 2-((1-((6-(4-Chlorophenoxy)pyridin-3-yl)methyl)-4-hydroxy-6-oxo-2,3-dihydropyridine-5-carbonyl)amino)acetic acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ≥ 125 mg/mL (~289.47 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.82 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 20.8 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: ≥ 2.08 mg/mL (4.82 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 20.8 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: ≥ 2.08 mg/mL (4.82 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 20.8 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.)