Sodium-dependent bile acid transporter (ASBT)

Bile acid homeostasis is vital for numerous metabolic and immune functions in humans. The enterohepatic circulation of bile acids is extremely efficient, with ~95% of intestinal bile acids being reabsorbed. Disturbing intestinal bile acid uptake is expected to substantially affect intestinal and systemic bile acid levels. Here, we aimed to predict the effects of apical sodium-dependent bile acid transporter (ASBT)-inhibition on systemic plasma levels. For this, we combined in vitro Caco-2 cell transport assays with physiologically based (PBK) modeling. We used the selective ASBT-inhibitor odevixibat (ODE) as a model compound. Caco-2 cells grown on culture inserts were used to obtain transport kinetic parameters of glycocholic acid (GCA). The apparent Michaelis-Menten constant (Km,app), apparent maximal intestinal transport rate (Vmax,app), and ODE’s inhibitory constant (Ki) were determined for GCA. These kinetic parameters were incorporated into a PBK model and used to predict the ASBT inhibition effects on plasma bile acid levels. GCA is transported over Caco-2 cells in an active and sodium-dependent manner, indicating the presence of functional ASBT. odevixibat (ODE) inhibited GCA transport dose-dependently. The PBK model predicted that oral doses of ODE reduced conjugated bile acid levels in plasma. Our simulations match in vivo data and provide a first proof-of-principle for the incorporation of active intestinal bile acid uptake in a bile acid PBK model. This approach could in future be of use to predict the effects of other ASBT-inhibitors on plasma and intestinal bile acid levels [2].

Odevixibat (A4250) (0.01% (w/w) in feed; 4 weeks) ameliorates sclerosing cholangitis and dramatically lowers serum levels of BA, alkaline phosphatase, and alanine aminotransferase, as well as the expression of pro-inflammatory and pro-fibrotic genes in the liver and bile duct proliferation in Mdr2-/-mice [1]. Additionally, while stimulating Ntcp and Cyp7a1, Odevixibat (A4250) considerably lowers bile flux and bile BA output, which coincides with reduced bsep transcription [1].

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Odevixibat (A4250) improved sclerosing cholangitis in Mdr2−/− mice and significantly reduced serum alanine aminotransferase, alkaline phosphatase and BAs levels, hepatic expression of pro-inflammatory (Tnf-α, Vcam1, Mcp-1) and pro-fibrogenic (Col1a1, Col1a2) genes and bile duct proliferation (mRNA and immunohistochemistry for cytokeratin 19 (CK19)). Furthermore, A4250 significantly reduced bile flow and biliary BA output, which correlated with reduced Bsep transcription, while Ntcp and Cyp7a1 were induced. Importantly A4250 significantly reduced biliary BA secretion but preserved HCO3− and biliary phospholipid secretion resulting in an increased HCO3−/BA and PL/BA ratio. In addition, A4250 profoundly increased fecal BA excretion without causing diarrhea and altered BA pool composition, resulting in diminished concentrations of primary BAs tauro-β-muricholic acid and taurocholic acid. [1]

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Selective ASBT inhibitor Odevixibat (A4250) improves cholestatic liver and bile duct injury in Mdr2−/− mice [1]
Odevixibat (A4250) was well tolerated in Mdr2−/− mice and had no impact on animal behavior or body weight after short- or long-term feeding (1 and 4 weeks, respectively; data not shown). In our study we focused on potential changes of cholestatic liver and biliary injury in Mdr2−/− mice at 8 weeks of age, a time point when bile duct injury is fully established with pronounced cholestasis in these mice. As determined by liver histology, A4250 ameliorated bile duct injury with pericholangitis and onion skin type fibrosis in Mdr2−/− mice after 4 weeks of feeding (Fig. 1A). Notably, serum ALT as marker of hepatocellular injury was significantly decreased already after 2 weeks (Fig. 1B), whereas serum markers of cholestasis (AP and BA) were significantly decreased after 4 weeks of A4250 feeding in Mdr2−/− mice (Fig. 1C and D). This indicates that longer treatment is required to observe the full impact on biliary injury. In line with serum liver enzymes and BAs, serum bilirubin also slightly but significantly decreased in Mdr2−/− mice after A4250 feeding (0.085 ± 0.0 vs. 0.16 ± 0.1 mg/dl). Interestingly, serum triglyceride levels remained unchanged, while serum cholesterol increased in Mdr2−/− mice after dietary A4250 supplementation (Supplementary Fig. 1). In line with the histological and biochemical findings, liver LW/BW and SW/BW% ratios were significantly decreased in Mdr2−/− mice after A4250 treatment (Fig. 1E), reflecting its overall beneficial effects on liver injury. Furthermore, A4250 significantly decreased proliferation of bile ducts (Fig. 2A) as determined by CK19 IHC staining and quantification (Fig. 2B) as well as assessment of mRNA expression (Fig. 2C). Altogether, these findings established a beneficial role of ASBT inhibition on cholestatic liver and bile duct injury in a mouse model of sclerosing cholangitis.

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Hepatic inflammation and periductal fibrosis are reduced by Odevixibat (A4250) in Mdr2−/− mice [1]
Since ongoing inflammation and reactive proliferation of bile ducts in cholangiopathies are associated with development of biliary fibrosis [3]. We next measured mRNA expression of Tnf-α, Mcp-1 and Vcam-1, the main pro-inflammatory cytokines involved in the pathogenesis of liver injury in Mdr2−/− mice (Fig. 3A) in addition to expression of biliary fibrosis markers such as Col1a1 and Col1a2 (Fig. 3B). Interestingly, transcription of pro-fibrogenic genes such as Col1a1 and Col1a2 was profoundly reduced in Odevixibat (A4250)-treated mice (Fig. 3B). Conversely, hepatic hydroxyproline content and α-SMA protein levels did not differ between the groups (Fig. 3C, 3D). In line with inhibited expression of pro-inflammatory cytokines and pro-fibrogenic genes, quantification of Sirius Red staining revealed significantly reduced peribiliary fibrosis and a tendency towards a reduction of overall fibrosis (Fig. 3E, F). Taken together, these data suggest an anti-inflammatory and moderate anti-fibrotic effect of A4250 over a 4 week treatment course in Mdr2−/− mice.

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Odevixibat (A4250) significantly alters bile acid homeostasis in Mdr2−/− mice [1]
Importantly, gene expression of Asbt as well as basolateral BA transporter Ost-α and Ost-β in the ileum of Mdr2−/− mice remained unchanged after Odevixibat (A4250) feeding, whereas expression of Fgf15, an important intestinal regulator of BA synthesis [15] and target of intracellular BA sensor FXR was profoundly reduced (Fig. 4A), thereby confirming efficient inhibition of ileal BA uptake. In line, A4250 feeding resulted in 3-fold enhanced transcription of the rate limiting enzyme for BA synthesis cholesterol 7 alpha-hydroxylase (Cyp7a1) and sinusoidal BA uptake transporter Ntcp in liver, while expression of the canalicular BA export pump Bsep was reduced (Fig. 4B). In addition, A4250 reduced gene expression of BA detoxifying enzymes Cyp3a11, Ugt1a1 and Ugt2b5 and sinusoidal export transporter Mrp3 without significant alterations of sinusoidal exporters Mrp4, Ost-α and Ost-β (Fig. 4B). Collectively, these findings reflect decreased hepatocellular BA load and compensatory changes of hepatic BA homeostasis.

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ASBT inhibition reduces biliary BA output and modifies biliary BA composition [1]
Odevixibat (A4250) significantly reduced bile flow (Fig. 5A), which was associated with significant reductions of biliary BA concentrations and output (Fig. 5B, C). Furthermore, Odevixibat (A4250) significantly altered biliary BA composition by reducing concentrations of primary BAs tauro-β-muricholic acid (TβMCA) and taurocholic acid (TCA) (Fig. 5D). In contrast, concentrations of the secondary BAs taurodeoxycholic acid (TDCA) and taurohyodeoxycholic acid (THDCA) were increased, whereas tauro-ω-muricholic acid (TωMCA) and tauroursodeoxycholic acid (TUDCA) were reduced (Fig. 5E). Of note, unconjugated BAs were not found in bile of chow or A4250-fed mice. Total fecal BA levels were markedly increased in mice subjected to A4250 feeding (Fig. 5F). Of note, anions suggesting the presence of sulphated BAs were not found in feces. Importantly, contrasting diminished BA output, biliary concentrations as well as output of protective HCO3− did not differ between the A4250-fed and chow-fed mice (Fig. 5B, C), whereas relative output of HCO3− and of PLs was significantly increased in A4250-fed Mdr2−/− mice as demonstrated by increased ratios of HCO3−/BA and PL/BA (Fig. 5B). Since non-micellar bound free BAs are believed to trigger cholangiopathy in Mdr2−/− mice, significant reduction of biliary BA output with simultaneous increase of HCO3− proportion may represent an important protective mechanism in toxic bile-mediated liver injury.

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Odevixibat (A4250) treatment does not promote diarrhea or gut inflammation in Mdr2−/− mice [1]
Despite markedly increased total fecal BA levels (Fig. 5F) mice did not develop diarrhea as determined by assessing stool quantity and consistency. As such, we observed no difference in stool weight between control group and Odevixibat (A4250) fed mice. (1.30 g stool per day in control mice vs. 1.38 g stool in A4250 fed mice). We also addressed whether A4250 promotes colon inflammation. Importantly, conventional histology showed no differences in structure as well as inflammatory cell infiltration of the colonic wall between chow and A4250-fed Mdr2−/− mice (Fig. 6A). Furthermore, IHC staining for the macrophage marker F4/80 (data not shown) and the proliferation marker Ki67 (Fig. 6B, E) showed no significant differences between experimental groups. In line with these findings, transcription of pro-inflammatory cytokine Tnf-α remained unchanged in the ileum of A4250-treated Mdr2−/− mice (Fig. 6D). Since GLP-1 released from entero-endocrine cells in response to increased intestinal BA concentrations may have direct protective effects on cholangiocytes [18], we next addressed whether GLP-1-mediated gut-liver signaling may contribute to A4250-mediated beneficial effects in cholestasis. Notably, GLP-1 IHC did not show differences between control and A4250 fed animals whereas ileal mRNA expression of pre-glucagon was reduced in A4250-fed Mdr2−/− mice (Fig. 6C and F), indicating that this mechanism is rather unlikely to contribute to A4250-mediated protection from cholestatic liver and bile duct injury.

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Odevixibat (A4250) reduces gallbladder size [1]
Since bile homeostasis plays a critical role in gallbladder physiology, we also studied the impact of reduced bile flow and biliary BA output on gallbladder morphology. Interestingly, thickness of gallbladder wall was increased in Odevixibat (A4250)-fed Mdr2−/− mice through increased thickness of muscularis propria and number of mucosal folds (Supplementary Fig. 2A, B). Importantly, no increase of inflammatory cell infiltration as reflected by F4/80 staining was detected in lamina propria, muscularis propria or epithelium (Supplementary Fig. 2C), thereby excluding an inflammation-mediated mechanism of gallbladder wall thickening. Since Fgf15 stimulates gallbladder filling through counteracting cholecystokinin [19], increased gallbladder wall thickness may reflect reduced Fgf15 signaling resulting in a contracted gallbladder in A4250-fed Mdr2−/− mice. Importantly, gallbladders of Mdr2−/− mice treated with A4250 were not filled with sediments or stones.

Bile acids regulate digestion and immune functions. Too little bile acid reuptake in the gut is related to several diseases, including inflammatory bowel disease. This study investigates how reducing bile acid absorption affects bile acid levels in humans using the drug odevixibat (ODE) as an example. odevixibat (ODE) reduces bile acid absorption by blocking the intestinal bile acid transporter protein in gut cells. The transport of a bile acid through a gut cell line commonly used to model the intestinal barrier was measured with and without ODE, and mathematical modeling was used to translate the laboratory results to whole-body effects. This combined approach accurately predicted the known effects of ODE on intestinal and bloodstream bile acid levels in humans. This novel approach could be used to predict the effects of other chemicals on intestinal bile acid absorption and intestinal and bloodstream bile acid levels instead of animal testing [2].

Animal/Disease Models: Eightweeks old Mdr2-/- (Abcb4-/-) mice (cholestatic liver injury and sclerosing cholangitis model) [1]
Doses: 0.01% (w/w) in feed Administration time: 4-week
Experimental Results: Cholestasis diminished in mouse liver and bile duct injury model.

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Odevixibat (A4250) (a specific ASBT inhibitor) was synthesized. Eight week old male Mdr2−/− mice received either control diet or a diet supplemented with 0.01% (w/w) A4250 either for 4 weeks or for 1 week. The 4 week treatment protocol was used for biochemical, molecular and histological data analysis, whereas the 1 week treated mice were subjected to bile flow measurement. [1]

Absorption, Distribution and Excretion
Following a single oral dose of 7.2 mg oudevizia in adults, the peak plasma concentration (Cmax) was 0.47 ng/mL, and the area under the curve (AUC0-24h) was 2.19 hng/mL. Odevizia was undetectable in plasma in most adult and pediatric patients after receiving the therapeutic dose. The recovery rate of oudevizia in feces was 82.9%, and in urine it was less than 0.002%. 97% of the recovered drug in feces was the unmetabolized parent compound. Odevizia was undetectable in plasma in most adult and pediatric patients after receiving the therapeutic dose. Therefore, the volume of distribution was not calculated. Odevizia was undetectable in plasma in most adult and pediatric patients after receiving the therapeutic dose. Therefore, clearance was not calculated. Metabolism/Metabolites Odevizia is primarily unmetabolized, but a small amount can be metabolized via in vitro monohydroxylation. Because the primary endpoint of clinical trials is to determine the structure of metabolites at concentrations exceeding 10% of the dose in plasma, urine, or feces, the exact structure of the metabolites has not yet been determined. Metabolites at such high concentrations have not yet been identified.

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Biological Half-Life
The mean half-life of 7.2 mg odeviziba orally in adults is 2.36 hours.

Hepatotoxicity
In trials of odevixibat in children with cholestatic liver disease, 8% to 11% of participants experienced serum ALT elevations exceeding three times the upper limit of normal, particularly with long-term treatment. However, children with progressive familial intrahepatic cholestasis (PFIC) often present with elevated serum ALT and AST, making it difficult to determine whether mild to moderate serum enzyme elevations are caused by odevixibat treatment or by spontaneous fluctuations due to the primary disease. Despite limited clinical experience with odevixibat, there have been no reports of clinically significant liver injury (with jaundice or liver decompensation) in children. Probability score: E (suspected but not confirmed cause of clinically significant liver injury). Protein Binding Due to the low systemic absorption rate of odevixibat, in vivo plasma protein binding studies were not possible. Odevixibat exhibits >99% protein binding in vitro.

[1]. Inhibition of intestinal bile acid absorption improves cholestatic liver and bile duct injury in a mouse model of sclerosing cholangitis.J Hepatol. 2016 Mar;64(3):674-81.

[2]. Intestinal in vitro transport assay combined with physiologically based kinetic modeling as a tool to predict bile acid levels in vivo. ALTEX. 2024 Jan 9;41(1):20-36.

Odevixibat (also known as A4250) is an ileal sodium/bile acid cotransporter inhibitor indicated for the treatment of pruritus symptoms in patients with progressive familial intrahepatic cholestasis (PFIC) lasting more than 3 months. Odevixibat is the first non-surgical treatment approved for PFIC. Previous treatments for PFIC included the use of bile acid sequestrants, such as ursodeoxycholic acid. Odevixibat received approval from the U.S. FDA on July 20, 2021, and from Health Canada on November 13, 2023, respectively. Odevixibat is an ileal bile acid transporter inhibitor. Its mechanism of action is as an ileal bile acid transporter inhibitor. Odevixibat is an oral ileal bile acid transporter inhibitor used to treat severe pruritus in patients with cholestatic liver disease, such as progressive familial intrahepatic cholestasis. Odeviziba is associated with transient elevations in serum enzymes, especially with long-term treatment, but has not been found to be associated with clinically significant liver damage with jaundice, despite limited experience with its use.
Drug Indications
The U.S. Food and Drug Administration (FDA) and Health Canada have approved odeviziba for the treatment of pruritus in patients with progressive familial intrahepatic cholestasis (PFIC) for 3 months and 6 months or more, respectively. It is also indicated for the treatment of cholestatic pruritus in patients with Aragile syndrome for 12 months and more. Odeviziba may be ineffective in patients with type 2 progressive familial intrahepatic cholestasis (PFIC) carrying the ABCB11 variant, as these patients lack a functional bile acid output pump.
Viviv is indicated for the treatment of progressive familial intrahepatic cholestasis (PFIC) in patients for 6 months and more (see Sections 4.4 and 5.1).
Treatment of Aragile Syndrome
Treatment of Progressive Familial Intrahepatic Cholestasis
Treatment of Biliary Atresia

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Mechanism of Action
Progressive familial intrahepatic cholestasis (PFIC) is a group of autosomal recessive genetic disorders that lead to cholestasis, fibrosis, and ultimately require liver transplantation. PFIC patients require liver transplantation or develop hepatocellular carcinoma within the first few years of life. Many patients experience severe itching. The exact mechanism of itching in progressive familial intrahepatic cholestasis (PFIC) is unclear, but studies suggest that lowering bile acid concentrations can alleviate itching. Patients with certain types of PFIC type 2, due to dysfunction or absence of their bile acid export pumps, are not expected to benefit from olevisziba treatment. Ileal sodium/bile acid cotransporters are transport glycoproteins responsible for the reabsorption of 95% of bile acids in the distal ileum. Olevisziba is a reversible inhibitor of ileal sodium/bile acid cotransporters. After one week, a once-daily dose of 3 mg odevizia reduced the area under the bile acid curve (AUC) by 56%. A once-daily dose of 1.5 mg odevizia reduced the bile acid AUC by 43%. Reduced bile acid reabsorption leads to weakened FXR stimulation, which in turn reduces FGF19 expression and the binding of FGF19 to FGF4R, thereby reducing the inhibitory effect on bile acid synthesis. Unabsorbed bile acids are further synthesized, which helps lower LDL cholesterol levels.

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Pharmacodynamics
Odevixibat (or A4250) is an ileal sodium/bile acid cotransporter inhibitor indicated for the treatment of pruritus in patients with progressive familial intrahepatic cholestasis (PFIC) lasting more than 3 months. Due to the once-daily dosing requirement, its duration of action is moderate. Odevixibat has a broad therapeutic index, with single-dose doses up to 10 mg and a maximum therapeutic dose of 6 mg daily. Patients should be informed of the risks of elevated liver function test results, diarrhea, and fat-soluble vitamin deficiency.

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Background and Objectives
Approximately 95% of bile acids (BA) excreted into bile are reabsorbed in the intestine and circulate back to the liver for further bile secretion. Therefore, pharmacological inhibition of the apical ileum sodium-dependent bile acid transporter (ASBT/SLC10A2) may help prevent bile acid-mediated cholestatic liver injury and bile duct injury.
Methods
Eight-week-old Mdr2−/− (Abcb4−/−) mice (a model of cholestatic liver injury and sclerosing cholangitis) were fed either a diet supplemented with A4250 (0.01% w/w)—a highly potent and selective ASBT inhibitor—or a normal diet. Liver injury was assessed by biochemical and histological methods after 4 weeks of A4250 treatment. Gene expression profiles involved in bile acid homeostasis, inflammation, and fibrosis in liver and ileum homogenates were detected by RT-PCR. Intestinal inflammation was assessed using RNA expression profiling and immunohistochemistry. Bile flow and composition, as well as bile acid profiles in bile and feces, were analyzed after one week of ASBT inhibitor administration.
Results
A4250 ameliorated sclerosing cholangitis in Mdr2−/− mice and significantly reduced serum alanine aminotransferase, alkaline phosphatase, and bile acid levels, as well as the expression of pro-inflammatory genes (Tnf-α, Vcam1, Mcp-1) and pro-fibrotic genes (Col1a1, Col1a2) in the liver, and bile duct proliferation (mRNA and immunohistochemical staining for cytokeratin 19 (CK19)). Furthermore, A4250 significantly reduced bile flow and bile acid (BA) output, which was associated with reduced Bsep transcription, and induced the expression of Ntcp and Cyp7a1. Importantly, A4250 significantly reduced bile acid secretion but retained the secretion of HCO3− and bile phospholipids, resulting in an increase in the HCO3−/BA and PL/BA ratios. In addition, A4250 significantly increased fecal bile acid excretion without causing diarrhea and altered the composition of the bile acid pool, leading to a decrease in the concentrations of the primary bile acids taurine-β-mouse cholic acid and taurocholic acid. [1]

C37H48N4O8S2

740.93

740.291

C, 59.98; H, 6.53; N, 7.56; O, 17.27; S, 8.65

501692-44-0

501692-44-0;2409081-01-0 (hydrate);Odevixibat HCl;

10153627

White to off-white solid powder

1.3±0.1 g/cm3

1.643

7.03

5

11

17

51

1230

2

CCCCC1(CN(C2=CC(=C(C=C2S(=O)(=O)N1)OCC(=O)N[C@H](C3=CC=C(C=C3)O)C(=O)N[C@@H](CC)C(=O)O)SC)C4=CC=CC=C4)CCCC

XULSCZPZVQIMFM-IPZQJPLYSA-N

InChI=1S/C37H48N4O8S2/c1-5-8-19-37(20-9-6-2)24-41(26-13-11-10-12-14-26)29-21-31(50-4)30(22-32(29)51(47,48)40-37)49-23-33(43)39-34(25-15-17-27(42)18-16-25)35(44)38-28(7-3)36(45)46/h10-18,21-22,28,34,40,42H,5-9,19-20,23-24H2,1-4H3,(H,38,44)(H,39,43)(H,45,46)/t28-,34+/m0/s1

(S)-2-((R)-2-(2-((3,3-dibutyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydrobenzo[f][1,2,5]thiadiazepin-8-yl)oxy)acetamido)-2-(4-hydroxyphenyl)acetamido)butanoic acid

AZD8294; A4250; AR-H064974; Odevixibat; 501692-44-0; A4250; AZD8294; AR-H064974; Odevixibat [USAN]; 2W150K0UUC;AZD-8294; A-4250; AR-H-064974; Bylvay

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)

DMSO : ~166.67 mg/mL (~224.95 mM)

Solubility in Formulation 1: ≥ 4.17 mg/mL (5.63 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 41.7 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: ≥ 4.17 mg/mL (5.63 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 41.7 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.)