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
A 7.2 mg single oral dose of odevixibat in adults reaches a Cmax of 0.47 ng/mL, with an AUC0-24h of 2.19 h\*ng/mL. The majority of adult and pediatric patients, given a therapeutic dose, do not have detectable plasma concentrations of odevixibat.
Odevixibat is 82.9% recovered in the feces and <0.002% recovered in the urine. The dose recovered in the feces is 97% unchanged parent compound.
The majority of adult and pediatric patients, given a therapeutic dose, do not have detectable plasma concentrations of odevixibat. Therefore, a volume of distribution has not been calculated.
The majority of adult and pediatric patients, given a therapeutic dose, do not have detectable plasma concentrations of odevixibat. Therefore, the clearance has not been calculated.

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Metabolism / Metabolites
Odevixibat is largely unmetabolized, however a small amount is metabolized _in vitro_ by mono-hydroxylation. The exact structure of the metabolite has not been characterized as a primary endpoint of the clinical trial was to characterize the structure of metabolites accounting for >10% of the dose in plasma, urine, or feces. No metabolites have been identified at such a high concentration.

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Biological Half-Life
A 7.2 mg oral dose of odevixibat has a mean half life of 2.36 hours in adults.

Hepatotoxicity
In trials of odevixibat in children with cholestatic liver diseases, serum ALT elevations of greater than 3 times ULN arose in 8% to 11% of treated participants and were particularly common with long term therapy. However, children with PFIC typically have serum ALT and AST elevations, and it was difficult to establish whether mild-to-moderate serum enzyme elevations were due to odevixibat therapy or to the spontaneous fluctuations that occur with the underlying disease. Clinically apparent liver injury with jaundice or hepatic decompensation has not been reported in children treated with odevixibat, although the total clinical experience with its use is limited.
Likelihood score: E* (suspected but unproven cause of clinically apparent liver injury).

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Protein Binding
Due to the low systemic abosrption of odevixibat, plasma protein binding studies could not be performed _in vivo_. Odevixibat is >99% protein bound _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, or A4250, is an ileal sodium/bile acid cotransporter inhibitor indicated for the treatment of pruritus in patients older than 3 months, with progressive familial intrahepatic cholestasis (PFIC). Odevixibat is the first approved non-surgical treatment option for PFIC. Previous therapies for PFIC included a bile acid sequestrant such as [ursodeoxycholic acid]. Odevixibat was granted FDA and Health Canada approval on 20 July 2021 and 13 November 2023 respectively.
Odevixibat is an Ileal Bile Acid Transporter Inhibitor. The mechanism of action of odevixibat is as an Ileal Bile Acid Transporter Inhibitor.
Odevixibat is an orally available inhibitor of the ilieal bile salt transporter which is used to treat severe pruritus in patients with cholestatic liver disease such as progressive familial intrahepatic cholestasis. Odevixibat is associated with transient serum enzyme elevations particularly with long term therapy but has not been linked to instances of clinically apparent liver injury with jaundice, although experience with its use has been limited.

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Drug Indication
Odevixibat is indicated for the treatment of pruritus in patients older than 3 months and 6 months with progressive familial intrahepatic cholestasis (PFIC) by the FDA and Health Canada respectively. It is also indicated for the treatment of cholestatic pruritus in patients 12 months of age and older with Alagille Syndrome. Odevixibat may not be effective in patients with PFIC type 2 with ABCB11 variants since these patients lack a functional bile salt export pump.
Bylvay is indicated for the treatment of progressive familial intrahepatic cholestasis (PFIC) in patients aged 6 months or older (see sections 4. 4 and 5. 1).
Treatment of Alagille syndrome
Treatment of Progressive Familial Intrahepatic Cholestasis
Treatment of biliary atresia

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Mechanism of Action
Progressive familiar intrahepatic cholestasis (PFIC) is a group of autosomal recessive disorders leading to cholestasis, fibrosis, and eventually a need for liver transplantation. Patients with PFIC require liver transplants or develop hepatocellular carcinomas in their first few years of life. Many of these patients experience severe pruritus. The exact mechanism of pruritus is PFIC is not known, but lower concentrations of bile acids have been shown to reduce pruritus. Patients with certain forms of PFIC type 2, associated with a non-functional or absent bile salt export pump, are not expected to benefit from odevixibat treatment. The ileal sodium/bile acid cotransporter is a transport glycoprotein responsible for reabsorption of 95% of bile acids in the distal ileum. Odevixibat is a reversible inhibitor of the ileal sodium/bile acid contransporter. Patients taking odevixibat for a week experienced a 56% reduction in bile acid area under the curve with a 3 mg once daily dose. A 1.5 mg daily dose lead to a 43% reduction in bile acid area under the curve. The decreased reabsorption of bile acids, leads to reduced stimulation of FXR, which reduces expression of FGF19, reducing binding of FGF19 to FGF4R, decreasing inhibition of bile acid synthesis. Further synthesis of bile acids that will not be reabsorbed in the intestine contributes to lowering low density lipoprotein 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 older than 3 months, with progressive familiar intrahepatic cholestasis (PFIC). It has a moderate duration of action as it is given once daily. Odevixibat has a wide therapeutic index as patients were given single doses up to 10 mg while the maximum therapeutic dose is 6 mg daily. Patients should be counselled regarding the risks of elevated liver function tests, diarrhea, and fat soluble vitamin defiencies.

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Background and Aims
Approximately 95% of bile acids (BAs) excreted into bile are reabsorbed in the gut and circulate back to the liver for further biliary secretion. Therefore, pharmacological inhibition of the ileal apical sodium-dependent BA transporter (ASBT/SLC10A2) may protect against BA-mediated cholestatic liver and bile duct injury.

Methods
Eight week old Mdr2−/− (Abcb4−/−) mice (model of cholestatic liver injury and sclerosing cholangitis) received either a diet supplemented with A4250 (0.01% w/w) – a highly potent and selective ASBT inhibitor – or a chow diet. Liver injury was assessed biochemically and histologically after 4 weeks of A4250 treatment. Expression profiles of genes involved in BA homeostasis, inflammation and fibrosis were assessed via RT-PCR from liver and ileum homogenates. Intestinal inflammation was assessed by RNA expression profiling and immunohistochemistry. Bile flow and composition, as well as biliary and fecal BA profiles were analyzed after 1 week of ASBT inhibitor feeding.

Results
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]

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.)