Antithrombotic; antithrombin III (AT III); heparin cofactor II (HC II)

Sulodexide decreased heparinase III-induced shedding of SDC1 in MLMECs [1]
SDC1 is a member of a small family of transmembrane proteoglycans that are mainly expressed on the cell surface, and the main marker of endothelial glycocalyx degradation. To reduce the effect of endothelial cell inflammation on the glycocalyx stimulated by LPS, we used heparinase III as a specific hydrolytic agent of the glycocalyx to explore the effect of glycocalyx shedding on endothelial cells. Although heparinase III is not predicted to cause SDC1 shedding based upon its expected site of activity at heparan sulfate molecules, reducing the amount of heparan sulfate by addition of bacterial heparinase III can elevate SDC1 shedding dramatically. Sulodexide is a glycosaminoglycan consist of heparan sulfate and dermatan sulfate with anti-inflammatory property, which reduces the release of LPS-stimulated inflammatory mediators from macrophages. Heparin and heparin derivatives, as the main components of sulodexide, are believed to bind acute-phase and complement proteins, cytokines, and growth factors. In addition, sulodexide has numerous antiproteolytic effects via modulation of serine and metalloprotease enzymes, and matrix metalloproteinases, which are also involved in shedding of the glycocalyx.

To test the effect of Sulodexide on the endothelial glycocalyx layer, we performed MLMECs and HUVECs culture conditions in presence of heparinase III. As expected, heparan sulfate was removed from the endothelium by heparinase III (Figures S2A, B ). Notably, the administration of heparinase III also induced the shedding of SDC1 from the membrane of MLMECs, and the administration of supplemental sulodexide significantly presented a rescue at different times (Figures 2A–C ). The same result was found in HUVECs ( Figures S2C, D ). Next, we assessed whether sulodexide increased SDC1 levels in MLMECs by upregulating the expression of the SDC1 gene. Therefore, we measured SDC1 mRNA expression under these conditions. However, we found no difference in the different treatment groups on SDC1 mRNA expression ( Figure S2E ). Thus, these data showed that sulodexide could improve heparinase III-induced SDC1 expression, but not by increasing SDC1 gene expression.

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Sulodexide prevented endothelial permeability through enhancing ZO-1 expression [1]
The effect of sulodexide on EC barrier function was assessed using a Transwell system ( Figure 3A ). We found that SDX decreased heparinase-III-induced endothelial permeability ( Figure 3B ). Sulodexide also prevented heparinase-induced endothelial cell permeability in HUVECs ( Figure S3A ). Interestingly, permeability improved in the SDX group compared to the control group. VE-cadherin and ZO-1 were important components that play crucial roles in the maintenance of EC barrier (24, 25). We aimed to determine whether endothelial barrier dysfunction due to SDC1 shedding is associated with ZO-1 and VE-cadherin. Notably, 2 h or 4 h after treatment with heparinase III, the levels of VE-cadherin and ZO-1 markedly decreased, as determined by western blot analysis ( Figures 3C, D ). Interestingly, the administration of supplemental sulodexide upregulated heparinase III-induced ZO-1 levels rather than VE-cadherin levels ( Figures 3C, D ; S3B, C ). Altogether, these data suggest that sulodexide can prevent glycocalyx shedding-induced endothelial permeability by increasing ZO-1 expression.

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Sulodexide improved permeability via abolishing activation of NF-κB signaling [1]
Under various pathophysiological conditions, NF-κB plays an important role in inflammatory phenotypic changes as a transcription factor in endothelial cells. After glycocalyx disruption, shear stress leads to the upregulation of ICAM-1 protein expression and increased NF-κB activation. However, it is unclear whether the expression of ZO-1 is mediated by the NF-κB pathway when heparinase III degrades the glycocalyx. To address this, we evaluated the levels of NF-κB/p-p65 and NF-κB/p65 in total. Phosphorylated p65 levels markedly increased within 15/30 min of heparinase treatment and decreased with the addition of sulodexide in MLMECs/HUVECs ( Figures S4A, B ). With the administration of sulodexide, phosphorylated p65 decreased after heparinase treatment in MLMECs/HUVECs ( Figures 4A, B ). Similarly, the administration of Bay 11-7082, an inhibitor of NF-κB, markedly attenuated heparinase III-induced increases in phosphorylated p65 and increased the expression of ZO-1 ( Figures 4C-F ). Collectively, our results revealed that the activation of NF-κB signaling contributes to the expression of ZO-1 when heparinase III degrades the glycocalyx in MLMECs/HUVECs. Notably, sulodexide can promote the remodeling of glycocalyx and improve permeability by preventing NF-κB/ZO-1 signaling overactivation ( Figure 4G ).

Inhibition of SDC1 shedding by Sulodexide improved lung injury and prevented death in septic mice [2]
We established a sepsis model using CLP and LPS ( Figure 5A ). First, we measured SDC1 levels in the plasma of mice with sepsis. We found that SDC1 was upregulated in mice with sepsis. With the administration of sulodexide, the SDC1 level was downregulated, and the survival rate of mice with sepsis improved ( Figures 5B-E ). However, sulodexide treatment did not reduce IL-6 levels during sepsis ( Figures 5B, D ). Taken together, these data suggested that sulodexide restored the survival rate and decreased SDC1 expression in the plasma of mice with sepsis. We evaluated the histology of the lungs in mice. Notably, in the LPS/CLP group, lung tissues were remarkably damaged, unlike those in the control group. Compared with the LPS/CLP group, there was a marked improvement in morphological features in the LPS+SDX/CLP+SDX group ( Figures 5F , S5A ). To determine pulmonary vascular permeability in mice, we measured the wet/dry (W/D) ratio of lung tissues. As expected, the LPS/CLP increase in W/D in the lung tissues were significantly inhibited by sulodexide ( Figures 5G , S5B ). Finally, we assessed glycocalyx damage by measuring SDC1 expression lung tissues fluorescent labeling of the lung tissue. In the sepsis model, SDC1 expression was lower than in the control group. Sulodexide helped preserve SDC1 expression in the lung tissues of the sepsis model ( Figures 5H, I , S5C ). Altogether, these data show that sulodexide prevented lung injury and sepsis-induced the shedding of endothelial glycocalyx in mice.

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Mice with DN showed progressive albuminuria and renal deterioration over time, accompanied by mesangial expansion, PKC and ERK activation, increased renal expression of TGF-β1, fibronectin and collagen type I, III and IV, but decreased glomerular perlecan expression. Sulodexide treatment significantly reduced albuminuria, improved renal function, increased glomerular perlecan expression and reduced collagen type I and IV expression and ERK activation. Intra-glomerular PKC-α activation was not affected by sulodexide treatment whereas glomerular expression of fibronectin and collagen type III was increased. MMC stimulated with 30 mM D-glucose showed increased PKC and ERK mediated fibronectin and collagen type III synthesis. Sulodexide alone significantly increased fibronectin and collagen type III synthesis in a dose-dependent manner in MMC and this increase was further enhanced in the presence of 30 mM D-glucose. Sulodexide showed a dose-dependent inhibition of 30 mM D-glucose-induced PKC-βII and ERK phosphorylation, but had no effect on PKC-α or PKC-βI phosphorylation.
Conclusions: Our data demonstrated that while Sulodexide treatment reduced proteinuria and improved renal function, it had differential effects on signaling pathways and matrix protein synthesis in the kidney of C57BL/6 mice with DN. [3]

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Sulodexide is a mixed glycosaminoglycan composed of heparin and dermatan sulfate. In this study, the anti-angiogenic effect of sulodexide was investigated using an oxygen-induced retinopathy (OIR) mouse model. The retinas of sham-injected OIR mice (P17) had a distinctive central area of nonperfusion, and this area was significantly decreased in sulodexide-injected mice. The number of neovascular tufts measured by SWIFT_NV and mean neovascular lumen number were significantly decreased in sulodexide-injected mice. Hyperbaric oxygen exposure resulted in increased levels of VEGF, MMP-2 and MMP-9, and when mice were treated with sulodexide, a dose-dependent reduction in VEGF, MMP-2 and MMP-9 levels was observed. Our results clearly demonstrate the anti-angiogenic effect of sulodexide and highlight sulodexide as a candidate supplementary substance to be used for the treatment of ocular pathologies that involve neovascularization. [4]

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Sulodexide is a heparinoid compound with wide-ranging pharmacological activities. However, the effect of sulodexide on liver fibrogenesis has not been reported. In this study, we aim to evaluate the therapeutic potential of sulodexide in mouse model of liver fibrosis and explore the underlying antifibrotic mechanisms. We found that sulodexide treatment significantly attenuated thioacetamide (TAA) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver fibrosis in mice. Transcriptome analysis revealed that sulodexide treatment downregulated fibrosis-related genes and liver sinusoidal endothelial cells (LSECs) capillarization-associated genes in fibrotic livers. Immunohistochemistry confirmed that the increased expression of LSEC capillarization-related genes (CD34, CD31 and Laminin) in liver fibrotic tissues was reduced by sulodexide treatment. Scanning electron microscopy showed that LSECs fenestrations were preserved upon sulodexide treatment. Quantitative real-time PCR and immunofluorescence demonstrated that the expression of mesenchymal markers was downregulated by sulodexide administration, suggesting sulodexide inhibited endothelial-mesenchymal transition of LSECs during liver fibrosis. Furthermore, sulodexide administration protected primary LSECs from endothelial dysfunction in vitro. In conclusion, sulodexide attenuated liver fibrosis in mice by restoration of differentiated LSECs, indicating that sulodexide treatment may present as a potential therapy for patients with liver fibrosis [5].

Cell treatment conditions [2]
The MLMECs were allocated into four groups: (i) control group; cells were treated with serum-free medium for 2 h; (ii) heparinase III group; cells were treated with 15 mU/mL heparinase III for 2 h, 4 h, or 8 h, (iii) SDX/Sulodexide group; cells were treated with 30 LSU/mL SDX for 2 h, and (iv) heparinase III+SDX group; cells were pretreated with 30 LSU/mL SDX for 2 h, then with 15 mU/mL heparinase III for 2 h, 4 h, or 8 h.

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Evaluation of endothelium barrier permeability [2]
MLMECs were cultured using the Transwell system (0.4 μm pore size polyester membrane inserts). Measurement of FD40 across the endothelium was used to evaluate endothelial barrier permeability. The MLMECs were treated with or without heparinase III (15 mU/mL) or SDX/Sulodexide (30 LSU/mL). We added 0.1 mg/mL of FD40 to the upper inserts and an equal amount of serum-free medium was added to the lower compartments of the Transwell system for 60 min. Fluorescence across the upper inserts was measured at excitation and emission wavelengths of 490 and 520 nm, respectively.

Endotoxemia model [2]
The mice were randomly allocated to four groups (n = 5/experiment): LPS+SDX, LPS, Sulodexide/SDX, and control. Within the groups, mice were injected intraperitoneally (ip.) with LPS (30 mg/kg body weight/mouse) and/or treated intragastrically (ig.) with Sulodexide (40 mg/kg/mouse). Equal amounts of saline or Sulodexide were injected into the control mice or mice in the SDX group. In the survival experiment, we recorded the mortality in each group three times a day for 120 h after LPS injection. For general anesthesia, 1% tribromoethanol was administered, and the mice were sacrificed 12 h later. Blood and lung samples were collected from the surviving mice. CLP-induced polymicrobial sepsis model [2]
We randomly grouped the mice into four (n = 5/experiment): control, CLP, SDX/Sulodexide, and CLP+SDX. The CLP-induced sepsis model was developed based on previous literature. Briefly, after anesthesia with 1% tribromoethanol, laparotomy was performed. Cecum was ligated with 4-0 silk to 1 cm and punctured with a 22-gauge needle. Subsequently, a small mound of feces was squeezed from the hole after removing the needle. The peritoneum was sutured with a 6-0 silk suture, and the skin was intermittently sutured with a 4-0 silk suture. The same operation was performed on the control mice without ligation and perforation. The CLP mice were and/or intragastric (ig.) sulodexide (40 mg/kg). An equivalent volume of normal saline (NS) was injected into the control mice. After surgery, all the mice were subcutaneously resuscitated in 40 mL/kg saline. All the mice were sacrificed 24 h later. Retro-orbital blood and lung samples were collected from the surviving mice.

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Male C57BL/6 mice were rendered diabetic with streptozotocin. After the onset of proteinuria, mice were randomized to receive Sulodexide (1 mg/kg/day) or saline for up to 12 weeks and renal function, histology and fibrosis were examined. The effect of Sulodexide on fibrogenesis in murine mesangial cells (MMC) was also investigated.
Male C57BL/6 mice at 6–8 weeks of age were fasted for 6 h prior to intra-peritoneal injection of streptozotocin (STZ, 50 mg/kg) in 10 mM citrate buffer, pH 4.5, administered on five consecutive days. Diabetes mellitus was confirmed by tail vein blood sampling of glucose concentration, measured with Accu-Chek Advantage II Glucostix test strips. Spot urine was tested weekly for albuminuria with QuantiChrom albumin assay kit until sacrifice. Mice with elevated blood glucose levels (>10 mM) and albuminuria (>100 mg/dl) on two separate occasions two days apart (defined as ‘baseline’ in the animal studies) were randomized to receive treatment with either saline (vehicle control) or sulodexide (1 mg/kg/day) by oral gavage for 2, 4, 8 or 12 weeks (6 mice per time-point for each group). After 2, 4, 8 and 12 weeks of treatment, mice were sacrificed, blood samples were obtained by cardiac puncture and the kidneys harvested, decapsulated and weighed. The left kidney was cut perpendicular to the long-axis and one half of the kidney was snap frozen in OCT followed by immersion in liquid nitrogen, while the second half was fixed in 10% neutral-buffered formalin followed by paraffin embedding. Renal cortical tissue from the right kidney was separated from the medulla and frozen at −80°C until mRNA isolation. Six diabetic mice that had just developed proteinuria were also sacrificed to obtain baseline values for clinical, histological and morphometrical parameters. Negative control groups included non-diabetic male C57BL/6 mice treated with either saline or sulodexide for 12 weeks. Serum creatinine and urea levels were measured using QuantiChrom creatinine and urea assay kits respectively. [3]

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Oxygen-induced retinopathy in mice [4]
ICR pups were randomly divided into three groups: a normoxia group (control group), an oxygen-exposed group (OIR group), and a Sulodexide group; each group had one nursing mother and 5-7 pups. Oxygen-induced retinopathy was induced in ICR pups, as described previously. For the OIR model, the newborn pups were transferred at post-natal day (P) 7 along with their mother to a chamber supplied with 75 ± 2% oxygen, under continual monitoring with a ProOx 110 oxygen controller for 120 h. On P12, the mice were returned to the room air and given daily intraperitoneal (IP) injections of vehicle (saline) or 5-15 mg/kg of sulodexide dissolved in the vehicle. The mice in the normoxia group were maintained in room air from birth until P17.

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Mouse models of liver fibrosis [5]
Liver fibrosis was induced by thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet. For the TAA model, mice were supplied with 400 mg/L TAA in drinking water for 16 weeks to establish the liver fibrosis mouse model. Sulodexide, known as Vessel Due F, was administrated to the mice by gastric gavage. Mice were randomly assigned to each of three groups (with six mice per group): (a) the control group, (b) the TAA + vehicle group, and (c) the TAA + sulodexide (SDX) group. All animals received food and water ad libitum. For the DDC model, mice were fed a diet containing 0.12 % w/w DDC for four weeks to induce liver fibrosis with cholangitis.

[1]. Andreozzi GM. Sulodexide in the treatment of chronic venous disease. Am J Cardiovasc Drugs. 2012 Apr 1;12(2):73-81.

[2]. Sulodexide improves vascular permeability via glycocalyx remodelling in endothelial cells during sepsis. Frontiers in immunology, 2023, 14: 1172892.

[3]. Sulodexide decreases albuminuria and regulates matrix protein accumulation in C57BL/6 mice with streptozotocin-induced type I diabetic nephropathy. PLoS One. 2013;8(1):e54501.

[4]. Sulodexide inhibits retinal neovascularization in a mouse model of oxygen-induced retinopathy. BMB reports, 2014, 47(11): 637.

[5]. Sulodexide attenuates liver fibrosis in mice by restoration of differentiated liver sinusoidal endothelial cell. Biomedicine & Pharmacotherapy, 2023, 160: 114396.

Background: Degradation of the endothelial glycocalyx is critical for sepsis-associated lung injury and pulmonary vascular permeability. We investigated whether Sulodexide, a precursor for the synthesis of glycosaminoglycans, plays a biological role in glycocalyx remodeling and improves endothelial barrier dysfunction in sepsis. Methods: The number of children with septic shock that were admitted to the PICU at Children's Hospital of Fudan University who enrolled in the study was 28. On days one and three after enrollment, venous blood samples were collected, and heparan sulfate, and syndecan-1 (SDC1) were assayed in the plasma. We established a cell model of glycocalyx shedding by heparinase III and induced sepsis in a mouse model via lipopolysaccharide (LPS) injection and cecal ligation and puncture (CLP). Sulodexide was administrated to prevent endothelial glycocalyx damage. Endothelial barrier function and expression of endothelial-related proteins were determined using permeability, western blot and immunofluorescent staining. The survival rate, histopathology evaluation of lungs and wet-to-dry lung weight ratio were also evaluated.
In summary, this study demonstrated a previously unknown role of SDC1 in prognosis for children with septic shock and promoting vascular permeability by inducing ZO-1 disruption mediated by NF-κB dependent signaling in ECs. Sulodexide administration may thus serve as a helpful treatment in sepsis by attenuating glycocalyx shedding and downstream EC signaling that promotes vascular leakage. [2]

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SSulodexide is a mixture of glycosaminoglycans that may reduce proteinuria in diabetic nephropathy (DN), but its mechanism of action and effect on renal histology is not known. We investigated the effect of sulodexide on disease manifestations in a murine model of type I DN.
In conclusion, we have demonstrated that Sulodexide treatment reduced albuminuria, improved serum levels of urea, restored perlecan expression and ameliorated selective renal histopathologic changes in male C57BL/6 DN mice that included reduced collagen type I and IV deposition, and ERK and PKC-βII activation. In contrast, sulodexide had no effect on PKC-α or PKC-βI activation, but increased glomerular but not tubulo-interstitial deposition of fibronectin and collagen type III. It is possible that an increase in glomerular expression of these matrix proteins and an inability to suppress PKC-α or PKC-βI activation during progressive disease may explain at least in part, why sulodexide showed no efficacy in recent clinical studies although further studies are warranted to confirm this. Whether sulodexide can provide renoprotection in sub-populations of DN patients with specific histopathology remains to be determined. [3]

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In conclusion, Sulodexide was shown to be effective, at least in part, in inhibiting retinal neovascularization in a mouse model of retinopathy. Sulodexide acts by inhibiting pro-angiogenic proteins including VEGF, MMP-1, and MMP-9. Because pathologic angiogenesis following retinal ischemia is one of the leading causes of blindness, drugs with low toxicity and potent anti-angiogenic activity are urgently needed. Our results show that sulodexide fits these criteria and can be suggested as a supplementary compound in the treatment of ocular pathologic angiogenesis. Detailed information on the pharmacokinetics of this substance in the eye will require further investigation. [4]

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In summary, we provided evidence for the efficacy of Sulodexide in treating two mouse models of liver fibrosis; sulodexide treatment ameliorated liver fibrosis in both TAA- and DDC-induced mouse models. Mechanistic studies revealed that sulodexide downregulated fibrosis-related signaling pathways and LSEC capillarization-related genes. Furthermore, sulodexide treatment preserved LSEC fenestrae and inhibited capillarization and EndMT in fibrotic livers. Consistent with these findings in vivo, sulodexide administration minimized LSEC capillarization and EndMT progression in primary LSECs in vitro. This suggested that the anti-fibrotic activity of sulodexide occurred through restoration of differentiated LSECs. Our findings provide the first insights into a potential mechanism of action for orally-administrated sulodexide in liver fibrosis. [5]

57821-29-1

Colorless to light yellow Solid-Liquid Mixture

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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