Osteoclast-mediated bone resorption

Here, researchers establish the antimalarial activities of risedronate, one of the most potent bisphosphonates clinically used to treat bone resorption diseases, against blood stages of Plasmodium falciparum (50% inhibitory concentration [IC50] of 20.3 ± 1.0 μM). They also suggest a mechanism of action for risedronate against the intraerythrocytic stage of P. falciparum and show that protein prenylation seems to be modulated directly by this drug. Risedronate inhibits the transfer of the farnesyl pyrophosphate group to parasite proteins, an effect not observed for the transfer of geranylgeranyl pyrophosphate[1].

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In this report researchers confirmed that risedronate, a nitrogen-containing bisphosphonate (N-BP), has a potent activity against the blood stages of P. falciparum in vitro. The IC50 established for parasite growth is in the range obtained for the same isolate (3D7) in previous studies. Researchers also showed that the inhibitory effect induced by risedronate can be partially reversed by the simultaneous addition of FPP or GGPP during P. falciparum culture treatment (Fig. 1). The restoration observed after the addition of GGPP is plausible, since Couto et al. previously demonstrated that P. falciparum is able to convert GGPP into FPP. In contrast, when we added IPP to the cultures, the parasites could not recover, suggesting that the inhibition of FPPS is a potential target for risedronate, which could also act by inhibiting GGPPS. Luckman et al. verified the same event of restoration after coincubating J774 macrophages with alendronate and FPP or GGPP, observing a partial prevention of apoptotic events.[1]
The results regarding the effect of risedronate on isoprenoid biosynthesis (Fig. 2) suggest the inhibition of FPPS. The RP-TLC profile for treated parasites shows that bands with Rf values equivalent to FOH and GGOH are decreased compared to the signal from untreated parasites, leading us to speculate that risedronate inhibits enzymes involved in FPP and/or GGPP synthesis. It is known that the major target of N-BPs, as risedronate, is FPPS (11); therefore, we assume the possible role of risedronate as a potent inhibitor of the isoprenoid pathway in P. falciparum by inhibiting FPPS and, consequently, blocking protein farnesylation and geranylgeranylation. In J774 macrophages, the drug inhibited protein prenylation, including Ras protein prenylation[1].

In vivo experiments demonstrate that risedronate leads to an 88.9% inhibition of the rodent parasite Plasmodium berghei in mice on the seventh day of treatment; however, risedronate treatment did not result in a general increase of survival rates.[1]
After determining the antiplasmodial effect of risedronate in vitro, we verified its efficacy in BALB/c mice infected with P. berghei strain ANKA. The administration of 20 and 25 mg/kg risedronate for 4 days led to decreases of parasitemia of 68.9% and 83.6%, respectively. On the seventh day of treatment the inhibitions were 63% and 88.9% with 20 and 25 mg/kg, respectively (Fig. 4A). After recovering the parasitemia, a dose-response curve was obtained for estimating the ID50 (dose causing 50% inhibition), equivalent to 17 ± 1.8 mg/kg after 7 days of treatment. Four days after the interruption of treatment (11 days postinfection), the parasitemias of the groups treated with 10, 15, 20, and 25 mg/kg/day were 15.3%, 15.9%, 15.2%, and 5.7%, respectively. Conversely, the group that received PBS presented parasitemia of 25.6%. Among the groups treated with risedronate, only the animals that received 25 mg/kg had a significant inhibition of 77.8% (see Table S1 in the supplemental material), demonstrating that even after treatment discontinuation, the parasitemia of the animals remained low in relation to that of the controls; however, parasite recrudescence was observed for all treated groups. By Kaplan-Meier survival analysis there was no difference between risedronate-treated mice and PBS-treated groups (Fig. 4B)[2].

Inhibition tests with risedronate and rescue assays.[1]
Risedronate was dissolved in sterile deionized water, resulting in a 25 mM stock solution. The inhibition tests were carried out with flat-bottomed microtiter plates using the following drug concentrations: 3,000, 300, 30, 3, 0.3, 0.03, and 0.003 μM. We employed a method described previously by Desjardins and coauthors, with some modifications, to determine risedronate 50% inhibitory concentrations (IC50s) for P. falciparum intraerythrocytic stages after 48 h of treatment. Briefly, synchronic ring-stage parasite cultures (5% hematocrit and 1% parasitemia) were exposed to increasing drug concentrations, and the parasitemia and parasite morphologies were determined with Giemsa-stained smears immediately before the start and at intervals of 24 to 96 h, instead of [3H]hypoxanthine incorporation. All tests were performed in triplicates for three independent experiments. The IC50, IC90 (± standard deviation), and 95% confidence interval (CI95%) values for growth inhibition were calculated by using Origin 8.1 software. For the rescue assays, FPP, GGPP, and IPP were solubilized in RPMI medium (5 mM stock solution), and different concentrations of each compound (100 nM to 1,000 nM) were then added simultaneously to synchronous P. falciparum cultures in the ring stage previously treated with 20 μM risedronate. Parasitemia was assessed every 24 h. Statistical analysis was performed by using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. A P value of <0.05 was considered statistically significant.

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Treatment with risedronate and metabolic labeling.[1]
Asynchronous cultures of P. falciparum were treated with 15 μM risedronate for 36 h and labeled with [1-3H]GGPP (3.125 μCi/ml) or [1-3H]FPP (3.125 μCi/ml) in normal RPMI 1640 medium for the last 12 h in the presence of drug. After labeling, ring, trophozoite, and schizont forms were purified on a 40%-70%-80% discontinuous Percoll gradient, followed by cell lysis in a solution containing ice-cold 10 mM Tris-HCl (pH 7.2), 150 mM NaCl, 2% (vol/vol) Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM iodoacetamide, 1 mM N-(p-tosyl-lysine)chloromethyl ketone, and 1 μg/ml leupeptin. After incubation for 15 min at 4°C, lysates were centrifuged at 10,000 × g for 30 min. Supernatants of parasites were stored in liquid N2 for subsequent SDS-PAGE analysis. For the analysis of isoprenoids, synchronic cultures in the ring stage were treated with 15 μM risedronate for 36 h and metabolically labeled with [1-14C]IPP for the last 12 h. After labeling, schizont-stage parasites were purified on a 40%-70%-80% discontinuous Percoll gradient as described above and freeze-dried prior to lipid extraction as described elsewhere previously (30). Risedronate at 15 μM was considered the ideal drug concentration to be used in our metabolic labeling experiments, since approximately 90% of the parasite population remained viable after 36 h of treatment.

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Reverse-phase thin-layer chromatography (RP-TLC).[1]
Similar amounts of schizont-stage pellets of untreated or risedronate-treated parasites labeled with [1-14C]IPP as described above were extracted with hexane for the subsequent separation of alcohols on reverse-phase Silica Gel 60 plates with acetone-H2O (6:1, vol/vol). Plates were sprayed with En3Hance and subjected to autoradiography for 45 days at −70°C. The standards were visualized with iodine vapor, and Rf values were determined. Hexane extracts of uninfected erythrocytes were used as a control group.

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Immunoprecipitation assays.[1]
Synchronic cultures in the ring stage were treated with 15 μM risedronate for 24 h and metabolically labeled with [1-3H]FPP or [1-3H]GGPP for an additional 12 h in the presence of the drug. After labeling, schizont-stage parasites were purified on a 40%-70%-80% discontinuous Percoll gradient as described above. Pellets of untreated and treated schizont-stage parasites were resuspended in immunoprecipitation buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% [vol/vol] Triton X-100, 0.5% [wt/vol] sodium deoxycholate, 0.1% [wt/vol] SDS, 5 μg/ml protease inhibitor cocktail [0.2 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 2 mM β-mercaptoethanol, chymostatin {5 mg/ml}, and 1 μg/ml leupeptin, antipain, and pepstatin A]) and then precleared with protein A-Sepharose beads. Schizont forms were then incubated with anti-human Ras or anti-Rap1/Krev-1 monoclonal immunoglobulins (1:20 dilution) for 2 h at 4°C. The antigen-antibody complex was precipitated by using 100 μl of a 10% protein A-Sepharose slurry. After five washes with phosphate-buffered saline (PBS), the bound antigen was released in SDS sample buffer and analyzed by SDS-PAGE and autoradiography (20). Densitometric analyses were performed by using Image J software.

Each male BALB/c mouse (3 to 4 weeks old) (n = 10 to 15 per group) was injected intraperitoneally (i.p.) with 106 blood-stage Plasmodium berghei strain ANKA parasites. Our laboratory previously established this parasite burden as the 50% lethal dose 14 days after inoculation. Risedronate treatment with different concentrations was initiated in 2 h after infection on day 0 and continued for 7 days. The drug was diluted in PBS and administered i.p. at 10, 15, 20, and 25 mg/kg of body weight/day. Parasitemia levels were monitored microscopically by examining Giemsa-stained thin blood smears on days 4, 7, 11, 14, and 17 postinfection. Throughout this period, the spontaneous death of each animal was computed. The percentage of parasitemia inhibition was calculated as follows: 100 − [(mean parasitemia for the treated group/mean parasitemia for the control group) × 100] (14). For comparisons of average parasitemias at different time points, analysis of variance was performed with a post hoc Mann-Whitney test for comparisons of the means. [1]

Absorption, Distribution and Excretion
Oral bioavailability is 0.63% and maximum absorption is approximately 1 hour after dosing. Administration half and hour before a meal reduces bioavailability by 55% compared to fasting and dosing 1 hour before a meal reduces bioavailability by 30%.
Risedronate is excreted by the kidneys and the unabsorbed dose is eliminated in the feces.
13.8 L/kg.
Mean renal clearance was 52mL/min and mean total clearance was 73mL/min.
/Absorption is/ rapid and independent of dose, occurring throughout the upper gastrointestinal tract. Mean oral bioavailability is 0.63% and is decreased when administered with food. Administration either 0.5 hour before breakfast or 2 hours after dinner reduces the extent of absorption by 55% compared to the fasting state (no food or drink for 10 hours before or 4 hours after administration). Administration 1 hour before breakfast reduces the extent of absorption by 30% compared with the fasting state. /Risedronate/
Studies in rats and dogs with intravenously administered single doses of radiolabeled risedronate showed that approximately 60% of the dose was distributed to bone. The mean steady-state volume of distribution is 6.3 L/kg of body weight in humans. /Risedronate/
After multiple oral dosing in rats, the uptake of risedronate in soft tissues was in the range of 0.001% to 0.01%.
Risedronate was detected in feeding pups exposed to lactating rats for a 24-hour period postdosing, indicating a small degree of lacteal transfer. /Risedronate/
Elimination: Fecal, unabsorbed drug (unchanged). Renal, unchanged, approximately 50% of the absorbed dose within 24 hours, 85% over 28 days. Mean renal clearance is 105 mL/minute and mean total clearance is 122 mL/min, the difference primarily reflecting nonrenal clearance or clearance due to absorption to bone. Note: Renal clearance is not concentration dependent and there is a linear relationship between renal clearance and creatinine clearance. /Risedronate/

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Metabolism / Metabolites
Risedronic acid is not likely not metabolized before elimination. The P-C-P group of bisphosphonates is resistant to chemical and enzymatic hydrolysis preventing metabolism of the molecule.
There is no evidence that risedronate is metabolized in humans or animals. /Risedronate/
No evidence found for metabolization of risedronate in humans or mammals.
Route of Elimination: Risedronate is excreted unchanged primarily via the kidney. Insignificant amounts (<0.1% of intravenous dose) of drug are excreted in the bile in rats.
Half Life: 1.5 hours

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Biological Half-Life
The initial half life of risedronic acid is approximately 1.5 hours, with a terminal half life of 561 hours.
Initial: Approximately 1.5 hours; Terminal exponential: 480 hours (which may represent the dissociation of risedronate from the surface of bone). /Risedronate/

Toxicity Summary
The action of risedronate on bone tissue is based partly on its affinity for hydroxyapatite, which is part of the mineral matrix of bone. Risedronate also targets farnesyl pyrophosphate (FPP) synthase. Nitrogen-containing bisphosphonates (such as pamidronate, alendronate, risedronate, ibandronate and zoledronate) appear to act as analogues of isoprenoid diphosphate lipids, thereby inhibiting FPP synthase, an enzyme in the mevalonate pathway. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprenoid lipids (FPP and GGPP) that are essential for the post-translational farnesylation and geranylgeranylation of small GTPase signalling proteins. This activity inhibits osteoclast activity and reduces bone resorption and turnover. In postmenopausal women, it reduces the elevated rate of bone turnover, leading to, on average, a net gain in bone mass.

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Because no information is available on the use of risedronate during breastfeeding, an alternate drug may be preferred, especially while nursing a newborn or preterm infant. However, absorption of risedronate by a breastfed infant is unlikely.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
~24%.

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Interactions
Antacids or Mineral Supplements Containing Divalent Cations: Pharmacokinetic interaction (decrease risedronate absorption) when risedronate is used concomitantly with antacids or mineral supplements containing divalent cations (e.g. aluminum, calcium, magnesium). /Risedronate/
Nonsteroidal Anti-inflammatory Agents /(SRP: NSAIDs)/: No evidence of increased adverse upper GI effects. /Risedronate/
Histamine H2 Receptor Antagonists, Proton Pump Inhibitors: No evidence of increased adverse upper GI effects. /Risedronate/

[1]. In vitro and in vivo antiplasmodial activities of risedronate and its interference with protein prenylation in Plasmodium falciparum. Antimicrob Agents Chemother. 2011 May;55(5):2026-31.

[2]. Treatment of osteoporosis by risedronate-- speed, efficacy and safety. Reumatizam. 2006;53(2):66-71.

Risedronic acid is a member of pyridines.
Risedronic acid is a third generation bisphosphonate that is used for the treatment of some forms of osteoperosis and Paget's disease. It functions by preventing resorption of bone.
Risedronic acid is a Bisphosphonate.
Risedronic Acid is a synthetic pyridinyl bisphosphonate, with antiresorptive activity. Upon administration, risedronic acid binds to hydroxyapatite crystals in bone and inhibits osteoclast-dependent bone resorption.
Risedronate is only found in individuals that have used or taken this drug. It is a bisphosphonate used to strengthen bone, treat or prevent osteoporosis, and treat Paget's disease of bone.The action of risedronate on bone tissue is based partly on its affinity for hydroxyapatite, which is part of the mineral matrix of bone. Risedronate also targets farnesyl pyrophosphate (FPP) synthase. Nitrogen-containing bisphosphonates (such as pamidronate, alendronate, risedronate, ibandronate and zoledronate) appear to act as analogues of isoprenoid diphosphate lipids, thereby inhibiting FPP synthase, an enzyme in the mevalonate pathway. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprenoid lipids (FPP and GGPP) that are essential for the post-translational farnesylation and geranylgeranylation of small GTPase signalling proteins. This activity inhibits osteoclast activity and reduces bone resorption and turnover. In postmenopausal women, it reduces the elevated rate of bone turnover, leading to, on average, a net gain in bone mass.
A pyridine and diphosphonic acid derivative that acts as a CALCIUM CHANNEL BLOCKER and inhibits BONE RESORPTION.
See also: Risedronate sodium hemi-pentahydrate (active moiety of); Risedronate sodium monohydrate (active moiety of).

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Drug Indication
Risedronic acid is indicated for the treatment of osteoperosis in men, treatment of Paget's disease, treatment and prevention of osteoperosis in postmenopausal women, and treatment and prevention of glucocorticoid-induced osteoperosis.
FDA Label

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Mechanism of Action
Risedronatic acid binds to bone hydroxyapatite. Bone resorption causes local acidification, releasing risedronic acid which is that taken into osteoclasts by fluid-phase endocytosis. Endocytic vesicles are acidified, releasing risedronic acid to the cytosol of osteoclasts where they induce apoptosis through inhbition of farnesyl pyrophosphate synthase. Inhibition of osteoclasts results in decreased bone resorption.
Risedronate binds to bone hydroxyapatite and, at the cellular level, inhibits osteoclasts. Although the osteoclasts adhere normally to the bone surface, they show evidence of reduced active resorption (e.g., lack of ruffled border). Evidence from studies in rats and dogs indicates that risedronate treatment reduces bone turnover (activation frequency, i.e., the number of sites at which bone is remodeled) and bone resorption at remodeling sites. /Risedronate/
Risedronate sodium, a synthetic pyridinyl bisphosphonate analog of pyrophosphate, is an inhibitor of osteoclast-mediated bone resorption. / Risedronate sodium/
... Nitrogen-containing bisphosphonates (such as pamidronate, alendronate, risedronate, ibandronate and zoledronate) appear to act as analogues of isoprenoid diphosphate lipids, thereby inhibiting FPP synthase, an enzyme in the mevalonate pathway. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprenoid lipids (FPP and GGPP) that are essential for the post-translational farnesylation and geranylgeranylation of small GTPase signalling proteins. Loss of bone-resorptive activity and osteoclast apoptosis is due primarily to loss of geranylgeranylated small GTPases. Identification of FPP synthase as the target of nitrogen-containing bisphosphonates has also helped explain the molecular basis for the adverse effects of these agents in the GI tract and on the immune system. /Risedronate/

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Therapeutic Uses
Bone resorption inhibitor.
Risedronate is indicated for the prevention and treatment of glucocorticoid-induction osteoporosis in men and women who are either initiating or continuing systemic glucocorticoid treatment for chronic diseases./Risedronate; Included in US product labeling/
Risedronate is indicated for the prevention of osteoporosis in postmenopausal women. It may be considered in postmenopausal women who are at risk of developing osteoporosis and for whom the desired clinical outcome is to maintain bone mass and to reduce the risk of fracture. /Risedronate; Included in US product labeling/
Risedronate is indicated for the treatment of post menopausal osteoporosis. It increases the bone mineral density and reduces the incidence of vertebral fractures and a composite endpoint of nonvertebral osteoporosis fractures. /Risedronate; Included in US product labeling/
Risedronate is indicated for the treatment of Paget's disease of bone (osteitis deformans) in patients with alkaline phosphatase concentrations that are at least two times the upper limit of normal, those who are symptomatic, or those at risk for future complications from the disease. Signs and symptoms of Paget's disease may include bone pain, deformity, and/or fractures; increased concentrations of N-telopeptide of I collagen, serum alkaline phosphatase, and/or urinary hydroxyproline; neurologic disorders associated with skull lesions and spinal deformities; and elevated cardiac output and other vascular disorders associated with increased vascularity of bones. /Risedronate; Included in US product labeling/

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Drug Warnings
Risendronate should not be used in patients with severe renal impairment (creatinine clearance less than 30 mL/minute). Adjustments in risedronate sodium dosage are not necessary in patients with mild-to-moderate renal impairment (a creatinine clearance of 30 mL/minute or greater) or in patients with hepatic impairment. /Risedronate/
Adverse upper GI effects (e.g. dysphagia, esophagitis, esophageal or gastric ulcer) have been reported in patients receiving bisphosphonates. In clinical studies, the incidence of such adverse upper GI effects in patients receiving risedronate was similar to that in patients receiving placebo. Data from postmarketing surveillance have occurred, albeit rarely, in patients receiving risedronate sodium 4mg to take risedronate with 180-240 mL of plain water to avoid lying down for 30 minutes following administration of the drug. To minimize risk of adverse upper GI effects, patients should be advised to take risedronate with 180 to 240 mL of plain water and to avoid lying down for 30 minutes following administration of the drug. /Risedronate sodium/
Osteonecrosis and osteomyelitis of the jaws have been reported in patients, principally in these with cancer, who have received bisphosphonates.
Hypocalcemia and other disturbances of bone and mineral metabolism must be corrected before risedronate therapy is initiated, and patients with osteoporosis or Paget's disease of bone should receive supplemental calcium and vitamin D if their daily dietary intake is adequate. /Risedronate/
For more Drug Warnings (Complete) data for RISEDRONIC ACID (10 total), please visit the HSDB record page.

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Pharmacodynamics
Risedronate is a pyridine-based bisphosphonate that inhibits bone resorption caused by osteoclasts.

C7H11NO7P2

283.11

283.001

C, 29.70; H, 3.92; N, 4.95; O, 39.56; P, 21.88

105462-24-6

Risedronic Acid-d4;1035438-80-2

5245

White to off-white solid powder

1.9±0.1 g/cm3

692.3±65.0 °C at 760 mmHg

252-262

372.5±34.3 °C

0.0±2.3 mmHg at 25°C

1.651

-2.94

5

8

4

17

339

0

IIDJRNMFWXDHID-UHFFFAOYSA-N

InChI=1S/C7H11NO7P2/c9-7(16(10,11)12,17(13,14)15)4-6-2-1-3-8-5-6/h1-3,5,9H,4H2,(H2,10,11,12)(H2,13,14,15)

(1-hydroxy-1-phosphono-2-pyridin-3-ylethyl)phosphonic acid

Risedronic acid; Risedronate; 105462-24-6; Atelvia; Ridron; Acido risedronico; Acide risedronique; Acidum risedronicum;

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)

0.1 M NaOH : ~11 mg/mL (~38.85 mM)
H2O : ~0.67 mg/mL (~2.37 mM)

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.)