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
The oral bioavailability is 0.63%, with peak absorption occurring approximately 1 hour after administration. Administration 30 minutes before a meal reduces bioavailability by 55% compared to fasting, and administration 1 hour before a meal reduces bioavailability by 30%. Risedronate sodium is excreted by the kidneys; unabsorbed doses are excreted in the feces. 13.8 L/kg. The mean renal clearance is 52 mL/min, and the mean total clearance is 73 mL/min. Absorption is rapid, dose-independent, and occurs throughout the upper gastrointestinal tract. The mean oral bioavailability is 0.63%, and co-administration with food reduces its bioavailability. Administration 0.5 hours before breakfast or 2 hours after dinner reduces absorption by 55% compared to a fasting state (no food or water for 10 hours before or 4 hours after administration). Administration 1 hour before breakfast reduces absorption by 30% compared to a fasting state. Studies in rats and dogs have shown that approximately 60% of the dose of radiolabeled sodium risedronate is distributed to bone after a single intravenous injection of radiolabeled sodium risedronate. In humans, the mean steady-state volume of distribution is 6.3 L/kg body weight. In rats, after multiple oral administrations, the absorption rate of sodium risedronate in soft tissues ranged from 0.001% to 0.01%. Sodium risedronate was detected in pups exposed to lactating rats within 24 hours of administration, indicating minimal milk transport. Sodium risedronate excretion: Excreted in feces; no absorption (unaltered). Excreted by the kidneys; no alteration of the drug; approximately 50% of the absorbed dose is excreted within 24 hours, and 85% within 28 days. The mean renal clearance is 105 mL/min, and the mean total clearance is 122 mL/min; the difference primarily reflects clearance not due to renal resorption or bone resorption. Note: Renal clearance is independent of drug concentration; renal clearance is linearly related to creatinine clearance. Risedronate Sodium

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Metabolism/Metabolites
Risedronate is unlikely to be metabolized before excretion. PCP bisphosphonates are resistant to chemical and enzymatic hydrolysis, thus preventing the metabolism of this molecule.
There is no evidence that risedronate sodium is metabolized in humans or animals. Risedronate Sodium
No evidence has been found that risedronate sodium is metabolized in humans or mammals.
Excretion Route: Risedronate sodium is primarily excreted unchanged via the kidneys. In rats, only a very small amount (<0.1% of the intravenous dose) of the drug is excreted via bile.
Half-life: 1.5 hours

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Biological Half-life
The initial half-life of risedronate is approximately 1.5 hours, and the terminal half-life is 561 hours. Initial duration: approximately 1.5 hours; terminal exponential decay: 480 hours (potentially representing the dissociation of risedronate from the bone surface). Risedronate

Toxicity Summary
Risedronate sodium's effects on bone tissue are partly based on its affinity for hydroxyapatite, a component of the bone mineral matrix. Risedronate sodium also targets farnesyl pyrophosphate (FPP) synthase. Nitrogenous bisphosphonates (such as pamidronate sodium, alendronate sodium, risedronate sodium, ibandronate sodium, and zoledronic acid sodium) appear to act as lipid analogs of isoprene diphosphate, thereby inhibiting an enzyme in the mevalonate pathway—FPP synthase. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprene lipids (FPP and GGPP), which are crucial for the post-translational farnesylation and geranylation of small GTPase signaling proteins. This activity inhibits osteoclast activity and reduces bone resorption and bone turnover. In postmenopausal women, it reduces elevated bone turnover rates, resulting in a net increase in bone mass on average.

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Effects during pregnancy and lactation
◉ Overview of use during lactation
Since there is currently no information on the use of risedronate sodium during lactation, alternative medications may be preferred, especially when breastfeeding newborns or premature infants. However, breastfed infants are unlikely to absorb risedronate sodium.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding rate
Approximately 24%.

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Interactions
Antacids or mineral supplements containing divalent cations: When risedronate sodium is used concomitantly with antacids or mineral supplements containing divalent cations (e.g., aluminum, calcium, magnesium), a pharmacokinetic interaction occurs (reducing the absorption of risedronate sodium). Risedronate Sodium
Nonsteroidal anti-inflammatory drugs (SRPs: NSAIDs): No evidence of increased upper gastrointestinal adverse reactions was found. Risedronate Sodium
Histamine H2 receptor antagonists, proton pump inhibitors: No evidence of increased upper gastrointestinal adverse reactions was found. Risedronate Sodium/

[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.

Risedronate belongs to the pyridine class of compounds. Risedronate is a third-generation bisphosphonate used to treat certain types of osteoporosis and Paget's disease. Its mechanism of action is the inhibition of bone resorption. Risedronate is a bisphosphonate. Risedronate is a synthetic pyridyl bisphosphonate with anti-bone resorption activity. After administration, risedronate binds to hydroxyapatite crystals in bone, inhibiting osteoclast-dependent bone resorption. Risedronate sodium is only present in individuals who have used or taken this drug. It is a bisphosphonate used to strengthen bones, treat or prevent osteoporosis, and treat Paget's disease. Risedronate sodium's action on bone tissue is partly based on its affinity for hydroxyapatite, a component of the bone mineral matrix. Risedronate sodium also targets farnesyl pyrophosphate (FPP) synthase. Nitrogenous bisphosphonates (such as pamidronate sodium, alendronate sodium, risedronate sodium, ibandronate sodium, and zoledronic acid sodium) appear to act as lipid analogs of isoprene diphosphate, thereby inhibiting an enzyme in the mevalonate pathway—FPP synthase. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprene lipids (FPP and GGPP), which are crucial for the post-translational farnesylation and geranylation of small GTPase signaling proteins. This activity inhibits osteoclast activity and reduces bone resorption and bone turnover. In postmenopausal women, it reduces elevated bone turnover and, on average, results in a net increase in bone mass. A pyridine and bisphosphonate derivative that acts as a calcium channel blocker, inhibiting bone resorption. See also: risedronate sodium hemipentahydrate (active ingredient); risedronate sodium monohydrate (active ingredient).

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Drug Indications
Risedronate is indicated for the treatment and prevention of osteoporosis in men, Paget's disease, and postmenopausal women, as well as glucocorticoid-induced osteoporosis.
FDA Label

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Mechanism of Action
Risedronate binds to bone hydroxyapatite. Bone resorption leads to local acidification, releasing risedronate, which is then absorbed by osteoclasts via liquid-phase endocytosis. Following acidification of the endocytic vesicles, risedronate is released into the osteoclast cytosol, where it induces osteoclast apoptosis by inhibiting farnesyl pyrophosphate synthase. This inhibition of osteoclast activity leads to reduced bone resorption.

Risedronate sodium binds to bone hydroxyapatite and inhibits osteoclasts at the cellular level. Although osteoclasts still adhere normally to the bone surface, they exhibit signs of reduced active resorption (e.g., lack of folded margins).
Studies in rats and dogs have shown that risedronate sodium treatment reduces bone turnover rate (activation frequency, i.e., the number of sites of bone remodeling) and bone resorption at remodeling sites. /Risedronate Sodium/
Risedronate sodium is a synthetic pyridyl bisphosphonate analog, a pyrophosphate analog, that inhibits osteoclast-mediated bone resorption. Risedronate Sodium/
...Nitrogenous bisphosphonates (such as pamidronate sodium, alendronate sodium, risedronate sodium, ibandronate sodium, and zoledronic acid sodium) appear to act as isoprene diphosphate lipid analogs, thereby inhibiting an enzyme in the mevalonate pathway—FPP synthase. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprene lipids (FPP and GGPP), which are crucial for the post-translational farnesylation and geranylation of small GTPase signaling proteins. Loss of bone resorption activity and osteoclast apoptosis are primarily attributed to the loss of geranylation of small GTPases. Identifying FPP synthase as the target of nitrogenous bisphosphonates also helps explain the molecular mechanisms by which these drugs cause adverse reactions in the gastrointestinal and immune systems. Risedronate Sodium /

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Therapeutic Use
Bone resorption inhibitor.
Risedronate sodium is indicated for the prevention and treatment of glucocorticoid-induced osteoporosis in men and women who have started or continue systemic glucocorticoid therapy for chronic disease. /Risedronate Sodium; included on the US product label/
Risedronate sodium is indicated for the prevention of osteoporosis in postmenopausal women. Risedronate sodium may be considered for postmenopausal women at risk of developing osteoporosis and whose expected clinical outcome is maintenance of bone mass and reduction of fracture risk. /Risedronate Sodium; included on the US product label/
Risedronate sodium is indicated for the treatment of postmenopausal osteoporosis. It increases bone mineral density and reduces the incidence of the composite endpoint of vertebral fractures and non-vertebral osteoporotic fractures. Rizedronate sodium; the US product label contains the following: Rizedronate sodium is indicated for the treatment of Paget's disease (osteodermatitis deformans) in patients with alkaline phosphatase concentrations at least twice the upper limit of normal, symptomatic patients, or patients at risk of developing complications of the disease. Signs and symptoms of Paget's disease may include bone pain, deformity and/or fracture; elevated N-terminal peptide of type I collagen, serum alkaline phosphatase and/or urinary hydroxyproline concentrations; neurological disorders associated with skull lesions and spinal deformities; and increased cardiac output and other vascular disorders associated with increased skeletal angiogenesis. Rizedronate sodium; the US product label contains the following:

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Drug Warning
Rizedronate sodium is contraindicated in patients with severe renal impairment (creatinine clearance less than 30 mL/min). No dose adjustment of risedronate sodium is necessary for patients with mild to moderate renal impairment (creatinine clearance ≥30 mL/min) or hepatic impairment. Risedronate Sodium/
Patients taking bisphosphonates have reported upper gastrointestinal adverse reactions (e.g., dysphagia, esophagitis, esophageal or gastric ulcers). Clinical studies have shown that the incidence of such upper gastrointestinal adverse reactions in patients taking risedronate sodium is similar to that in patients taking placebo. Post-marketing surveillance data show that, although rare, patients taking 4 mg risedronate sodium should take it with 180-240 mL of water and avoid lying down for 30 minutes after taking the medication. To minimize the risk of upper gastrointestinal adverse reactions, patients should be advised to take risedronate sodium with 180 to 240 mL of water and avoid lying down for 30 minutes after taking the medication. Risedronate Sodium
Osteonecrosis of the jaw and osteomyelitis have been reported in patients receiving bisphosphonate treatment (primarily cancer patients).
Before initiating risedronate sodium treatment, hypocalcemia and other bone and mineral metabolism disorders must be corrected; patients with osteoporosis or Paget's disease should supplement with calcium and vitamin D if their daily dietary intake is adequate. Risedronate Sodium
For more complete data on risedronate (10 in total), please visit the HSDB records page.

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Pharmacodynamics
Risedronate sodium is a pyridine bisphosphonate that inhibits osteoclast-induced bone resorption.

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.)