Target: Muscarinic acetylcholine receptors (M1, M2, M3, M4, M5). Tiotropium Bromide hydrate (BA-679 BR) is a muscarinic receptor antagonist with prolonged M3 receptor antagonism (dissociates extremely slowly from M3 receptors) and rapid dissociation from M2 receptors.[1]

In vitro activity: Tiotropium bromide is a potent muscarinic antagonist with equal affinity for M1-, M2- and M3-receptors and is approximately 10-fold more potent than ipratropium bromide. Tiotropium bromide has a potent inhibitory effect against cholinergic nerve-induced contraction of guinea-pig and human airways, that has a slower onset than atropine or ipratropium bromide. Tiotropium bromide, ipratropium bromide and atropine all increase ACh release on neural stimulation and that this effect is washed out equally quickly for the three antagonists. Tiotropium bromide dissociates slowly from M3-receptors (on airway smooth muscle) but rapidly from M2 autoreceptors (on cholinergic nerve terminals). Tiotropium significantly inhibits the release of chemotactic substances by AM, MonoMac6 and A549 cells. Tiotropium bromide inhibits the Th2 cytokine production from PBMCs.
Kinase Assay: Tiotropium Bromide hydrate is an anticholinergic and bronchodilator and a muscarinic receptor antagonist. Target: mAChR Tiotropium bromide (Ba 679 BR) is a novel potent and long-lasting muscarinic antagonist that has been developed for the treatment of chronic obstructive airways disease (COPD). Binding studies with [3H]tiotropium bromide in human lung have confirmed that this is a potent muscarinic antagonist with equal affinity for M1-, M2- and M3-receptors and is approximately 10-fold more potent than ipratropium bromide. In vitro tiotropium bromide has a potent inhibitory effect against cholinergic nerve-induced contraction of guinea-pig and human airways, that has a slower onset than atropine or ipratropium bromide. tiotropium bromide dissociates slowly from M3-receptors (on airway smooth muscle) but rapidly from M2 autoreceptors (on cholinergic nerve terminals). Tiotropium bromide is a quaternary ammonium derivative that binds to muscarinic receptors. However, although tiotropium binds with high affinity to muscarinic receptors of M1-, M2- and M3-subtypes, it dissociates very slowly from M1- and M3-receptors but more rapidly from M2-receptors, thereby giving it a unique kinetic selectivity.

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In Vitro: Tiotropium Bromide hydrate (BA-679 BR) at 20 nM significantly inhibited the release of chemotactic substances by human alveolar macrophages (AM), MonoMac6 cells (myelo-monocytic cell line), and A549 cells (bronchial epithelial cell line) after stimulation with acetylcholine (ACh, 100 µM). The secretion of chemotactic mediators was suppressed by more than 70% after co-incubation with Tiotropium Bromide hydrate (BA-679 BR). Tiotropium Bromide hydrate (BA-679 BR) alone did not influence granulocyte migration. The ACh-induced chemotactic activity was mainly attributed to leukotriene B4 (LTB4), as the selective LTB4 receptor antagonist BIL260 inhibited the migration. ACh did not induce the release of interleukin-8 (IL-8) or monocyte chemotactic protein-1 (MCP-1) from these cells. Tiotropium Bromide hydrate (BA-679 BR) alone did not change chemotactic activity, arguing for direct competition with ACh for binding to muscarinic receptors. Preincubation with Tiotropium Bromide hydrate (BA-679 BR) did not increase the observed effect. Cell viability was higher than 90% in all experiments after incubation with Tiotropium Bromide hydrate (BA-679 BR). No release of chemotactic mediators was detected in fibroblasts incubated with ACh and Tiotropium Bromide hydrate (BA-679 BR). [1]

Tiotropium bromide significantly reduces airway inflammation and the Th2 cytokine production in bronchoalveolar lavage fluid (BALF) in both acute and chronic mouse models of asthma. Tiotropium bromide significantly decreases the goblet cell metaplasia, thickness of airway smooth muscle, and airway fibrosis in a mouse model of asthma. Tiotropium bromide reduces the levels of TGF-beta1 in BALF in a chronic model. Tiotropium inhibits the increase in airway smooth muscle mass, myosin expression, and contractility in a guinea pig model of ongoing allergic asthma. Tiotropium abrogates the LPS-induced increase in neutrophils, goblet cells, collagen deposition and muscularised microvessels in a guinea pig model of chronic obstructive pulmonary disease (COPD), but has no effect on emphysema.

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Cell Assay:
1. Cell culture: Lung fibroblasts were obtained from healthy lung tissue, minced, digested with collagenase IV, and cultured in Iscove's modified Dulbecco's medium with 10% foetal calf serum (FCS), glutamine, and antibiotics. A549 bronchial epithelial cells and MonoMac6 myelo-monocytic cells were cultured in growth medium (GM) and subdivided every 3 days. Alveolar macrophages (AM) were obtained from bronchoalveolar lavage (BAL) of non-smoker patients without lung disease; BAL fluid was filtered, centrifuged, and cells resuspended. Only preparations with <3% granulocytes and <10% lymphocytes were used. [1]
2. Conditioned media preparation: Fibroblasts (2×10^5 cells), A549 cells (2×10^5 cells), MonoMac6 cells (3.2×10^5 cells/ml), or AM (1×10^6 cells/ml) were incubated in Ham's F12 medium with different concentrations of ACh (1-100 µM) for various time periods (4-72 h). Tiotropium Bromide hydrate (BA-679 BR) (20 nM) was added as indicated. Supernatants were collected, centrifuged, and frozen. Cell viability was measured after incubation using propidium iodide and flow cytometry (viability >90%). [1]
3. Granulocyte isolation: Heparinised blood from healthy donors was subjected to density gradient centrifugation with Ficoll/Paque. The erythrocyte/granulocyte pellet was resuspended in polyvinylalcohol, incubated for sedimentation, and remaining erythrocytes lysed with ammonium chloride. Purity (>95% CD62L-positive, CD3/CD19/CD14-negative) was determined by flow cytometry. Cells were resuspended in ice-cold RPMI at 2×10^7 cells/ml. [1]
4. Chemotaxis assay: A 24-well transwell chamber with 3 µm pore diameter was used. Conditioned medium was placed in the bottom chamber, and isolated granulocytes (100 µl of 2×10^6 cells) in the upper chamber. After 30 min incubation at 37°C in 5% CO2, non-migrated cells were wiped off, filters formalin-fixed, stained with crystal violet, and bound stain extracted with acetic acid. Migration was measured photometrically at 570 nm. Spontaneous migration (GM alone) set to 100%; fMLP (1×10^-6 M) was positive control. For LTB4 receptor blocking, BIL260 (100 nM) was added to the upper chamber with granulocytes. [1]
5. Cytokine measurement: Commercially available ELISA kits were used to detect human IL-8 and MCP-1 in culture supernatants. [1]

acute and chronic mouse models of asthma

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Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
While there is currently no published data on the use of tiotropium bromide during lactation, its concentration in maternal serum is extremely low, and the drug in breast milk is not absorbed by the infant. The risk to breastfed infants from maternal inhalation of tiotropium bromide is very small. International guidelines agree that breastfeeding can continue during tiotropium bromide treatment.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Respir Med.2007 Nov;101(11):2386-94;Clin Exp Allergy.2010 Aug;40(8):1266-75.

Tiotropium bromide hydrate is a hydrated form of tiotropium bromide monohydrate. It is used for maintenance therapy of airflow obstruction in patients with chronic obstructive pulmonary disease (COPD). It is a muscarinic receptor antagonist and bronchodilator. Tiotropium bromide is a scopolamine derivative and cholinergic antagonist with bronchodilatory effects. It is used to treat COPD. See also: Tiotropium bromide monohydrate (note moved to).

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Additional Info:
- Background: Chronic obstructive pulmonary disease (COPD) is associated with chronic inflammation, and exacerbations are often due to excessive inflammation. Parasympathetic nerves release acetylcholine (ACh) which activates muscarinic receptors on airway smooth muscle and submucosal glands, causing bronchoconstriction and mucus secretion. Non-neuronal actions of ACh have been shown to induce release of chemotactic factors from epithelial cells and macrophages. [1]
- Proposed mechanism: Tiotropium Bromide hydrate (BA-679 BR) suppresses ACh-induced release of chemotactic mediators (mainly LTB4) from alveolar macrophages and epithelial cells via blockade of muscarinic M3 receptors, thereby potentially reducing neutrophil migration and chronic inflammation in the lung. This may contribute to the reduced exacerbation frequency observed in clinical studies with Tiotropium Bromide hydrate (BA-679 BR) in COPD patients. [1]
- Clinical relevance: The reduction in exacerbation rates in COPD with Tiotropium Bromide hydrate (BA-679 BR) is traditionally attributed to inhibition of vagal-mediated bronchoconstriction and mucus secretion, but this study suggests an additional anti-inflammatory action. [1]
- Receptor expression: The study found that lung tissue, A549 cells, and MonoMac6 cells express all five muscarinic receptor subtypes (M1-M5) with predominance of M3. Lung fibroblasts predominantly express M2 receptor mRNA (226-fold higher than M3). The absence of pro-inflammatory action of ACh on fibroblasts may be due to low M3 receptor expression. [1]

C19H22NO4S2.BR.XH2O

490.43

489.027

139404-48-1

136310-93-5 [Tiotropium Bromide (BA679 BR)]

11431811

Typically exists as solid at room temperature

218-220ºC

2

8

5

28

564

4

[H][C@@]12[C@@H](O3)[C@@H]3[C@@](C[C@H](OC(C(C4=CC=CS4)(O)C5=CC=CS5)=O)C2)([H])[N+]1(C)C.O.[Br-]

DQHNAVOVODVIMG-RGECMCKFSA-M

InChI=1S/C19H22NO4S2.BrH/c1-20(2)12-9-11(10-13(20)17-16(12)24-17)23-18(21)19(22,14-5-3-7-25-14)15-6-4-8-26-15;/h3-8,11-13,16-17,22H,9-10H2,1-2H3;1H/q+1;/p-1/t11?,12-,13+,16-,17+;

(1R,2R,4S,5S,7s)-7-(2-hydroxy-2,2-di(thiophen-2-yl)acetoxy)-9,9-dimethyl-3-oxa-9-azatricyclo[3.3.1.02,4]nonan-9-ium bromide

BA 679BR; PUR-0200; BA 679BR; BA-679BR; BA679BR; PUR0200; PUR 0200; Tiotropium bromide, trade name: Spiriva.

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)

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.)