Trypsin-like serine proteases; Tryptase (Ki = 20 μM); Trypsin (Ki = 21 μM)

Benzamidine hydrochloride (50 μM) decreases fibroblasts' [3H]thymidine synthesis, indicating coupling to the active catalytic site. Tryptase's capacity to promote the synthesis of collagen is diminished by benzoamidine hydrochloride [2].

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Variability in the structures of bound amidine inhibitors [1]
Comparison of the structures of the inhibitor complexes of uPA, tPA, trypsin and thrombin denoted by the bold entries in Table 1 reveals a remarkable diversity in the binding of such small inhibitors. The complexes can be divided roughly into two groups based on the orientations of the aromatic planes of the bound inhibitors. The inhibitors in the first set are rotated by ∼20° from those in the second set around their long symmetry axis. Consequently the amidines in the first set are rotated around the amidine-aromatic bond by ∼–20° from those in the second set in order to maintain hydrogen-bonding interactions at S1. The first set includes trypsin–, thrombin– and uPA–4-iodobenzo[b]thiophene-2-carboxamidine, uPA–thieno[2,3-b]pyridine-2-carboxamidine, and uPA– and tPA–benzamidine; the second set trypsin– and thrombin–Benzamidine, and trypsin–thieno[2,3-b]pyridine-carboxamidine. The aromatic amidine dihedrals for the bound inhibitors range from –20° to +18° (Table 2).

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Diversity in the S1 site architectures of the trypsin-like serine proteases complexes [1]
Differences in the bound structure of Benzamidine or of other small amidine inhibitors among the trypsin-like protease complexes reflect subtle but significant changes in the corresponding S1 site architectures. In uPA–benzamidine there are shifts (of ∼0.5 Å) in the position of OγSer190, OηTyr228, and water1 from the respective locations in trypsin–benzamidine (Figure 2a, Table 3a). In trypsin–benzamidine (and in many other trypsin–amidine complexes) water1 makes four hydrogen bonds. In the hydrogen-bonding scheme of Figure 2a, water1 accepts hydrogen bonds from OγSer190, (3.14 ± 0.06 Å) and from N1benzamidine (3.04 ± 0.06 Å), and donates hydrogen bonds to OTrp215 (3.06 ± 0.06 Å) and to OVal227 (2.87 ± 0.05 Å; Table 2). In uPA–benzamidine, however, the latter hydrogen bonds are absent (water1–OTrp215=3.4 Å, water1–OVal227=3.3 Å). Their absence is associated with shorter N1–OγSer190 and water1–OγSer190 hydrogen bonds (2.5 Å and 2.8 Å) than in the other trypsin and uPA complexes (Table 2). The difference in hydrogen-bonding interactions involving water1 between uPA– and trypsin–benzamidine is not seen between uPA– and trypsin–4-iodobenzo[b]thiophene-2-carboxamidine. In both of the latter complexes water1 makes hydrogen bonds with OγSer190, N1 and OVal/Phe227, but not with OTrp215 (Table 2).

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A dramatic increase in the depth of the S1 site of uPA compared with that of thrombin is apparent in the comparison of the structures of uPA– and thrombin–Benzamidine(Figure 2b). Bound benzamidine is shifted by 0.5 Å, and the Asp189 sidechain is correspondingly shifted by ∼0.6 Å (Table 3a). The relative positions of the bound inhibitors and of the Asp189 sidechain are significantly different in many of the comparisons that involve a common bound inhibitor. The largest differences occur between uPA and thrombin. By contrast, the structures of the S1 sites of uPA and tPA are highly similar (Figure 3c) and have the lowest overall rms deviations from one another, 0.29 Å (Table 3a). Benzamidine binds in a similar relative position and orientation, and with a similar planarity to these two related proteases (Figure 3c). The high similarity of the uPA– and tPA–benzamidine complexes underscores the effect of residue 190 on inhibitor potency. The most obvious difference between the complexes, the absence of the Ser190 sidechain and of the associated hydrogen bond to benzamidine in the tPA complex, is strongly implicated in the 7.7-fold decrease in potency. [1]

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Structural comparison of inhibitor complexes with corresponding inhibitor-free enzymes [1]
The B factors of protein groups making hydrogen bonds with trypsin (Oδ1,2Asp189, OγSer190 and OGly219,) decrease significantly upon inhibitor binding (Table S1b), and the OγSer190 atom undergoes a large shift, 0.5 Å in trypsin–Benzamidine (Table 3b). Binding of benzamidine to thrombin decreases the B factors of Nϵ2His57, Oδ1,2Asp189 and CβAla190 (Table S1b). Binding of inhibitors to thrombin incurs greater and more extensive structural changes than does binding of the same inhibitors to trypsin. The rms deviations between the superimposed inhibitor-binding sites of thrombin–benzamidine and apo-thrombin is 0.27 Å, compared with 0.14 Å for trypsin–benzamidine/apo-trypsin (Table 3b). Binding of benzamidine induces a major contraction of the S1 site of thrombin, by 0.8 Å, along the CαGlu192–CαTrp215 vector, compared with only 0.2 Å for trypsin (Table S1a). In thrombin the contraction is reflected in a 0.6 Å change in the position of CαGlu192 (Table 3b).
The water (water1) that mediates inhibitor binding at S1 in the complexes is bound in a similar location in the structures of apo-trypsin (Figure 4a), apo-thrombin (Figure 4b) and apo-factor Xa. In apo-trypsin water1 makes hydrogen bonds with OγSer190, OVal227, OTrp215, as in trypsin–Benzamidine, and trypsin–thieno[2,3-b]pyridine-2-carboxamidine, and makes a fourth hydrogen bond with another ordered water (Figure 4a). Although there is a moderate increase in mobility of this water (29 Å2) compared with that in the complexes (B=18–25 Å2), it is well ordered in apo-trypsin in terms of both location and orientation.

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Preincubation of tryptase with either leupeptin or Benzamidine hydrochloride reduced [3 H]thymidine incorporation in fibroblasts (Fig. 1 A), suggesting dependency on an active catalytic site. Using the same concentrations of protease inhibitors, the activity of tryptase (18 and 36 mU/ml) towards the substrate BAPNA was inhibited by 90% in the case of leupeptin, and by 75% for benzamidine hydrochloride. Leupeptin and benzamidine hydrochloride alone at the concentrations used did not significantly affect [3 H]thymidine incorporation in fibroblasts (Fig. 1 B). Since all the tryptase preparations used in this study contained heparin in a 1:1 ratio (wt/wt) in order to maintain enzymatic activity, the effect of heparin alone on cell proliferation was assessed. Heparin at the concentrations employed had no significant effect on [3 H]thymidine incorporation, although there was a tendency for inhibition at high concentrations of heparin (Fig. 1 B). [2]

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[3 H]proline incorporation. A 2.5-fold increase in collagen synthesis determined by the incorporation of [3 H]proline was detected in fibroblast supernatants following incubation with tryptase (Fig. 3 A). Leupeptin and Benzamidinehydrochloride reduced the ability of tryptase to stimulate collagen synthesis. As was the case with the cell proliferation study, the response appeared to be reduced at 120 mU/ml tryptase, although no cytotoxic effects were evident, and the cell monolayer appeared intact as visualized under the microscope. Since not all the collagen synthesized is released into the supernatant, and a proportion may remain cell-associated, the cell monolayer was assayed for collagen synthesis in a similar manner, and the data was presented in Fig. 3 B. Very little collagen remained cell-associated, indicating that almost all the collagen synthesized by fibroblasts had been released into the supernatant. A small increase in proline incorporation was noted in lysates from cells incubated with 15 and 30 mU/ml tryptase, although the increase observed at 30 mU/ml was not reduced in the presence of benzamidine hydrochloride. It is possible that this amount may represent residual collagen associating nonspecifically with the cell surface. Leupeptin and benzamidine hydrochloride alone at the concentrations employed, and heparin at 10, 50, and 100 mg/ml, did not have any significant effects on collagen synthesis (data not shown). [2]

Human thrombin–acetyl-hirudin and co-complexes. [1]
Thrombin and acetyl-hirudin were used. Thrombin-acetyl-hirudin was prepared as described previously. Thrombin (1.0 mg/ml in 50 mM HEPES, 50% glycerol, pH 7.0) was incubated with 1.0 mM acetyl-hirudin in the presence or absence of amidine inhibitors for 1 h at 4°C. The solution contained 5 equivalents of 4-iodobenzo[b]thiophene-2-carboxamidine, 10 mM Benzamidine, or was saturated in [2,3-b]thienopyridine-2-carboxamidine (∼2 mM). Glycerol was removed during concentration to ∼10 mg/ml of the complexes with a centricon 10 (Amicon). Crystals of thrombin-acetyl-hirudin with or without co-bound small molecule inhibitors, pH 7.3 or 7.8, space group C2 (a=71.2, b=71.8, c=72.7 Å, β=100.7°) were grown in hanging drops by vapor diffusion after streak seeding. The structure without an inhibitor at the S1 site is referred to as apo-thrombin. The drops were made from 3 ml complex and 3 ml reservoir solution (0.10 M HEPES, 0.30 M NaCl, 22% (by volume) PEG 5K monomethyl ether, pH 7.5 or 8.2). Large single crystals of dimensions >0.2 mm in each dimension grew within 1 week.

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Bovine trypsin complexes and inhibitor-free trypsin. [1]
Trypsin was crystallized as described previously. A structure of P3121 trypsin–Benzamidine was determined at 1.20 Å resolution, in 2.02 M MgSO4radical dot7 H2O, 100 mM MES, 1.0 mM CaCl2, pH 7.5. The carboxamidine complexes were prepared by soaking trypsin–Benzamidine crystals in synthetic mother liquor saturated in the inhibitors. The soaking solutions were replaced 4 times, about once a day. For the complexes determined at pH 8.2 the soaking solutions contained 1.73 M MgSO4radical dot7 H2O, 150 mM Tris, 1 mM CaCl2 and 2% dimethylsulfoxide (DMSO). For the 4-iodobenzo[b]thiophene-2-carboxamidine complex at pH 5.5, the soaking solution was 85% saturated sodium citrate, 1 mM CaCl2, 2.0% DMSO, saturated with inhibitor, pH 5.5. The pH was adjusted with saturated citric acid. For the thieno[2,3-b]pyridine-2-carboxamidine complex, pH 5.5, the soaking solution was 1.73 M MgSO4radical dot7 H2O, 150 mM MES, 1 mM CaCl2, 2% DMSO. To prepare inhibitor-free trypsin crystals, trypsin–Benzamidine crystals were soaked at the target pH values for several weeks in 1.84 M MgSO4radical dot7 H2O, 150 mM MES or Tris, 1.0 mM CaCl2, during which the soaking solutions were periodically replaced.

Cell proliferation assay. Confluent fibroblasts were detached from culture flasks with a nonenzymatic cell dissociating solution and seeded into a 96-well microtiter plate at a density of 105 cells per ml in MEM containing 10% FCS. At confluence, the medium was replaced with serum-free (SF) medium consisting of MEM supplemented with 5 mg/ml bovine pancreas insulin, 5 mg/ml transferrin, and 5 ng/ml sodium selenite. After 24 h of serum deprivation, purified tryptase was added following its dialysis in the presence of heparin (1: 1; wt/wt; to stabilize enzymatic activity) for 24 h at 48C against phosphate-buffered saline (PBS). In experiments with protease inhibitors, tryptase with added heparin was incubated in the presence or absence of leupeptin or Benzamidine hydrochloride for 1 h at 48C, after which the BAPNA cleaving activity of tryptase was assayed to determine the percentage of inhibition achieved. Control tryptase samples to which no inhibitor was added were incubated under the same conditions. The inhibitors were used at concentrations which in preliminary experiments had been shown to be nontoxic to the fibroblasts, and without effect on thymidine incorporation. After the various additions were made, the cells were incubated at 378C for 32 h with the addition of 1 mCi of methyl-[3 H]thymidine per well for the last 8 h to measure DNA synthesis. The cells were harvested and counted in scintillant. In addition, fibroblasts were seeded into 24- well plates and incubated with two optimal doses of tryptase for 72 h, and total cell number was determined by counting in a Neubauer hemocytometer after staining with Trypan Blue. [2]

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Collagen assay. Collagen was assayed by measuring the incorporation of [3 H]proline into collagen as described previously. Fibroblasts were plated at a density of 105 per ml into 96-well microtiter plates, and grown to confluence in MEM (proline- and hydroxyproline-free) with 10% FCS and antibiotics. Confluent cells were then serum-starved for 48 h before the various additions were made. Purified tryptase with heparin (1:1, wt/wt) was dialyzed against MEM and used in the following studies. The time course of collagen production was determined by incubating the cells with 25 mU/ml of tryptase (with heparin) or with medium alone in the presence of 1 mCi of [ 3 H]proline for 3, 6, 24, and 48 h. Total protein synthesis (both collagenous and non-collagenous) was determined by precipitating all the proteins in 100 ml cell supernatant onto glass fiber filters and counting in a scintillant. Noncollagenous proteins were assayed in a second 100-ml aliquot of the same supernatant after digestion with 40 mg of purified bacterial collagenase for 2 h at 378C, as described by Peterkofsky and Diegelman. The precipitated collagenase-resistant proteins were defined as noncollagenous. A preliminary experiment had shown that digestion of collagenous proteins in fibroblast supernatants with this concentration of collagenase was essentially complete after 2 h (data not shown). The difference between total and noncollagenous protein counts was taken to reflect the amount of collagen synthesized. As a control, supernatant samples were incubated for the same period with purified trypsin (Worthington) to check for specificity of digestion by collagenase (data not shown). In subsequent experiments, fibroblasts were incubated for 48 h either with medium alone or with purified tryptase (with heparin) to determine the dose response to tryptase. Levels of cell-associated collagen were assayed in the cell monolayer as described above. Cells were rinsed two times with PBS, trypsinized, and lysed in 200 ml of cold lysis buffer (20 mM Tris HCl, pH 7.4, containing 150 mM NaCl, 1 mM MgCl2, 0.1 mM ZnCl2, 1 mM EGTA, 1 mM PMSF, 1 mg/ml leupeptin, 5 mM Benzamidine hydrochloride, and 1% Nonidet P-40). [2]

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To investigate if the action of tryptase was mediated via its catalytic site, cells were incubated with either purified tryptase (with heparin), or with tryptase (heparin) that had been preincubated with leupeptin or Benzamidine hydrochloride as described above. As controls, collagen synthesis was assayed in supernatants from fibroblasts incubated with leupeptin or benzamidine hydrochloride alone, or with various concentrations of transforming growth factor b (TGF-b) and heparin. Since TGF-b is a potent stimulator of collagen synthesis in fibroblasts (1) and fibroblasts are known to release this cytokine, it was necessary to determine if tryptase-induced collagen synthesis may be mediated via enhanced TGF-b release. An experiment was conducted in which cells were incubated with tryptase (with heparin) in the presence of either a neutralizing TGF-b antibody or an irrelevant rabbit antibody. In addition, cells were incubated with antibody alone (with heparin) or with the irrelevant rabbit antibody at the same concentrations. Towards the latter part of this study, purified preparations of the monoclonal antibody to tryptase (AA5) became available in sufficient quantities to allow immunoaffinity purification Mast Cell Tryptase Stimulates Collagen Synthesis 1315 of tryptase. An additional experiment using immunoaffinity purified tryptase was conducted to confirm the effect of tryptase on collagen synthesis [2].

mouse LD50 intraperitoneal 580 mg/kg BRAIN AND COVERINGS: MENINGEAL CHANGES; BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); LUNGS, THORAX, OR RESPIRATION: OTHER CHANGES Biology of Reproduction., 20(1045), 1979 [PMID:113041]

[1]. Structural basis for selectivity of a small molecule, S1-binding, submicromolar inhibitor of urokinase-type plasminogen activator. Chem Biol. 2000 Apr;7(4):299-312.

[2]. Mast cell tryptase stimulates the synthesis of type I collagen in human lung fibroblasts. J Clin Invest. 1997 Mar 15;99(6):1313-21.

[3]. Structure-activity relationships for the inhibition of acrosin by benzamidine derivatives. J Med Chem. 1978 Nov;21(11):1132-6.

[4]. Proteolytic enzymes. VI. Aromatic amidines as competitive inhibitors of trypsin. J Biochem. 1971 May;69(5):893-9.

Background: Urokinase-type plasminogen activator (uPA) is a protease associated with tumor metastasis and invasion. Inhibitors of uPA may have potential as drugs for prostate, breast and other cancers. Therapeutically useful inhibitors must be selective for uPA and not appreciably inhibit the related, and structurally and functionally similar enzyme, tissue-type plasminogen activator (tPA), involved in the vital blood-clotting cascade. Results: We produced mutagenically deglycosylated low molecular weight uPA and determined the crystal structure of its complex with 4-iodobenzo[b]thiophene 2-carboxamidine (K(i) = 0.21 +/- 0.02 microM). To probe the structural determinants of the affinity and selectivity of this inhibitor for uPA we also determined the structures of its trypsin and thrombin complexes, of apo-trypsin, apo-thrombin and apo-factor Xa, and of uPA, trypsin and thrombin bound by compounds that are less effective uPA inhibitors, benzo[b]thiophene-2-carboxamidine, thieno[2,3-b]-pyridine-2-carboxamidine and Benzamidine. The K(i) values of each inhibitor toward uPA, tPA, trypsin, tryptase, thrombin and factor Xa were determined and compared. One selectivity determinant of the benzo[b]thiophene-2-carboxamidines for uPA involves a hydrogen bond at the S1 site to Ogamma(Ser190) that is absent in the Ala190 proteases, tPA, thrombin and factor Xa. Other subtle differences in the architecture of the S1 site also influence inhibitor affinity and enzyme-bound structure. Conclusions: Subtle structural differences in the S1 site of uPA compared with that of related proteases, which result in part from the presence of a serine residue at position 190, account for the selectivity of small thiophene-2-carboxamidines for uPA, and afford a framework for structure-based design of small, potent, selective uPA inhibitors. [1]

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The structures of thrombin– and tPA–Benzamidine provide insight into the poor benzamidine potencies for these proteases (Ki=320 μM and 750 μM, respectively, Table 1). When bound to thrombin and tPA, benzamidine not only lacks a hydrogen bond with residue 190 (alanine in these cases), but also has considerable conformational strain (the phenyl-amidine dihedral is –7° in thrombin, and –3° in tPA). An example of how structural features other than residue 190 can influence inhibitor affinity can be seen by comparing the structures and associated Ki values for uPA– and trypsin–benzamidine, both of which have serine at position 190. The S1 site of uPA is deeper than that of trypsin; the OγSer190–CαSer195 distance is 0.5 Å shorter in trypsin–benzamidine than in uPA–benzamidine. The S1 site of uPA is also wider, by 0.7 Å, along the CαAsp189–NGly226 vector (Table S1a). These enlargements in the S1 pocket of uPA compared with that of trypsin result in the loss of the water1–OPhe227 and water1–OTrp215 hydrogen bonds in uPA–benzamidine compared with trypsin–benzamidine. The shift in the position of water1 in uPA–benzamidine, by 0.5 Å, from its location in trypsin–benzamidine, is accompanied by an unfavorable change in the phenyl-amidine dihedral, from –20 ± 2° in trypsin–benzamidine, to 5° in uPA–benzamidine. The dihedral change corresponds to a calculated increase in conformational strain of 1.9 kcal/mol, larger than the actual decrease in binding energy of 0.9 kcal/mol calculated from the 4.6-fold decrease in affinity (Table 1). Favorable changes that may partially offset the conformational strain in uPA-bound Benzamidine include the significantly shorter (by 3.3 σ and 5.0 σ, respectively) N1–OγSer190 and water1–OγSer190 hydrogen bonds (Table 2). [1]

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Mast cell activation is a characteristic feature of chronic inflammation, a condition that may lead to fibrosis as a result of increased collagen synthesis by fibroblasts. We have investigated the potential of tryptase, the major protease of human mast cells, to stimulate collagen synthesis in the human lung fibroblast cell line MRC-5. Tryptase was isolated from human lung tissue by ion-exchange and affinity chromatography. At concentrations of 18 and 36 mU/ml, tryptase stimulated both an increase in cell numbers, and a fivefold increase in DNA synthesis as determined by methyl-[3H]thymidine incorporation. Similar concentrations of tryptase resulted in a 2.5-fold increase in collagen synthesis as determined both by incorporation of [3H]proline into collagen, and by assay of hydroxyproline concentrations in the supernatants. There was also a twofold increase in collagenolytic activity in the culture medium after tryptase treatment, indicating that the increase in collagen synthesis was not a consequence of decreased collagenase production. All of these actions of tryptase were reduced in the presence of the protease inhibitors leupeptin and Benzamidine hydrochloride, indicating a requirement for an active catalytic site. SDS-PAGE and autoradiographic analysis of the [3H]collagen produced by the cells revealed it to be predominantly type I collagen. Our findings suggest that the release of tryptase from activated mast cells may provide a signal for abnormal fibrosis in inflammatory disease. [2]

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A series, consisting of 52 benzamidine derivatives, was evaluated for inhibitory activity against homogeneous boar sperm acrosin. All of the compounds in the series proved to be more potent than benzamidine (Ki = 4.0 x 10(-6) M), with one of the derivatives, alpha-(4-amidino-2,6-diiodophenoxy)-3-nitrotoluene (compound 16), showing outstanding potency with a Ki value of 4.5 X 10(-8) M. Although all of the derivatives were effective acrosin inhibitors, structural specificity was observed within homologous groups of compounds. The information gained from this preliminary study should prove extremely beneficial in the design and synthesis of future acrosin inhibitors.[3]

C7H8N2

120.15

618-39-3

Benzamidine hydrochloride;1670-14-0;Benzamidine hydrochloride hydrate;206752-36-5

Solid powder

N([H])([H])/C(/C1C([H])=C([H])C([H])=C([H])C=1[H])=N\[H]

Benzylamine

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.)