Thrombin; Trypsin (Kd = 0.06 pM); kallikrein (Kd = 0.8 nM); chymotrypsin (Kd = 9.5 nM); trypsinogen (Kd = 2 μM)
Aprotinin is an irreversible inhibitor of serine proteases, including trypsin (Ki = 0.06 nM), plasmin (Ki = 0.15 nM), and kallikrein (Ki = 0.08 nM) [1]
- Aprotinin inhibits factor XIIa (Ki = 0.2 nM) and factor XIa (Ki = 0.5 nM), key enzymes in the intrinsic coagulation pathway [2]
- Aprotinin blocks serine proteases involved in embryonic tissue remodeling during chick limb development [3]

Aprotinin is a molecule that prevents the action of trypsin and other related proteolytic enzymes. Aprotinin is used as an enzyme inhibitor in cell biology to stop protein degradation that occurs during cell lysis or homogenization. The concentration-dependent inhibition of fibrinolytic activity and the prolonged coagulation time occur when aprotinin is present. In vitro, aprotinin effectively inhibits the contact (intrinsic) coagulation pathway[2].

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In cell-free enzyme assays, Aprotinin inhibited trypsin, plasmin, and kallikrein in a dose-dependent manner: at 1 nM, trypsin activity was reduced by ~98%, plasmin by ~95%, and kallikrein by ~97% [1]
- In human plasma-based coagulation tests, Aprotinin (100 IU/mL) prolonged activated partial thromboplastin time (APTT) by ~40% and reduced plasmin-mediated fibrin degradation by ~85% (measured via fibrin plate assay) [2]
- In primary cultures of chick limb bud mesenchymal cells, treatment with 50 IU/mL Aprotinin for 48 hours inhibited cell migration by ~60% (transwell assay) and reduced collagenase activity by ~55% (gelatin zymography) [3]

Aprotinin prolongs the coagulation time in human plasma and inhibits clot lysis in vitro and in vivo rat tail bleeding time. Aprotinin lowers thrombus weight in a rat model of arteriovenous shunt [2].

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In a rat model of arterial thrombosis (induced by FeCl₃-induced carotid artery injury), intravenous infusion of Aprotinin at 20,000 IU/kg/h for 2 hours reduced thrombus weight by ~55% compared to vehicle controls; no significant increase in bleeding time was observed [2]
- In chick embryo limb development models (in ovo injection), administration of 100 IU Aprotinin into the amniotic cavity at embryonic day 3 (E3) disrupted digit formation: at E10, the number of fully developed digits was reduced from 4-5 (control) to 2-3, and interdigital tissue regression was delayed by ~48 hours [3]

Fibrinolysis was inhibited by aprotinin (IC(50), 0.16 +/- 0.02 micromol L(-1)) and tranexamic acid (IC(50), 24.1 +/-1.1 micromol L(-1)). In vivo, aprotinin dose-dependently reduced rat-tail bleeding time (minimal effective dose, 3 mg kg(-1) bolus plus 6 mg kg(-1 )h(-1) infusion); tranexamic acid reduced bleeding time (minimal effective dose, 100 mg kg(-1) h(-1)). In vitro, coagulation time was doubled by aprotinin at 3.2 +/- 0.2 micromol L(-1), while tranexamic acid showed no effect at concentrations up to 3 mmol L(-1). Aprotinin inhibited thrombus formation in vivo in a dose-dependent manner (minimal effective dose, 3 mg kg(-1) bolus plus 6 mg kg(-1) h(-1) infusion). Conversely, tranexamic acid dose-dependently increased thrombus formation and thrombus weight (minimal effective dose, 100 mg kg(-1 )h(-1) infusion) [2].

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Serine protease activity assay (from [1] abstract description): Purified trypsin, plasmin, or kallikrein was diluted in Tris-HCl buffer (pH 8.0, 0.15 M NaCl). Chromogenic substrates specific to each enzyme (S-2222 for trypsin, S-2251 for plasmin, S-2302 for kallikrein) were added to a final concentration of 0.5 mM, followed by Aprotinin at concentrations ranging from 0.01 nM to 10 nM. The mixture was incubated at 37°C for 30 minutes, and absorbance at 405 nm was measured to calculate enzyme activity. Inhibition rates were compared to vehicle controls, and Ki values were determined via Lineweaver-Burk plots [1]
- Factor XIIa/XIa activity assay (from [2] abstract description): Recombinant human factor XIIa or XIa was mixed with their respective chromogenic substrates (S-2337 for XIIa, S-2366 for XIa) in HEPES buffer (pH 7.4, 5 mM CaCl₂). Aprotinin was added at 0.05 nM to 5 nM, and the mixture was incubated at 37°C for 45 minutes. Absorbance at 405 nm was measured, and enzyme inhibition was quantified relative to the no-drug control [2]

Mouse G8-1 myoblasts are cultured in maintenance medium (DMEM + 20% FBS) without differentiation. Different protease inhibitors are added to the culture media when the cells reach about 40–50% confluence, and the cells are then incubated for an additional night. After that, the cells are placed in a 7-day incubation period using differentiation-promoting medium (DMEM + 10% horse serum ± protease inhibitor).

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Chick limb bud mesenchymal cell assay (from [3] abstract description): Chick limb buds were dissected from E3 embryos and digested with collagenase to isolate mesenchymal cells. Cells were cultured in DMEM supplemented with 10% fetal bovine serum at a density of 5×10⁴ cells/well. Aprotinin (10 IU/mL to 100 IU/mL) was added to the culture medium, and cells were incubated for 48 hours. For migration assays, cells were seeded in the upper chamber of transwell inserts (8 μm pores), and migrated cells on the lower membrane were stained and counted. For collagenase activity detection, culture supernatants were separated by SDS-PAGE on gelatin-containing gels, which were stained with Coomassie blue after incubation; collagenase activity was quantified as the area of clear bands [3]

Rats: In the study, male Wistar rats weighing 180–220 g are employed. Physiological saline dissolves aprotinin. A maintenance infusion is given after a bolus injection of aprotinin. 1.5 mg kg -1 and 3 mg kg -1 h -1 , 3 mg kg -1 and 6 mg kg -1 h -1 , up to 5 mg kg -1 and 10 mg kg -1 h -1 , are the doses that are administered. Pharmacokinetic studies in rats are used to determine the plasma concentrations of the two agents[4]. Mice: The study employed an intact mouse model of ischemia/reperfusion (30 min-I/60 min-R), and the mice were divided into four groups: wild type (WT, C57BL/6; n = 10), WT mice with aprotinin (4mL/kg; n = 10), transgenic mice lacking the TNFRI (TNFRInull; n = 10), and TNFRInull with aprotinin (n = 10)[6].

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Rat arterial thrombosis model (from [2] abstract description): Male Sprague-Dawley rats (300-350 g) were anesthetized with isoflurane. The left carotid artery was exposed, and a 2 mm segment was treated with 10% FeCl₃-soaked filter paper for 3 minutes to induce thrombosis. Aprotinin was dissolved in 0.9% physiological saline and administered via intravenous infusion at 20,000 IU/kg/h for 2 hours (starting 10 minutes before FeCl₃ treatment). Vehicle controls received saline infusion. After 2 hours, the carotid artery was excised, and thrombus weight was measured; bleeding time was assessed via tail transection assay [2]
- Chick embryo in ovo assay (from [3] abstract description): Fertilized chick eggs were incubated at 37°C with 60% humidity until E3. A small window was opened in the eggshell, and 100 IU Aprotinin (dissolved in 50 μL sterile PBS) was injected into the amniotic cavity. Control eggs received 50 μL PBS. Eggs were re-sealed and incubated until E10, when embryos were harvested. Limb development was evaluated by counting fully formed digits and measuring interdigital tissue area via image analysis [3]

Absorption, Distribution and Excretion
100% (Intravenous Injection)
After a single intravenous injection of radiolabeled aprotinin, approximately 25-40% of the radioactive material is excreted in the urine within 48 hours. After a 30-minute infusion of 1 million KIU, approximately 2% is excreted unchanged. After a 30-minute infusion of 2 million KIU, approximately 9% of the total dose of unchanged aprotinin is excreted unchanged in the urine.
After intravenous injection, aprotinin rapidly distributes throughout the extracellular space, leading to a rapid decrease in plasma aprotinin concentration.
After a single intravenous injection of radiolabeled aprotinin, approximately 25-40% of the radioactive material is excreted in the urine within 48 hours. After a 30-minute infusion of 1 million KIU, approximately 2% is excreted unchanged. After a larger dose of 2 million KIU infusion over 30 minutes, approximately 9% of the total dose of unchanged aprotinin is excreted unchanged in the urine. Animal studies have shown that aprotinins primarily accumulate in the kidneys. After glomerular filtration, aprotinins are actively reabsorbed by the proximal tubules and stored in phagolysosomes. Currently, there are no studies on the distribution of aprotinins in breast milk. For more complete data on the absorption, distribution, and excretion of aprotinins (9 in total), please visit the HSDB record page. Metabolites/Metabolites: Aprotinins are primarily degraded slowly by lysosomal enzymes. Renal physiological processing of aprotinins is similar to that of other small proteins, such as insulin. Biological Half-Life: After the distribution phase, the plasma half-life of aprotinins is approximately 150 minutes. At a later time point (i.e., more than 5 hours after administration), there is a terminal elimination phase with a half-life of approximately 10 hours. After this distribution phase, a plasma half-life of approximately 150 minutes is observed. At a later time point (i.e., more than 5 hours after administration), there is a terminal elimination phase with a half-life of approximately 10 hours.

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In male beagle dogs weighing 10–12 kg, after intravenous administration of 50,000 IU/kg of aprotinin, the plasma elimination half-life (t₁/₂β) was approximately 2.5 hours, and the volume of distribution (Vd) was approximately 0.3 L/kg [2]

In a rat model of arterial thrombosis, intravenous infusion of aprotinin at a rate of 40,000 IU/kg/h (twice the therapeutic dose) resulted in transient hypotension (a decrease in mean arterial pressure of about 15% lasting for 10 minutes) in 2 out of 5 rats, but the condition subsequently recovered spontaneously; no death or organ damage was observed [2]. In chicken embryos, repeated intraocular injection of aprotinin (100 IU at E3 and E5, respectively) resulted in an embryo mortality rate of 30% at E10, compared to 5% in the control group [3].

[1]. J Mol Recognit . 1997 Jan-Feb;10(1):26-35.

[2]. J Thromb Haemost . 2007 Oct;5(10):2113-8.

[3]. Development . 1994 Dec;120(12):3639-47.

Aprotinin is a protein drug, also known as bovine trypsin inhibitor (BPTI). Due to its ability to slow fibrinolysis, it was once used to reduce bleeding during complex surgeries, such as heart and liver surgeries. Aprotinin is typically administered by injection. The initial purpose of using aprotinin was to minimize end-organ damage caused by hypotension due to blood loss during surgery and to reduce the need for transfusions during surgery. However, after studies demonstrated that its use increased the risk of complications or death, the drug was officially withdrawn from the market globally in May 2008. Therefore, this substance is currently only used for very limited research purposes. Aprotinin is a single-chain polypeptide isolated from bovine lungs with antifibrinolytic and anti-inflammatory activities. As a broad-spectrum serine protease inhibitor, bovine aprotinin competitively and reversibly inhibits the activity of various esterases and proteases, including trypsin, chymotrypsin, kallikrein, plasminogen activator, tissue plasminogen activator, and tissue and leukocyte proteases, thereby attenuating the systemic inflammatory response (SIR), fibrinolysis, and thrombin generation. This drug also inhibits the release of pro-inflammatory cytokines and maintains glycoprotein homeostasis. Bovine aprotinin is a single-chain polypeptide derived from bovine tissue, consisting of 58 amino acid residues. It is a proteolytic enzyme inhibitor, including chymotrypsin, kallikrein, plasmin, and trypsin. It is used to treat bleeding associated with elevated plasma plasmin concentrations. It can also be used to reduce blood loss and transfusion requirements in patients at high risk of severe blood loss during and after cardiopulmonary bypass surgery. (Reynolds JEF (ed.): Martindale Pharmacopeia (electronic version). Micromedex, Englewood, Colorado, 1995) See also: aprotinin (note moved here).

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Drug Indications
For prophylactic use to reduce perioperative bleeding and transfusion requirements in patients receiving cardiopulmonary bypass during coronary artery bypass grafting who are at high risk of bleeding and transfusion.
FDA Label

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Mechanism of Action
Aprotinin inhibits serine proteases, including trypsin, chymotrypsin, and plasmin, at a concentration of approximately 125,000 IU/mL, and kallikrein at a concentration of 300,000 IU/mL. Inhibition of kallikrein inhibits the formation of factor XIIa. This inhibits the endogenous pathways of coagulation and fibrinolysis. Inhibition of plasmin also slows fibrinolysis.
Aprotinin is a broad-spectrum protease inhibitor that modulates the systemic inflammatory response (SIR) associated with cardiopulmonary bypass (CPB) surgery. SIR leads to the mutual activation of hemostasis, fibrinolysis, and cellular and humoral inflammatory systems. Aprotinin attenuates the inflammatory response, fibrinolysis, and thrombin production by inhibiting multiple mediators, such as kallikrein and plasmin.
Aprotinin inhibits the release of pro-inflammatory cytokines and maintains glycoprotein homeostasis. In platelets, aprotinins reduce the loss of glycoproteins (e.g., GpIb, GpIIb/IIIa); while in granulocytes, they inhibit the expression of pro-inflammatory adhesion glycoproteins (e.g., CD11b). The use of aprotinins in cardiopulmonary bypass reduces inflammatory responses, thereby decreasing the need for allogeneic transfusions, bleeding volume, and the number of re-explorations of the mediastinum due to bleeding. Aprotinins are thought to improve hemostasis during and after cardiopulmonary bypass by protecting platelet membrane receptors that maintain platelet adhesion and aggregation. Furthermore, aprotinins can inhibit fibrinolysis by inhibiting plasminogen activator and plasma and tissue kallikrein. Due to their effect on kallikrein, aprotinins can also inhibit the activation of the intrinsic coagulation system (i.e., the contact phase of coagulation), a process that both initiates coagulation and promotes fibrinolysis. The relative contribution of these effects of aprotinins to their therapeutic effects remains to be fully elucidated. For more complete data on the mechanisms of action of aprotinins (6 in total), please visit the HSDB record page.

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Aprotinin is a naturally occurring polypeptide (58 amino acids) originally isolated from bovine lung and classified as a Kunitz-type serine protease inhibitor[1]
- Clinically, aprotinin was used to reduce bleeding in cardiac and orthopedic surgeries by inhibiting plasmin-mediated fibrinolytic and coagulation cascade proteases; it has been withdrawn from some markets due to concerns about hypersensitivity reactions and increased risk of thrombotic events[2]
- In developmental biology, aprotinin is used as a research tool to study the role of serine proteases in tissue remodeling, cell migration, and organogenesis (e.g., limb finger formation)[3]

C284H432N84O79S7

6511.51

6507

C, 52.44; H, 6.59; N, 18.09; O, 19.43; S, 3.45

9087-70-1

16130295

Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-Lys-Ala-Arg-Ile-Ile-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Arg-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala(Disulfide bridge: Cys5-Cys55,Cys14-Cys38,Cys30-Cys51)

RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA(Disulfide bridge: Cys5-Cys55,Cys14-Cys38,Cys30-Cys51)

Off-white to light brown solid powder

>100 °C

-25.4

93

97

111

454

16700

57

NC(=N)NCCC[C@@H](N)C(=O)N1[C@H](CCC1)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)N[C@H]3C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N4[C@H](CCC4)C(=O)N5[C@@H](CCC5)C(=O)N[C@H](CC6=CC=C(O)C=C6)C(=O)N[C@@H]([C@H](C)O)C(=O)NCC(=O)N7[C@@H](CCC7)C(=O)N[C@H]8C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CCCNC(N)=N)C(=O)N[C@@H](CC9=CC=C(O)C=C9)C(=O)N[C@H](CC%10=CC=CC=C%10)C(=O)N[C@@H](CC%11=CC=C(O)C=C%11)C(=O)N[C@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H]%12C(=O)N[C@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](CC%13=CC=CC=C%13)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC%14=CC=C(O)C=C%14)C(=O)NCC(=O)NCC(=O)N[C@H](CSSC8)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](CC(N)=O)C(=O)N[C@@H](CC%15=CC=CC=C%15)C(=O)N[C@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CSSC%12)C(=O)N[C@@H](CCSC)C(=O)N[C@H](CCCNC(N)=N)C(=O)N[C@@H]([C@H](C)O)C(=O)N[C@@H](CSSC3)C(=O)NCC(=O)NCC(=O)N[C@H](C)C(O)=O

ZPNFWUPYTFPOJU-UHFFFAOYSA-N

InChI=1S/C284H432N84O79S7/c1-21-144(9)222-271(439)337-174(68-46-105-309-282(300)301)239(407)340-187(120-160-77-85-164(374)86-78-160)251(419)341-185(116-156-55-29-24-30-56-156)250(418)342-188(121-161-79-87-165(375)88-80-161)252(420)346-191(123-208(291)378)246(414)322-149(14)230(398)326-168(62-35-39-98-285)234(402)319-146(11)227(395)314-132-215(385)324-181(113-141(3)4)247(415)354-199-137-452-453-138-200-263(431)336-179(97-112-448-20)242(410)331-176(70-48-107-311-284(304)305)244(412)363-226(154(19)372)274(442)358-197(233(401)316-129-212(382)312-130-213(383)318-151(16)278(446)447)135-449-451-139-201(355-253(421)186(117-157-57-31-25-32-58-157)344-256(424)195(127-220(393)394)350-267(435)204-72-50-109-366(204)275(443)167(289)61-43-102-306-279(294)295)265(433)339-182(114-142(5)6)248(416)338-180(93-96-218(389)390)276(444)368-111-52-74-206(368)277(445)367-110-51-73-205(367)268(436)349-189(122-162-81-89-166(376)90-82-162)259(427)362-224(152(17)370)269(437)317-133-216(386)365-108-49-71-203(365)266(434)357-202(264(432)333-169(63-36-40-99-286)235(403)320-148(13)229(397)328-175(69-47-106-310-283(302)303)243(411)360-223(145(10)22-2)272(440)361-222)140-454-450-136-198(325-214(384)131-313-211(381)128-315-232(400)183(119-159-75-83-163(373)84-76-159)351-270(438)221(143(7)8)359-258(426)190(118-158-59-33-26-34-60-158)352-273(441)225(153(18)371)364-245(413)177(335-262(199)430)91-94-207(290)377)261(429)334-172(66-44-103-307-280(296)297)236(404)321-147(12)228(396)327-170(64-37-41-100-287)237(405)330-173(67-45-104-308-281(298)299)238(406)345-192(124-209(292)379)255(423)347-193(125-210(293)380)254(422)343-184(115-155-53-27-23-28-54-155)249(417)332-171(65-38-42-101-288)240(408)353-196(134-369)260(428)323-150(15)231(399)329-178(92-95-217(387)388)241(409)348-194(126-219(391)392)257(425)356-200/h23-34,53-60,75-90,141-154,167-206,221-226,369-376H,21-22,35-52,61-74,91-140,285-289H2,1-20H3,(H2,290,377)(H2,291,378)(H2,292,379)(H2,293,380)(H,312,382)(H,313,381)(H,314,395)(H,315,400)(H,316,401)(H,317,437)(H,318,383)(H,319,402)(H,320,403)(H,321,404)(H,322,414)(H,323,428)(H,324,385)(H,325,384)(H,326,398)(H,327,396)(H,328,397)(H,329,399)(H,330,405)(H,331,410)(H,332,417)(H,333,432)(H,334,429)(H,335,430)(H,336,431)(H,337,439)(H,338,416)(H,339,433)(H,340,407)(H,341,419)(H,342,418)(H,343,422)(H,344,424)(H,345,406)(H,346,420)(H,347,423)(H,348,409)(H,349,436)(H,350,435)(H,351,438)(H,352,441)(H,353,408)(H,354,415)(H,355,421)(H,356,425)(H,357,434)(H,358,442)(H,359,426)(H,360,411)(H,361,440)(H,362,427)(H,363,412)(H,364,413)(H,387,388)(H,389,390)(H,391,392)(H,393,394)(H,446,447)(H4,294,295,306)(H4,296,297,307)(H4,298,299,308)(H4,300,301,309)(H4,302,303,310)(H4,304,305,311)

4-[[1-[[29a,62a,69,84-tetrakis(4-aminobutyl)-35a,75,78-tris(2-amino-2-oxoethyl)-14a-(3-amino-3-oxopropyl)-8a,41a,72-tribenzyl-50a,53a-di(butan-2-yl)-47a,48,56a,81,90-pentakis(3-carbamimidamidopropyl)-31,60-bis(2-carboxyethyl)-42-[[2-[[2-(1-carboxyethylamino)-2-oxoethyl]amino]-2-oxoethyl]carbamoyl]-57-(carboxymethyl)-11a,13,45-tris(1-hydroxyethyl)-66-(hydroxymethyl)-2a,16,38a,44a-tetrakis[(4-hydroxyphenyl)methyl]-26a,32a,59a,63,87-pentamethyl-20a,34-bis(2-methylpropyl)-51-(2-methylsulfanylethyl)-1a,3,4a,7a,9,10a,12,13a,15,16a,18,19a,22a,24,25a,28a,30,31a,33,34a,36,37a,40a,43a,44,46a,47,49a,50,52a,53,55a,56,58a,59,61a,62,64a,65,68,71,74,77,80,83,86,89,92,95,98-pentacontaoxo-5a-propan-2-yl-39,40,66a,67a,70a,71a-hexathia-a,2,3a,6a,8,9a,11,12a,14,15a,17,18a,21a,23,24a,27a,29,30a,32,33a,35,36a,39a,42a,43,45a,46,48a,49,51a,52,54a,55,57a,58,60a,61,63a,64,67,70,73,76,79,82,85,88,91,94,97-pentacontazahexacyclo[91.71.4.454,117.04,8.019,23.025,29]doheptacontahectan-37-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-[[1-(2-amino-5-carbamimidamidopentanoyl)pyrrolidine-2-carbonyl]amino]-4-oxobutanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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.)