Src HuH7 (GI50 = 9 nM); Src PLC/PRF/5 (IC50 = 13 nM); Src Hep3B (IC50 = 26 nM); Src HepG2 (IC50 = 60 nM)
Src kinase; tubulin (microtubules). [1]

One Src inhibitor that targets the Src substrate pocket is tirbanibulin (KX2-391). When compared to four hepatic cell carcinoma (HCC) cell lines, namely Huh7 (GI50=9 nM), PLC/PRF/5 (GI50=13 nM), Hep3B (GI50=26 nM), and HepG2 (GI50=60 nM), tirbanibulin (KX2-391) exhibits steep dose-response curves[1]. It has been discovered that tirbanibulin (KX2-391) inhibits some leukemia cells, such as those originating from chronic leukemia cells with the T3151 mutation, that are resistant to currently marketed medications. In SYF/c-Src527F and NHH3T3/c-Src527F cells, tirbanibulin (KX2-391) is assessed in Src-driven cell growth assays and displays GI50 values of 39 nM and 23 nM, respectively[2].

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Tirbanibulin (KX2391; KX-O1) inhibited in vitro growth of ER/PR/HER2-negative breast cancer cells MDA-MB-231, MDA-MB-157, and MDA-MB-468 with concentrations from 10-250 nmol/L. At 25 nmol/L, it induced significant apoptosis in MDA-MB-468 cells whereas dasatinib at 250 nmol/L did not. [1]
In combination with paclitaxel (5 nmol/L) at suboptimal concentrations, Tirbanibulin (KX2391; KX-O1) (25 nmol/L) resulted in synergistic growth inhibition (43.2% inhibition at 24h) compared to single agents (11.2% and 14.1% respectively). Combination index values <1 indicated synergism across concentration ranges. [1]
Combination of Tirbanibulin (KX2391; KX-O1) with paclitaxel enhanced apoptosis two-fold compared to single drug treatments as measured by DNA fragmentation ELISA. [1]
Tirbanibulin (KX2391; KX-O1) inhibited invasion of MDA-MB-231 cells in Boyden chamber Matrigel invasion assay: at 50 nmol/L, invasion decreased by 52.9% after normalization to cell number. [1]
In 3D-on top assay, Tirbanibulin (KX2391; KX-O1) at 25 and 50 nmol/L reduced invasive stellate structures, with almost complete inhibition at 50 nmol/L. [1]
In scratch wound healing assay, Tirbanibulin (KX2391; KX-O1) at 5, 10, and 25 nmol/L decreased migration by 11.7%, 15.6%, and 39.9% respectively. [1]
Tirbanibulin (KX2391; KX-O1) at 200 nmol/L for 24h markedly inhibited microtubule formation in MDA-MB-231 cells in vitro as shown by alpha-tubulin immunofluorescence. [1]

Tirbanibulin (KX2-391) taken orally has been demonstrated in pre-clinical animal models of cancer to decrease primary tumor growth and to suppress metastasis[2].

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Oral administration of Tirbanibulin (KX2391; KX-O1) (1 and 5 mg/kg, BID for 28 days) inhibited growth of MDA-MB-231 xenograft tumors by 35.3% and 79.0% respectively; in MDA-MB-157 xenografts, tumor volume reduced by 23.5% and 42.0% respectively. [1]
IHC analysis of MDA-MB-231 tumors showed that Tirbanibulin (KX2391; KX-O1) significantly inhibited phosphorylation of Src (Y416) and downstream FAK (Y861) without affecting total protein levels. [1]
Tirbanibulin (KX2391; KX-O1) increased TUNEL-positive apoptotic cells by 3-4 fold, reduced Ki67-positive proliferating cells from 58.7% to 37.3%, and reduced microvessel density (CD31-positive vessels) compared to vehicle. [1]
Combination of Tirbanibulin (KX2391; KX-O1) (5 mg/kg BID) with paclitaxel (3 mg/kg once/week) reduced MDA-MB-231 tumor volume by 93.0% with complete regression in 2/5 mice; in MDA-MB-157, tumor volume reduced by 71.1%. [1]
In larger established tumors (~300 mm^3), Tirbanibulin (KX2391; KX-O1) (15 mg/kg once daily) alone reduced tumor volume by 48.2%, paclitaxel (20 mg/kg once/week) reduced by 35.4%, and combination reduced by 90.6% with tumor regression. [1]
Immunofluorescence of tumors showed that Tirbanibulin (KX2391; KX-O1) disrupted microtubule network (diffuse staining), while paclitaxel stabilized microtubules; combination resulted in fragmented microtubules. [1]
Tirbanibulin (KX2391; KX-O1) alone or in combination with paclitaxel significantly reduced micrometastasis to lung and liver as measured by qPCR for human chromosome 17. No detectable human cells in lung or liver from KX-01 treated groups; paclitaxel alone did not significantly reduce metastasis. [1]

Tirbanibulin (KX2-391) is a Src inhibitor that targets the substrate pocket of Src.
Tirbanibulin (KX2-391) exhibits sharp dose-response curves for four hepatic cell cancer (HCC) cell lines: Hep3B (GI50=26 nM), HepG2 (GI50=60 nM), PLC/PRF/5 (GI50=13 nM), and Huh7 (GI50=9 nM).

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Regularly cultured and maintained in basal medium containing 2% fetal bovine serum (FBS) at 37°C and 5% CO₂, liver cell lines such as Huh7, PLC/PRF/5, Hep3B, and HepG2 (NutriCyte, Buffalo, NY) are used. 1.5% FBS-containing basal medium is used to seed cells at 4.0×10³/190 μL and 8.0×10³/190 μL per well of a 96-well plate. KX2-391, at concentrations ranging from 6,564 to 0.012 nM in triplicates, is added after these are cultivated for an additional night at 37°C and 5% CO₂. Incubation lasts three days for treated cells. Day 3 then sees the addition of 10 microliters of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL) to each well, followed by a 4-hour incubation period. With 10% SDS in diluted HCl, the formazan product is dissolved. Using a BioTek Synergy HT multiplatform microplate reader, optical density at 570 nm is discovered. Parallel experiments using KX2-391 are carried out for comparison of potency and activity. A statistical program called GraphPad Prism 5 is used to calculate growth inhibition curves, 50% inhibition concentration (GI50), and 80% inhibition concentration (GI80). The data are presented in the optical density at wavelength of 570 nm (OD570) signal format and normalized to represent the percentage of maximum response.

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MTT assay: Cells were seeded in 96-well plates, incubated with vehicle or varying concentrations of Tirbanibulin (KX2391; KX-O1) (5-250 nmol/L) for 48h. MTT solution (5 mg/mL) was added for 4h, then formazan dissolved in 10% SDS in dilute HCl, OD570 measured. [1]
Apoptosis assay: DNA fragmentation ELISA performed 24h after drug incubation. [1]
Invasion assay: Boyden chamber with Matrigel-coated filters. Cells pre-incubated with Tirbanibulin (KX2391; KX-O1) (10,25,50 nmol/L) for 24h, then seeded in upper chamber. After 22h, invaded cells on lower surface were fixed, stained, counted. Invasion normalized to cell viability. [1]
3D-on top assay: Cells cultured in Matrigel mixture (growth factor reduced Matrigel with DMEM). Tirbanibulin (KX2391; KX-O1) added at time of plating, replaced every 2 days for 4 days. Photomicrographed to assess stellate structures. [1]
Scratch assay: Monolayer scratched with pipette tip, then incubated with vehicle or Tirbanibulin (KX2391; KX-O1) (5,10,25 nmol/L) for 24h. Wound closure measured by ImageJ. [1]
Immunofluorescence for alpha-tubulin: Cells incubated with Tirbanibulin (KX2391; KX-O1) (200 nmol/L) for 24h, fixed, stained with anti-alpha-tubulin antibody conjugated with Alexafluor-488, imaged with confocal microscope. [1]
MTT assay on liver cancer cell lines: Huh7, PLC/PRF/5, Hep3B, HepG2 cells seeded at 4,000 or 8,000 per well in 96-well plates with 1.5% FBS, treated with Tirbanibulin (KX2391; KX-O1) (0.012 to 6564 nM) for 3 days. MTT added, formazan dissolved in SDS/HCl, OD570 measured. GI50 values were nanomolar for all cell lines. [2]
MTT assay on Src-driven cells: NIH3T3/c-Src527F and SYF/c-Src527F cells seeded in 96-well plates, incubated with Tirbanibulin (KX2391; KX-O1) (5-point 2-fold serial dilution) for 2-3 days. MTT added, then 20% SDS/0.01M HCl added, OD570 measured. GI50 values: 0.023 μM and 0.039 μM respectively. [3]

Mouse bearing MDA-MB-231 tumors; Oral gavage; 1, 5mg/kg dose
Xenograft procedures and KX-01 oral dosing were as described (11). Briefly, mammary fat pad tumors were established by injecting 5×106 MDA-MB-231 cells in 150μl of PBS-Matrigel mixture (1:2) orthotopically and bilaterally into the mammary fat pads of female NUDE mice (two tumors/mouse). Treatments were started when tumors reached ∼80-100mm3. The first study used MDA-MB-231 xenografts and was performed using vehicle (ultra-pure water) and two doses of KX-01 (1, 5mg/kg) administered twice/day (BID) by oral gavage (using metal 22g feeding needle) for 28 days. A similar experiment was performed with MDA-MB-157 xenografts (another ER/PR/HER2 negative model) to assess KX-01 response. A second study was performed to test combination of KX-01 with paclitaxel on tumor growth. MDA-MD-231 tumor xenograft bearing mice were treated with vehicle or KX-01 (5mg/kg) BID, paclitaxel by intraperitoneal injection (IP) once/week, or combination of KX-0+paclitaxel. Treatments were for 40 days for all groups. A third study used MDA-MB-157 xenografts with the same combination treatment. A fourth study tested the effect of KX-01 or combination with paclitaxel for 24 days on larger MDA-MB-231 tumors (∼300mm3). Tumors were allowed to reach ∼300mm3 before beginning treatments. In this experiment mice were treated with KX-01 at a higher dose of 15mg/kg, and mice were treated once/day instead of twice/day. Paclitaxel was used at a dose of 20mg/kg IP once/week. In all experiments, tumor caliper measurements were taken twice/week and tumor volume was by calculated by the formula: 0.523×LM2 (where L-large diameter, M-small diameter). At the end the experiments animals were sacrificed and tumors and mouse organs removed. Tissues were either stored in 10% neutral buffered formalin for paraffin embedding, or snap frozen for measurement of chromosome-17 by real-time PCR, and embedded for frozen sectioning for CD-31 staining. Immunohistochemistry (IHC) was performed as described on paraffin-embedded tumor tissues [3].

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Female athymic NUDE mice (BALB/c, 4-5 weeks old) were housed in sterile cages. Mammary fat pad tumors were established by orthotopic injection of 5x10^6 MDA-MB-231 cells in 150 μL PBS-Matrigel mixture (1:2) bilaterally. Treatments started when tumors reached ~80-100 mm^3. Tirbanibulin (KX2391; KX-O1) was dissolved in ultra-pure water and administered twice daily (BID) by oral gavage (using metal 22g feeding needle) at doses of 1 or 5 mg/kg for 28 days. Paclitaxel was administered intraperitoneally (IP) once/week at 3 mg/kg. For larger tumor study, Tirbanibulin (KX2391; KX-O1) was given at 15 mg/kg once daily, and paclitaxel at 20 mg/kg IP once/week. Tumor volumes measured twice/week with calipers and calculated as 0.523 x L x M^2. At study end, animals sacrificed and tumors/organs collected. [1]

Absorption, Distribution and Excretion
Tibanibulin exhibits good oral bioavailability. Steady-state concentrations of tibanibulin are reached within 72 hours following topical application of 54 to 295 mg to the face or scalp. Five days after the first dose, the mean Cmax was 0.34 ± 0.30 ng/mL in the facial topical application group and 0.18 ± 0.10 ng/mL in the scalp topical application group. The mean AUC24 was 5.0 ± 3.9 h × ng/mL in the facial topical application group and 3.2 ± 1.9 h × ng/mL in the scalp topical application group. The median time to peak concentration (Tmax) was approximately 7 hours. Limited information is available regarding the elimination pathway of tibanibulin. Limited information is available regarding the volume of distribution of tibanibulin. In a mouse HT29 xenograft study, the tissue/plasma ratio of tibanibulin was 1.52. Limited information is available regarding the clearance rate of tebanibulin. Metabolism/Metabolites: In vitro studies have shown that tebanibulin is primarily metabolized by CYP3A4, with a small amount metabolized by CYP2C8. In adult subjects with actinic keratosis, the metabolites KX2-5036 and KX2-5163 were detected; these are pharmacologically inactive metabolites, with peak plasma concentrations of 0.09 ng/mL and 0.12 ng/mL, respectively. Biological Half-Life: The half-life is approximately 4 hours.

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Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Tibanibulin topical application during lactation has not been studied.
However, serum concentrations are extremely low after topical application, and the drug binds to plasma proteins at a rate as high as 88%, therefore the drug content in breast milk may be extremely low. If the mother needs to use tebanibulin, there is no need to discontinue breastfeeding.
Do not apply tebanibulin to the breast or nipple, and ensure that the infant's skin does not come into direct contact with the treated area.
◉ Effects on Breastfed Infants
No relevant published information was found as of the revision date.
◉ Effects on Lactation and Breast Milk
No relevant published information was found as of the revision date.

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Protein Binding
Tibanibulin binds to plasma proteins at a rate of 88%, and the degree of plasma protein binding is independent of drug concentration within the drug concentration range of 0.01 to 10 µg/mL.

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No noticeable toxicity or significant body weight reduction was observed in either drug alone or combination treatment groups (Suppl.Fig.3A). In the high-dose combination study (KX-01 15 mg/kg + paclitaxel 20 mg/kg), approximately 10% body weight reduction was observed but without noticeable toxicity (initial mean body weight 19.4g, final 17.6g; Suppl.Fig.3C). [1]

[1]. Lau GM, et al. Expression of Src and FAK in hepatocellular carcinoma and the effect of Src inhibitors on hepatocellular carcinoma in vitro. Dig Dis Sci, 2009, 54(7), 1465-1474.
[2]. Fallah-Tafti A, et al. Thiazolyl N-benzyl-substituted acetamide derivatives: synthesis, Src kinase inhibitory and anticancer activities. Eur J Med Chem, 2011, 46(10), 4853-4858.

[3]. Peptidomimetic Src/pretubulin inhibitor KX-01 alone and in combination with paclitaxel suppresses growth, metastasis in human ER/PR/HER2-negative tumor xenografts. Mol Cancer Ther. 2012 Sep; 11(9): 1936–1947.

Tibanibulin (trade names KX-O1 or KX2-391) is a dual inhibitor of Src kinase and tubulin. On December 14, 2020, tibanibulin was approved by the U.S. Food and Drug Administration (FDA) for the treatment of actinic keratosis of the face or scalp. Its trade name is Klisyri. Actinic keratosis is a chronic disease characterized by lesions that have the potential to develop into invasive squamous cell carcinoma, with a 1% risk of malignant transformation within 10 years. Tibanibulin blocks molecular pathways that promote the proliferation, survival, and metastasis of malignant cells. Tibanibulin has shown antitumor activity both in vitro and in vivo and has been studied for the treatment of various cancers, such as prostate and breast cancer. Tibanibulin is a microtubule inhibitor. Its physiological effects are achieved by inhibiting microtubules. Tibanibulin is a small-molecule Src kinase inhibitor with high oral bioavailability and potential antitumor activity. Unlike other Src kinase inhibitors that bind to ATP-binding sites, tebanibulin specifically binds to the peptide substrate binding site of Src kinase; inhibition of kinase activity may lead to primary tumor growth arrest and metastasis inhibition. Src tyrosine kinase is upregulated in many tumor cells and plays an important role in tumor cell proliferation and metastasis. Drug Indications Tebanibulin is indicated for the topical treatment of actinic keratosis of the face or scalp. Clicidide is indicated for the topical treatment of non-hyperkeratotic, non-hypertrophic actinic keratosis (Olsen grade 1) of the face or scalp in adults. Mechanism of Action Src tyrosine kinase regulates normal cell growth: Src kinase expression is upregulated during the anagen phase of the normal hair growth cycle. Furthermore, Src tyrosine kinase is a key regulator of cancer cell proliferation, survival, angiogenesis, migration, invasion, and metastasis. Compared to adjacent normal tissue, Src is frequently highly expressed in a variety of epithelial tumors, including colon cancer, breast cancer, and pancreatic cancer. In human actinic keratosis, characterized by proliferative precancerous skin lesions, Src expression and activity are also enhanced. The pathogenesis of actinic keratosis typically involves skin inflammation, oxidative stress, immunosuppression, impaired apoptosis, mutations, dysregulation of keratinocyte growth and proliferation, and tissue remodeling. In vitro studies have shown that Src plays a dominant role in the early stages of human skin tumorigenesis, rather than in later stages of tumor progression. The exact mechanism of action of tebanibulin as a topical treatment for actinic keratosis is not fully elucidated; however, its primary mechanism of action is through the inhibition of rapidly proliferating cells. Tebanibulin is a non-ATP-competitive Src kinase inhibitor and a tubulin polymerization inhibitor. Tebanibulin binds to the peptide substrate binding site of Src, which is the primary target of tebanibulin. By blocking downstream signaling pathways, tebanibulin inhibits the migration, proliferation, and survival of cancer cells. Tubulin is responsible for cell migration, protein transport, and mitosis: tebanibulin binds directly to the colchicine binding site of β-tubulin, inducing tubulin depolymerization. Furthermore, some studies hypothesize that inhibition of Src kinase may also help inhibit microtubule polymerization. At low nanomolar concentrations, tebanibulin reversibly induces G2/M phase cell cycle arrest in a dose-dependent manner. By inhibiting microtubule polymerization, tebanibulin can also induce mitotic catastrophe.

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Pharmacodynamics
In clinical trials involving patients with actinic keratosis of the face or scalp, compared to 5-13% in the placebo group, 44-54% of patients in the tebanibulin treatment group experienced complete clearance of actinic keratosis lesions at the treatment site after 57 days. Actinic keratosis is a chronic precancerous lesion characterized by the proliferation of lesions and neoplastic keratinocytes. Tebanibulin exerts its antiproliferative effect by inhibiting microtubule polymerization and Src kinase signaling. In numerous preclinical animal models of cancer, tebanibulin inhibited the growth and metastasis of primary tumors. In a human triple-negative breast cancer xenograft model (i.e., estrogen receptor (ER)/progesterone receptor (PR)/human epidermal growth factor receptor 2 (HER2) negative tumors), tebanibulin suppressed tumor growth and metastasis. Tebanibulin has also been shown to restore functional ERα expression in ERα-negative breast tumors. When tebanibulin is used in combination with tamoxifen and paclitaxel, it synergistically inhibits tumor growth in breast cancer cell lines. In a clinical trial involving patients with advanced solid tumors, dose-limiting toxicities of tebanibulin included elevated liver transaminases, neutropenia, and fatigue.

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Tirbanibulin (KX2391; KX-O1) has a dual mechanism of action: inhibition of Src kinase activity and disruption of microtubule polymerization. This dual activity may provide additional antitumor activity compared to compounds with Src inhibition alone. It synergizes with paclitaxel in ER/PR/HER2-negative breast cancer, potentially allowing lower paclitaxel doses. It also inhibits metastasis. [1]
In hepatocellular carcinoma (HCC) patient samples, Src and FAK are overexpressed. Tirbanibulin (KX2391; KX-O1) showed potent antiproliferative activity against HCC cell lines (Huh7, PLC/PRF/5, Hep3B, HepG2) with nanomolar GI50 values, and was more potent than Dasatinib. It may be an attractive treatment for HCC. [2]
Tirbanibulin (KX2391; KX-O1) (KX2-391) is a highly selective non-ATP Src kinase inhibitor targeting the substrate binding site, with tubulin polymerization inhibition as a second mechanism. It is in Phase 2 testing for solid tumors. It inhibits leukemia cells resistant to current drugs, including T315I mutation. In preclinical models, it inhibits primary tumor growth and suppresses metastasis, and synergizes with chemotherapeutic agents. Structure-activity relationship studies show that replacing pyridine with thiazole reduces activity, indicating pyridine is a pharmacophore. [3]

C26H29N3O3

431.53

431.22

C, 71.92; H, 6.52; N, 10.06; O, 11.50

897016-82-9

Tirbanibulin dihydrochloride;1038395-65-1;Tirbanibulin Mesylate;1080645-95-9

23635314

White to off-white solid powder

1.2±0.1 g/cm3

680.9±55.0 °C at 760 mmHg

365.6±31.5 °C

0.0±2.1 mmHg at 25°C

1.588

1.97

1

5

9

32

540

0

O=C(CC1C=CC(C2C=CC(OCCN3CCOCC3)=CC=2)=CN=1)NCC1C=CC=CC=1

YYLKKYCXAOBSRM-JXMROGBWSA-N

InChI=1S/C20H20N4O/c25-20(24-13-11-21-12-14-24)16-8-5-15(6-9-16)7-10-19-17-3-1-2-4-18(17)22-23-19/h1-10,21H,11-14H2,(H,22,23)/b10-7+

(E)-(4-(2-(1H-indazol-3-yl)vinyl)phenyl)(piperazin-1-yl)methanone

KX 01, KX2-391; KX-01, KX-2-391; Tirbanibulin; KX2391; KX-2391; Tirbanibulin free base; KXO1; KX 2-391; KX 2391

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.82 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 4% DMSO+30% PEG 300+ddH2O:5 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)