DDX5 (p68) – a multifunctional DEAD-box RNA helicase and master regulator oncoprotein. FL118 binds directly to DDX5 with high affinity (KD = 34.4 nM) and induces its dephosphorylation and degradation via the ubiquitin-proteasome pathway. This leads to downstream inhibition of survivin, Mcl-1, XIAP, cIAP2, c-Myc, and mutant Kras [2].
Cytoglobin (CYGB) – FL118 upregulates CYGB expression, which mediates its anti-proliferative and anti-migratory effects in ovarian cancer cells [1].

The proliferation of ES-2 and SK-O-V3 cells is inhibited by FL118 (0-200 nM; 24, 48, and 72 hours) [1]. The migration of ES-2 and SK-O-V3 cells is inhibited by FL118 (0-100 nM; 0 and 24 hours) [1]. FL118 (0-100 nM; 48 hours) has an impact on cytoglobin (CYGB) expression levels [1]. In ovarian cancer cells, FL118 (10 and 100 nM; 48 h) alters the expression levels of vimentin and E-cadherin and suppresses the PI3K/AKT/mTOR signaling pathway [1]. In 6 and 24 hours, FL118 (0-100 nM) dephosphorylates and breaks down DDX5 [2]. Through control of DDX5, FL118 (0-500 nM; 24, 48, 72 hours) modulates survivin, McL-1, XIAP, cIAP2, c-MYc, and mKras [2]. Significant cytotoxic efficacy against three tumor cell lines (A549, MDA-MB-231, and RM-1 cells) was demonstrated by FL118 (0-1 μM, 24 hours) [3]. FL118 (0–10 nM, 48 hours) causes A549 cells to undergo apoptosis and enhances the synthesis of PARP cleavage [3]. A549 cells are primarily arrested in the G2/M phase by FL118 (0–10 nM, 48 hours) [3].

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FL118 inhibits the proliferation of A549 (lung), MDA-MB-231 (breast), and RM-1 (mouse prostate) cancer cells with IC50 values ranging from 2.32 nM to 4.53 μM. In A549 cells, IC50 = 8.94 ± 1.54 nM; in MDA-MB-231 cells, IC50 = 24.73 ± 13.82 nM; in RM-1 cells, IC50 = 69.19 ± 8.34 nM. In comparison, irinotecan showed IC50 values of 9.14 μM (A549) and 7.82 μM (MDA-MB-231) [3].
In ovarian cancer ES-2 and SK-OV-3 cells, FL118 inhibits cell proliferation and migration in a dose- and time-dependent manner. At 100 nM, FL118 significantly reduces cell viability and migration distance after 24-72 h. FL118 upregulates cytoglobin (CYGB) mRNA and protein expression in a dose-dependent manner, and CYGB knockdown partially reverses FL118's anti-proliferative and anti-migratory effects. FL118 also inhibits PI3K/AKT/mTOR signaling (increases p-AKT and p-mTOR) and modulates EMT markers (increases E-cadherin, decreases vimentin) [1].
In HCT-8 colon cancer cells, FL118 (1-100 nM) inhibits survivin, Mcl-1, XIAP, cIAP2, and c-Myc expression. It is approximately 25-fold more potent than topotecan in inhibiting cell growth and colony formation. FL118 is not a substrate of P-gp/MDR1 or ABCG2 efflux pumps, and its efficacy is not affected by these transporters. FL118 exhibits high Caco-2 permeability (Papp = 6.76-8.86 × 10⁻⁶ cm/s) with a very low efflux ratio (1.0-1.2) [4].

FL118 inhibits antitumor activity at doses of 5 and 10 mg/kg, taken once a week for 20 days [1]. Intraperitoneally administered FL118 (0-1.5 mg/kg, five times per diem) efficiently eradicates human colon and head and neck tumors resistant to topotecan or irinotecan [4]. FL118 has good pharmacokinetic properties when given intravenously at a dose of 1.5 mg/kg [4]. Female SCID mice's FL118 pharmacokinetic parameters [4]. Sample FaDu SW620 Plasma T1/2 (hr) 6.852 12.75 1.788 Tmax (hr) 0.167 0.167 0.167 Cmax (ng/g, mL) 115 158 43 AUC (hr*ng/g) 413 842 82 AUC∞ (hr*ng/g ) 448 897 104 AUC% Extrap (%) 7.74 6.17 21.7 Vz (g/kg) (ml/kg) 33052 30742 36849 Cl (g/hr/kg) (ml/hr/kg) 3343 1671 14287

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In mice bearing irinotecan-resistant FaDu head-and-neck tumors, FL118 (1.5 mg/kg IP, q2d × 5) effectively eliminated resistant tumors; some regressed tumors that relapsed responded to a second cycle of FL118 treatment [4].
In mice bearing topotecan-resistant FaDu tumors, FL118 (1-1.5 mg/kg IP, q2d × 5) effectively eliminated resistant tumors [4].
In HCT116 colon carcinoma xenografts, FL118 (10 mg/kg IP, once daily for 14 days) inhibited tumor volume by 76%, superior to 5-FU (41% inhibition) and vorinostat (26% inhibition) [4].
In human PDAC patient-derived xenograft (PDX) tumors, FL118 (2.5 mg/kg oral, weekly × 4) regressed PDX19015 and PDX17624 tumors with high DDX5 expression, while PDX12872 with low DDX5 expression showed less sensitivity. Combination with low-dose gemcitabine (40 mg/kg) eliminated PDX12872 tumors [2].
In CRC xenografts, FL118 (5 mg/kg oral, weekly × 4) effectively inhibited tumor growth in high DDX5-expressing SW620 and SW837 tumors, while low DDX5-expressing SW480 and SW948 tumors showed poor sensitivity [2].
In ES-2 ovarian cancer xenografts, FL118 (5 or 10 mg/kg oral, once weekly for 20 days) showed superior antitumor activity compared to topotecan (2 mg/kg, 5×/week). At 10 mg/kg, 60% of tumors were completely abolished. FL118 upregulated CYGB expression in tumor tissues [1].
In a mouse intratracheal pharmacodynamic model, FL118 inhibited IL-6-induced lung pSTAT3 (JAK1/JAK2) with an ED50 of approximately 3 μg/animal [4].
In a mouse ear dermal model, 4% topical FL118 reduced IL-23-induced ear swelling by 48% over 11 days [4].

For FL118 binding to DDX5, isothermal titration calorimetry (ITC) was performed using purified Flag-DDX5 protein (10 μM) and FL118 (100 μM) in 1× PBS (pH 7.4) with 8% DMSO. The compound was titrated into the protein cell by 20 injections over 60 min (one injection per 3 min) at 25°C. The binding affinity (KD) was calculated as 34.4 nM. For Top1, the KD was 315 nM; no binding to BSA was detected [2].
For FL118 affinity purification, FL118 was coupled to agarose resin beads via a diaminodipropylamine linker through a Mannich reaction. Cancer cell lysates were passed through the FL118 affinity column and a control column in parallel. After stringent washing, bound proteins were eluted with 8 M urea, concentrated, and separated by SDS-PAGE. The ~70 kDa protein band was identified as DDX5 by mass spectrometry [2].
For Caco-2 permeability assay, Caco-2 cell monolayers were grown on collagen-coated polycarbonate membranes. FL118 (1 μM) was added to apical or basolateral side with or without valspodar (1 μM, P-gp inhibitor). Samples were taken at 120 min and analyzed by LC-MS/MS. Apparent permeability (Papp) and efflux ratio were calculated. FL118 showed high permeability (Papp = 6.76-8.86 × 10⁻⁶ cm/s) and low efflux ratio (1.0-1.2) [4].

Western Blot Analysis [1]
Cell Types: ES-2 and SK-O-V3 cell lines
Tested Concentrations: 10 and 100 nM
Incubation Duration: 48 h
Experimental Results: Effectively inhibited the activation of PI3K/AKT/mTOR signaling pathway in ovarian cancer cells and also inhibited Migration of ES-2 and SK-O-V3 cells.

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Cell migration assay[1]
Cell Types: ES-2 and SK-O-V3 Cell lines
Tested Concentrations: 0, 10 and 100 nM
Incubation Duration: 0 and 24 hrs (hours)
Experimental Results: Inhibition of migrating cells of ES-2 and SK-O-V3 dose dependent.

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RT-PCR[1]
Cell Types: ES-2 and SK-O-V3 cell lines
Tested Concentrations: 0, 10 and 100 nM
Incubation Duration: 48 hrs (hours)
Experimental Results: Promoted CYGB expression.

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Cell proliferation assay[1]
Cell Types: ES-2 and SK-O-V3 Cell lines
Tested Concentrations: 0, 1, 10, 50, 100 and 200 nM
Incubation Duration: 24, 48 and 72 hrs (hours)
Experimental Results: Inhibition of ES-2 and SK-O-V3 cells in a time- and dose-dependent manner.

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Western Blot Analysis[2]
Cell Types: SW620 and Mia Paca-2
Tested Concentrations: 0, 10 and 100 nM
Incubation Duration: 6 and 24 hrs (hours)
Experimental Results: Induced dephosphorylation of DDX5 through the ubiquitin-proteasome degradation pathway and degraded DDX5 time-dependently.

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Western Blot Analysis[2]
Cell Types: PDAC Panc1, CRC HCT-8, SW620, Mia Paca-2, Panc-1, HCT-8 cell lines
Tested Concentrations: 0, 10, 100 and 500 nM
Incubation Duration: 24, 48, 72 h
Experimental Results: Controled the expression of survivin, Mcl-1, XIAP, cIAP2, c-Myc and mKras by regulated DDX5, as an upstream master regulator in cancer development and malignant networks.

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cell cytotoxicity assay[3]
Cell Types: A549, MDA-MB-231, RM-1
Tested Concentrations: 0-1 μM
Incubation Duration: 24 h
Experimental Results: demonstrated cytotoxicity in A-549 (human lung carcinoma), MDA-MB-231 (human breast carcinoma) and RM-1 (mouse prostate carcinoma), with IC50 values of 8.94 ± 1.54 , 24.73 ± 13.82, and 69.19 ± 8.34 nM, respectively.

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Apoptosis analysis[3]
Cell Types: A549 cells
Tested Concentrations: 0, 2.5, 5, 10 nM
Incubation Duration: 48 h
Experimental Results: Resulted in the downregulation of survivin. Increased the production of PARP cleavage in a concentration-dependent manner, which is the hallmark of apoptosis. Induced apoptosis in A549.

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Cell cycle analysis[3]
Cell Types: A549 cells
Tested Concentrations: 0, 2.5, 5, 10 nM
Incubation Duration: 48 h
Experimental Results: Increased G2/M cell population in a concentration-dependent manner, and arrested A549 cells mainly at the G2/M phase.

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For cytotoxicity, cells were seeded in 96-well plates (2,000-7,000 cells/well), treated with various concentrations of FL118 for 24-72 h, and viability was measured by MTT or resazurin assays. IC50 values were calculated using Logit approach [4][3][1].
For colony formation, HCT-8 cells (100 cells/well in 12-well plates) were treated with FL118 or topotecan for 2 or 6 h, then washed and cultured for 2 weeks. Colonies were stained with crystal violet and counted [4].
For western blot, cells were lysed in RIPA buffer, proteins separated by SDS-PAGE, transferred to nitrocellulose or PVDF membranes, and probed with antibodies against DDX5, survivin, Mcl-1, XIAP, cIAP2, c-Myc, mutant Kras, p-AKT, p-mTOR, E-cadherin, vimentin, PARP, caspase-3, and loading controls (actin, GAPDH, tubulin). Blots were visualized by ECL [4][2][1].
For siRNA knockdown, cells were transfected with CYGB-specific siRNA or negative control using Lipofectamine RNAiMAX. After 48 h, cells were treated with FL118 (100 nM) and analyzed for proliferation (MTT) and migration (scratch wound assay) [1].
For CRISPR-Cas9 knockout of DDX5, Panc-1 and Mia Paca-2 cells were electroporated with DDX5 sgRNA-Cas9 ribonucleoprotein complexes. Knockout clones were validated by western blot and PCR/T7 endonuclease assay [2].
For scratch wound assay, cells were plated in 6-well plates, grown to 80-90% confluence, scratched with a 200 μL pipette tip, washed with PBS, and treated with FL118 (10-100 nM) in 1% FBS medium for 24 h. Migration distance was measured [1].

Animal/Disease Models: Female BALB/c nude mice[1]
Doses: 5 and 10 mg/kg
Route of Administration: po (oral gavage); 5 mg/kg once weekly; 10 mg/kg once weekly; 20-day
Experimental Results: demonstrated Bitot Potecan has better anti-tumor activity and dose-dependently inhibits the growth of ES-2 tumors by upregulating the expression level of CYGB.

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Animal/Disease Models: SCID (severe combined immunodeficiency) mice (ten weeks old, female, 20-25 g, 5 mice per cage) bearing human SW620 (colon) and FaDu (head and neck) xenograft tumors [4]: 0, 0.75, 1, 1.5 mg/kg
Doses: IP, once every other day, five times as one cycle (if the tumor relapses, mice are treated with FL118 for the second or third cycle)
Experimental Results: Elimination and acquisition of iritinib can detect human xenograft tumors or topotecan resistance and was effective after multiple cycles of treatment without developing FL118 resistance.

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Animal/Disease Models: SCID (severe combined immunodeficiency) mice harboring human SW620 (colon) and FaDu SCID (severe combined immunodeficient) mouse harboring human SW620 (colon) and FaDu (h

Following a single IV dose of FL118 (1.5 mg/kg) in SCID mice, FL118 rapidly accumulated in tumor tissues with a long half-life (6.85 h in FaDu tumors, 12.75 h in SW620 tumors) but was quickly cleared from circulation (plasma half-life = 1.788 h). Cmax in tumor was 1154 ng/g (FaDu) and 1588 ng/g (SW620); Cmax in plasma was 438 ng/mL. Tumor AUC was 3448-4289 hr·ng/g; plasma AUC was 1042 hr·ng/mL. FL118 concentration in tumor tissue at 10 min post-injection was >2.5-fold higher than in plasma [4].
FL118 has a cLogP of 4.4 and log D of 3.9 (shake flask method). It contains a weakly basic imidazole group (pKa = 4.5) and a weakly acidic fluorophenol group (pKa = 8.7). TPSA = 126 Ų [4].
In rat, IV (0.5 mg/kg): CL = 48 mL/min/kg, CLunbound >48,000 mL/min/kg, Vss = 0.8 L/kg, terminal T1/2 = 2.1 h, oral bioavailability <5%. In dog, IV (0.1 mg/kg infusion): CL = 18 mL/min/kg, CLunbound >18,000 mL/min/kg, Vss = 1.0 L/kg, terminal T1/2 = 2.0 h. Plasma protein binding ≥99.9% [4].
In vitro metabolism in human hepatocytes: major metabolites produced by direct glucuronidation and P450 metabolism; phenol-conjugated metabolites show no JAK inhibition. Consistent across rat and dog [4].

The maximum tolerated dose (MTD) of FL118 in mice for daily ×5 or q2d ×5 schedules is approximately 1.5 mg/kg. For weekly ×4 schedule, MTD is 10 mg/kg [4][2].
In exploratory 7-day IV toxicology studies in rats and dogs, no adverse test article-related findings were observed. A 36-fold margin was achieved in rat over total plasma AUC based on projected human clinical lung dose (200 μg), and a 55-fold margin in dog plasma AUC. BioLum Ames assay showed no genetic toxicity with or without metabolic activation; CHO in vitro micronucleus test also negative. No hERG inhibition at 1 μM (<1% inhibition). No cough signal in mouse Aδ fiber sensory nerve preparation up to 2 mg [4].
In ovarian cancer xenograft study, FL118 at 10 mg/kg (oral, weekly ×4) caused significant body weight loss compared to control, while 5 mg/kg showed no statistical difference. Topotecan (2 mg/kg, 5×/week) also caused body weight loss but not statistically different from control [1].
In PDX models, FL118 at 2.5 mg/kg (1/4 MTD) was well tolerated with minimal body weight changes. Combination with gemcitabine (40 mg/kg) did not increase toxicity [2].

[1]. FL118, a novel anticancer compound, inhibits proliferation and migration of ovarian cancer cells via up-regulation of cytoglobin in vivo and in vitro. Translational Cancer Research, 2017, 6(6):1294-1304.

[2]. FL118, acting as a 'molecular glue degrader', binds to dephosphorylates and degrades the oncoprotein DDX5 (p68) to control c-Myc, survivin and mutant Kras against colorectal and pancreatic cancer with high efficacy. Clin Transl Med. 2022 May;12(5):e881.

[3]. Synthesis of novel 10,11-methylenedioxy-camptothecin glycoside derivatives and investigation of their anti-tumor effects in vivo. RSC Adv. 2019 Apr 9;9(20):11142-11150.

[4]. FL118, a novel camptothecin analogue, overcomes irinotecan and topotecan resistance in human tumor xenograft models. Am J Transl Res. 2015 Oct 15;7(10):1765-81.

FL118 is a novel camptothecin analogue containing a unique methylenedioxy group at positions 10 and 11 of the A-ring. It was identified through high-throughput screening using a survivin promoter-driven luciferase reporter system. Unlike irinotecan/SN-38 and topotecan, FL118 is not a substrate of ABCG2/BCRP or P-gp/MDR1 efflux pumps, and its antitumor activity is independent of Top1 expression. FL118 selectively inhibits multiple cancer-associated anti-apoptotic proteins (survivin, Mcl-1, XIAP, cIAP2) and functions as a "molecular glue degrader" that binds to, dephosphorylates, and degrades the DDX5 oncoprotein via the proteasome pathway without affecting DDX5 mRNA. DDX5 is a master regulator controlling survivin, Mcl-1, XIAP, cIAP2, c-Myc, and mutant Kras [2].
FL118 shows broad-spectrum preclinical antitumor activity against colon, head-and-neck, lung, pancreatic, ovarian, and other cancers. It overcomes irinotecan and topotecan resistance in human tumor xenograft models. FL118 is currently in clinical development [4][2].

C21H16N2O6

392.36200

392.101

C, 64.28; H, 4.11; N, 7.14; O, 24.47

135415-73-5

(R)-FL118;151636-76-9

72403

Light brown to brown solid powder

1.64g/cm3

812.1ºC at 760mmHg

445ºC

1.808

1

7

1

29

852

1

CC[C@@]1(C2=C(COC1=O)C(=O)N3CC4=C(C3=C2)N=C5C=C6C(=CC5=C4)OCO6)O

RPFYDENHBPRCTN-NRFANRHFSA-N

InChI=1S/C21H16N2O6/c1-2-21(26)13-5-15-18-11(7-23(15)19(24)12(13)8-27-20(21)25)3-10-4-16-17(29-9-28-16)6-14(10)22-18/h3-6,26H,2,7-9H2,1H3/t21-/m0/s1

(5S)-5-ethyl-5-hydroxy-7,18,20-trioxa-11,24-diazahexacyclo[11.11.0.02,11.04,9.015,23.017,21]tetracosa-1(13),2,4(9),14,16,21,23-heptaene-6,10-dione

FL118; (S)-FL118; FL118; (S)-FL-118; FL 118; 10,11-(Methylenedioxy)-20(S)-camptothecin); FL-118

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)

DMSO : ~1 mg/mL (~2.55 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.)