HIF-1 (HIF-1α/HIF-1β heterodimer) binding to the hypoxia-responsive element (HRE) core sequence 5'-RCGTG-3' [1]
HIF1α [2]

Treatment with echinomycin (0-10 nM; 16 hours; U251 cells) markedly and dose-dependently reduced the production of VEGF mRNA produced by hypoxia. With an EC50 of 1.2 nM, echinomycin highly potently and dose-dependently suppresses the hypoxic stimulation of luciferase expression in U251-HRE [1].

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In a DNA-binding ELISA using recombinant HIF-1α-bHLH-PAS and HIF-1β-bHLH-PAS proteins, Echinomycin dose-dependently inhibited HIF-1 DNA-binding activity with nearly complete inhibition at 320 nmol/L [1].
- In EMSA, Echinomycin (up to 10 μmol/L) specifically inhibited HIF-1 DNA-binding activity but did not inhibit AP-1 (c-Fos/JunD) or NF-κB (p65) binding to their consensus sequences. It also completely inhibited binding of nuclear proteins from MCF-7 cells to an E-box oligonucleotide [1].
- Echinomycin (0.5 μmol/L) completely abrogated HIF-1 binding to the HRE of the VEGF promoter in ChIP assays in U251 cells, but did not affect c-Fos binding to the cyclin D1 promoter or p65 binding to the ICAM-1 promoter [1].
- Echinomycin did not affect HIF-1α protein accumulation in U251 cells under hypoxia; instead, HIF-1α levels were slightly increased [1].
- In U251-HRE cells, Echinomycin inhibited hypoxia-induced luciferase expression with an EC50 of approximately 1.2 nmol/L. The inhibition was partially reversible upon drug washout (94%, 80%, and 42% recovery after 4 h treatment at 5, 10, and 20 nmol/L, respectively) [1].
- Echinomycin (0-10 nmol/L) dose-dependently inhibited hypoxia-induced VEGF mRNA expression in U251 cells (5-fold induction reduced significantly at P<0.01) [1].
- In a 22K oligonucleotide microarray, Echinomycin (20 nmol/L, 20 h) down-regulated 276 genes under normoxia and 451 genes under hypoxia by ≥2-fold; 14 out of 17 known HIF-1-inducible genes were inhibited by >3-fold [1].
- In mouse TGB lymphoma cells, Echinomycin abrogated colony-forming unit (CFU) activity of cancer stem cells (c-Kit+Sca-1+) with an IC50 of 29.4 pM (as measured by HRE-GFP reporter). Normal mouse hematopoietic progenitor cells (HPC) were approximately 100-fold less sensitive [2].
- Echinomycin (20 pM, 16 h) selectively increased apoptosis in c-Kit+Sca-1+ lymphoma cells (Annexin V+ from 1.8% to 10%) but had no effect on c-Kit-Sca-1- cells [2].
- In seven primary human AML samples, Echinomycin inhibited CFU activity with IC50 values between 50-120 pM. HIF1α shRNA silencing reduced sensitivity to echinomycin [2].
- Echinomycin selectively induced apoptosis in the CD34+CD38- AML stem cell subset compared to bulk AML cells (data shown as percentage of Annexin V+ after subtraction of vehicle control) [2].
- Echinomycin (20 pM) did not affect c-Myc function at concentrations used [2].
- DNA thermal melting analysis showed that Echinomycin increased the Tm of HRE oligonucleotide from 51.5°C to 65°C (26.2% increase), while only a 5.5°C increase for NF-κB and 5°C increase for AP-1 oligonucleotides [1].

Through selective depletion of cancer stem cells (CSCs), treatment with Echinomycin (10 μg/kg; i.v.; for 40 days) efficiently eradicates human acute myeloid malignancies and murine lymphoma in xenogeneic models. AML, or leukemia. HIF1α prevents the negative feedback loop in the Notch pathway, which keeps mice lymphoma CSCs alive [2].

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In a syngeneic mouse lymphoma model (B10.BR mice injected i.p. with 1×10^6 cultured TGB lymphoma cells), treatment with Echinomycin (10 μg/kg/injection, i.p., every 2 days for a total of 5 injections starting 14 days after tumor injection) resulted in complete tumor elimination and long-term survival (all treated mice lived until euthanasia at 134 or 252 days with no sign of tumor) [2].
- In a xenogeneic NOD-SCID mouse model of human AML (irradiated mice injected i.v. with 8×10^6 peripheral blood cells from AML patients), Echinomycin treatment (10 μg/kg/injection, daily for 5 consecutive days followed by 2 days rest, repeated once, starting 15 days after transplantation) completely eliminated human CD45+ cells from AML150 and dramatically reduced human leukemia burden from AML71 in bone marrow at day 40 [2].
- In the same AML71 model, Echinomycin reduced the percentage of CD34+CD38- cells (putative AML stem cells) by >10-fold within the human CD45+ compartment. Bone marrow from echinomycin-treated mice failed to reinitiate leukemia upon serial transplantation into secondary recipients (0/5 recipients developed leukemia), whereas bone marrow from vehicle-treated mice readily induced leukemia [2].

HIF-1 DNA-binding ELISA: Biotin-labeled double-stranded oligonucleotide containing a canonical HRE was immobilized on streptavidin-coated plates. Recombinant HIF-1α-bHLH-PAS (10 pmol) and HIF-1β-bHLH-PAS (6 pmol) were mixed in buffer (25 mmol/L Tris-HCl pH7.6, 100 mmol/L KCl, 0.2 mmol/L EDTA, 20% glycerol, 5 mmol/L DTT) and preincubated for 5 min at room temperature. The mixture was added to the plate and incubated for 1 h at room temperature. The protein complex was detected using an anti-His tag monoclonal antibody (1:2,000) followed by anti-mouse HRP-conjugated antibody (1:10,000). After adding TMB substrate for 10 min, the reaction was stopped with 0.5 mol/L H2SO4 and absorbance read at 450 nm with reference at 655 nm. Compounds were screened at 10 μmol/L. Competition experiments used 50 pmol non-biotinylated oligonucleotides [1].
- Electrophoretic Mobility Shift Assay (EMSA): Recombinant HIF-1α-bHLH-PAS (10 pmol) and HIF-1β-bHLH-PAS (6 pmol), p65 (100 ng), or c-Fos (10 pmol) plus JunD (10 pmol) were mixed in 40 μL of buffer (25 mmol/L Tris-HCl pH7.6, 100 mmol/L KCl, 0.2 mmol/L EDTA, 20% glycerol, 5 mmol/L DTT, 200 ng calf thymus DNA). After 5 min preincubation at room temperature, 1 μL of 32P-labeled probe (~50,000 cpm) was added and incubated on ice for 20 min. For competition, 100-fold excess unlabeled WT or mutant oligonucleotides were used. For supershift, monoclonal anti-HIF-1α or polyclonal anti-HIF-1α antibodies were added. Oligonucleotide sequences: WT HRE (5'-GTGCTACGTGCTGCTACG-3'), mutant HRE (5'-GTGCTAAAAGCTGCTAG-3'), NF-κB (5'-AGTTGAGGGGACTTCCAGGGC-3'), AP-1 (5'-CTAGTGATGAGTCAGCCGGGATC-3'), WT Myc/Max E-box (5'-AGTTGACCACGTGGTCTGGG-3'), mutant Myc/Max (5'-AGTTGACTAAAAGGTCTGGG-3') [1].
- DNA Thermal Melting Analysis: Double-stranded oligonucleotides (0.5 μmol/L) containing HRE, NF-κB binding site, or AP-1 binding site were prepared in 10 mmol/L Tris pH8.0, 1 mmol/L EDTA, with or without 1 μmol/L Echinomycin (0.1% DMSO). Thermal transition curves were obtained by measuring absorbance at 260 nm as a function of temperature. Melting temperatures (Tm) were calculated using software [1].
- Ethidium Bromide Displacement Assay: Echinomycin caused approximately 50% displacement of ethidium bromide from HRE oligonucleotide at 3 μmol/L, whereas no significant displacement from AP-1 oligonucleotide was observed even at 10 μmol/L [1].

RT-PCR[1]
Cell Types: U251 Cell
Tested Concentrations: 0 nM, 0.625 nM, 1.25 nM, 5 nM, 10 nM
Incubation Duration: 16 hrs (hours)
Experimental Results: Dramatically inhibited hypoxia-induced VEGF mRNA expression in a dose-dependent manner.

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Chromatin Immunoprecipitation (ChIP): U251 cells were treated with hypoxia plus desferrioxamine (400 μmol/L) in the presence or absence of Echinomycin (0.5 μmol/L) for 6 h. Cells were crosslinked, lysed, and sonicated. Immunoprecipitation was performed using 2 μg of anti-HIF-1α monoclonal antibody, anti-c-Fos polyclonal antibody, anti-p65 polyclonal antibody, or isotype-matched irrelevant antibodies. PCR primers: VEGF promoter (forward 5'-CCTTTGGGTTTTGCCAGA-3', reverse 5'-CCAAGTTTGTGGAGCTGA-3', 135 bp product); cyclin D1 promoter (forward 5'-CTACACCCCAAACAAAACCA-3', reverse 5'-TAACCGGGAGAAACACACCT-3', 163 bp); ICAM-1 promoter (forward 5'-CGTGATTCAAGCTTAGCCTG-3', reverse 5'-TTATTTCCGGACTGACAGGG-3', 176 bp). PCR products were run on 3% agarose gel and stained with ethidium bromide [1].
- Western Blot: U251 cells were treated under normoxia or hypoxia for 16 h with or without Echinomycin (0.5 μmol/L). Cells were lysed in buffer (50 mmol/L Tris-HCl pH7.4, 1% NP40, 150 mmol/L NaCl, protease inhibitors). Lysates were centrifuged at 13,000 rpm for 5 min at 4°C. Supernatants were subjected to SDS-PAGE and immunoblotted with polyclonal anti-HIF-1α (1:200), anti-cyclin D1 (1:200), and anti-actin (1:3,000) antibodies [1].
- Luciferase Assay: U251-HRE cells (stably transfected with pGL2-TK-HRE) and U251-pGL3 control cells were seeded in 24-well plates and incubated under normoxia or hypoxia for 16 h with increasing concentrations of Echinomycin (0-1000 nmol/L). Luciferase activity was measured and normalized to protein concentration. For reversibility, cells were treated with echinomycin (5, 10, 20 nmol/L) for 4 h, washed three times, and then incubated for 36 h under normoxia or hypoxia [1].
- Real-time PCR for VEGF mRNA: U251 cells were cultured under normoxia or hypoxia for 16 h with increasing concentrations of Echinomycin (0-10 nmol/L). Total RNA was extracted, reverse transcribed, and subjected to TaqMan real-time PCR using human VEGF primers (forward 5'-TACCTCCACCATGCCAAGTG-3', reverse 5'-ATGATTCTGCCCTCCTCCTTC-3') and probe (5'-FAM-TCCCAGGGTGCACCATGGGC-TAMRA-3'). 18S rRNA was used as internal control [1].
- Microarray Analysis: U251 cells were treated with Echinomycin (20 nmol/L) under normoxia or hypoxia for 20 h. Total RNA was reverse transcribed with amino-allyl modified dUTP, coupled to Cy3 (control) or Cy5 (treated), and hybridized to human 22K oligonucleotide microarrays. Arrays were scanned and data analyzed [1].
- Colony Formation Unit (CFU) Assay for Mouse Lymphoma CSC: TGB lymphoma cells were cultured in methylcellulose medium. Echinomycin was added at indicated concentrations. Colonies were counted after culture. IC50 for CSC was 29.4 pM. For normal HPC, bone marrow cells were similarly assayed [2].
- CFU Assay for Human AML: Primary AML cells (2.5×10^5/mL) were pretreated with Echinomycin for 24 h, then plated at 10^5/well for CFU assay. Colonies were counted after 7-10 days. IC50 values ranged 50-120 pM across 7 AML samples [2].
- Apoptosis Assay (Annexin V): Cultured TGB lymphoma cells were treated with Echinomycin (20 pM) or vehicle for 16 h, then stained with c-Kit, Sca-1, and Annexin V. Percentage of Annexin V+ cells was determined in c-Kit+Sca-1+ vs c-Kit-Sca-1- subsets [2].
- Apoptosis Assay for Human AML: Primary AML cells were cultured overnight, then treated with Echinomycin (0-200 pM) for 30 h in medium containing cytokines (CSF, GM-CSF, IL-3). Cells were stained with CD34, CD38, Annexin V, and DAPI. Annexin V+ DAPI+/- cells were quantified [2].
- HRE-GFP Reporter Lentiviral Assay: A lentiviral reporter containing triple HRE elements upstream of a minimal TATA box and EGFP was transduced into TGB lymphoma cells. GFP+ cells (indicating HIF activity) were analyzed by flow cytometry. Echinomycin reduced the percentage of c-Kit+GFP+ cells with IC50 of 29.4 pM [2].
- shRNA Knockdown and Complementation: TGB lymphoma cells were transduced with lentivirus expressing HIF1α shRNA (sh-1: 5'-ctagagatgcagaagact-3'; sh-2: 5'-gagagaaatgcttacaca-3') or scrambled control. After blasticidin selection, CFU, c-Kit+Sca-1+ cell percentage, and Hes1 expression were analyzed. Complementation was performed with human HIF1α cDNA resistant to shRNA [2].

Animal/Disease Models: NOD-SCID (severe combined immunodeficient) mouse received 1.8Gy irradiation and intravenous (iv) (iv)injection of peripheral blood cells from AML-71 and AML-150 patients [2]
Doses: 10 μg/kg
Route of Administration: intravenous (iv) (iv)injection; intravenous (iv) (iv)injection. Results lasting 40 days: Effective eradication of mouse lymphoma and serially transplantable human AML in xenogeneic models by preferential elimination of CSCs.

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Syngeneic mouse lymphoma model (therapeutic study): B10.BR mice (immune competent) were injected intraperitoneally (i.p.) with 1×10^6 cultured TGB lymphoma cells. Fourteen days after tumor injection, mice received Echinomycin at 10 μg/kg/injection i.p. every 2 days for a total of 5 injections (days 14, 16, 18, 20, 22). Control mice received vehicle only (DMSO or similar). Mice were monitored daily for survival. Moribund mice were euthanized. All treated mice survived until euthanasia at 134 or 252 days with no tumor upon necropsy [2].
- Xenogeneic human AML model in NOD-SCID mice: NOD-SCID mice received 1.8 Gy total body irradiation. On the same day, they were injected intravenously (i.v.) with 8×10^6 peripheral blood cells from AML patients (AML71 or AML150). Fifteen days after transplantation, mice were treated with Echinomycin at 10 μg/kg/injection i.p. daily for 5 consecutive days, followed by 2 days rest, and then the cycle was repeated once (total 10 injections over 14 days). Control mice received vehicle only. Forty days after the last treatment, mice were sacrificed, and bone marrow cells were collected for flow cytometry analysis (human CD45, CD34, CD38, CD14, CD15, CD33, CD19) [2].
- Serial transplantation of human AML cells: Bone marrow cells from vehicle-treated or Echinomycin-treated primary AML71 xenograft mice were pooled and injected into secondary NOD-SCID recipients (1.5×10^6 cells/mouse) one day after irradiation. Recipients received no further treatment and were sacrificed at 8 weeks to analyze human cell reconstitution [2].

In U251 cells, Echinomycin showed IC50 >5 μmol/L in sulforhodamine B (SRB) assay under both normoxic and hypoxic conditions at 24 h, indicating no significant effect on cell viability at concentrations used for HIF-1 inhibition [1].
- In mouse colony formation assays, normal hematopoietic progenitor cells (HPC) from bone marrow were approximately 100-fold less sensitive to Echinomycin than lymphoma cancer stem cells (CSC) [2].
- In human AML, Echinomycin selectively induced apoptosis in CD34+CD38- stem cell subset compared to bulk leukemia cells (at concentrations up to 200 pM) [2].

[1]. Echinomycin, a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity. Cancer Res. 2005 Oct 1;65(19):9047-55.

[2]. Targeting HIF1α eliminates cancer stem cells in hematological malignancies. Cell Stem Cell. 2011 Apr 8;8(4):399-411.

Quinomycin C is a cyclic condensate. Echinoxam is a cytotoxic quinoxaline antibiotic isolated from Streptomyces echinatus. It binds to DNA and inhibits RNA synthesis. Echinoxam has been reported to exist in Streptomyces triostinicus, Streptomycetaceae, and Streptomyces echinatus, and relevant data exist. Echinoxam is a quinoxaline antitumor antibiotic isolated from Streptomyces echinatus. Echinoxam inserts into two sites on DNA simultaneously in a sequence-specific manner, thereby inhibiting DNA replication and RNA synthesis. (NCI04) A cytotoxic quinoxaline antibiotic isolated from Streptomyces echinatus that binds to DNA and inhibits RNA synthesis. See also: Echinoxam (note moved to).

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Echinomycin (NSC-13502) is a cyclic peptide of the quinoxaline antibiotic family originally isolated from Streptomyces echinatus. It binds to DNA in a sequence-specific fashion, with strong binding sites containing the central 2-bp sequence 5'-CG-3', and key recognition elements 5'-ACGT-3' and 5'-TCGT-3' [1].
- The compound was identified from a high-throughput screen of the NCI 140,000 small-molecule library using a HIF-1-targeted cell-based assay. Among 128 compounds tested in a HIF-1 DNA-binding ELISA, 20 inhibited by >50% at 10 μmol/L, and echinomycin was one of the most potent and specific inhibitors [1].
- Echinomycin inhibits HIF-1 DNA binding by intercalating into the HRE core sequence, thereby preventing the HIF-1 heterodimer from binding to DNA, rather than interfering with HIF-1α/HIF-1β dimerization or protein stability [1].
- In mouse lymphoma CSC, HIF1α is constitutively active under normoxia due to downregulation of VHL. Echinomycin targets this pathway and eliminates CSC by repressing a negative feedback loop in the Notch pathway (antagonizing Hes1 autoregulation) [2].
- In human AML, CD34+CD38- stem cells show elevated HIF1α and GLUT1 expression. Echinomycin effectively targets these cells and prevents serial transplantation, suggesting elimination of leukemia-initiating cells [2].
- The compound had previously entered clinical trials as a bifunctional intercalating agent but was abandoned. The remarkable efficacy in targeting cancer stem cells at picomolar concentrations suggests its re-evaluation for cancer therapy based on the cancer stem cell concept [2].

C51H64N12O12S2

1101.265

1100.42

512-64-1

3197

White to off-white solid powder

1.41 g/cm3

1427.2ºC at 760 mmHg

817ºC

1.536

1.826

4

18

7

77

2200

0

AUJXLBOHYWTPFV-UHFFFAOYSA-N

InChI=1S/C51H64N12O12S2/c1-25(2)38-49(72)74-22-36(59-42(65)34-21-53-30-17-13-15-19-32(30)57-34)44(67)55-28(6)46(69)63(10)40-48(71)62(9)39(26(3)4)50(73)75-23-35(58-41(64)33-20-52-29-16-12-14-18-31(29)56-33)43(66)54-27(5)45(68)60(7)37(47(70)61(38)8)24-77-51(40)76-11/h12-21,25-28,35-40,51H,22-24H2,1-11H3,(H,54,66)(H,55,67)(H,58,64)(H,59,65)

N,N'-((1R,4S,7R,11S,14R,17S,20R,24S)-11,24-diisopropyl-2,4,12,15,17,25-hexamethyl-27-(methylthio)-3,6,10,13,16,19,23,26-octaoxo-9,22-dioxa-28-thia-2,5,12,15,18,25-hexaazabicyclo[12.12.3]nonacosane-7,20-diyl)bis(quinoxaline-2-carboxamide)

Quinomycin A Antibiotic A 654ISK 302BEchinomycin NSC 526417NSC 13502

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 (e.g. under nitrogen), 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)

DMSO : ~5.3 mg/mL (~4.81 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.)