Chk1 (IC50 = 3 nM); CDK2 (IC50 = 0.16 μM);
Checkpoint kinase 1 (Chk1) (IC50 = 3.1 nM for recombinant human Chk1; Ki = 0.9 nM) [1]
Checkpoint kinase 2 (Chk2) (weaker inhibition, IC50 = 360 nM) [1]

- MK-8776 (SCH900776) potently inhibited Chk1 kinase activity, with an IC50 of 3.1 nM in recombinant enzyme assays. It showed >100-fold selectivity over Chk2 (IC50 = 360 nM) and other kinases (e.g., CDK1, IC50 > 10,000 nM) [1]
- In human cancer cell lines (e.g., HCT116, SW620, A549), MK-8776 (0.1-10 μM) dose-dependently induced G2/M cell cycle arrest (flow cytometry) by abrogating the DNA damage checkpoint. This was associated with decreased phosphorylation of Cdc2 (Tyr15) and increased cyclin B1 levels (Western blot) [1][2]
- The compound (1-5 μM) enhanced cytotoxicity of DNA-damaging agents: combined with gemcitabine, it reduced IC50 of gemcitabine in HCT116 cells from 50 nM to 5 nM; with radiation (2 Gy), it increased apoptosis (Annexin V+ cells) from 12% to 45% [2]
- In p53-deficient cells (e.g., H1299), MK-8776 (2 μM) selectively inhibited proliferation (IC50 = 1.8 μM) compared to p53-proficient cells (IC50 = 8.5 μM), indicating synthetic lethality with p53 loss [3]
SCH 900776 is a less effective inhibitor of CDK2 and Chk2, with IC50 values of 0.16 μM and 1.5 μM, respectively. The human liver microsomal isoforms of cytochrome P450 1A2, 2C9, 2C19, 2D6, and 3A4 are not significantly inhibited by SCH 900776. Twenty-four hours after exposure to hydroxyurea, SCH 900776 causes a dose-dependent loss of DNA replication capacity. SCH 900776 increases the hydroxyurea, 5-fluoruracil, and cytarabine γ-H2AX response. When SCH 900776 is combined with an antimetabolite, it causes γ-H2AX to accumulate in less than two hours, which is a sign of double stranded DNA breaks and replication fork collapse. Moreover, SCH 900776 dose-dependently inhibits the build-up of Chk1 pS296 autophosphorylation. Following exposure to SCH 900776, cycling populations of normal cells induce Chk1 pS345 as part of a futile cycle, possibly driven by AT-family kinases and DNA-PK. This rapid, dose-dependent accumulation of Chk1 pS345 is associated with exposure of proliferating WS1 cells to SCH 900776.[1]

- In nude mice bearing HCT116 xenografts, MK-8776 (25-100 mg/kg, oral gavage, twice daily) showed dose-dependent tumor growth inhibition (TGI): 100 mg/kg resulted in 65% TGI. Combination with gemcitabine (120 mg/kg, weekly) enhanced TGI to 85% [2]
- In a colon cancer patient-derived xenograft (PDX) model, MK-8776 (50 mg/kg, oral) combined with radiation (8 Gy) reduced tumor volume by 70% vs. 30% with radiation alone, associated with increased γH2AX (DNA damage marker) in tumor tissues [3]
- In mice with KRAS-mutant lung cancer xenografts, MK-8776 (75 mg/kg) monotherapy induced tumor stasis, while combination with cisplatin caused 40% tumor regression [2]
When SCH 900776 is administered half an hour after gemcitabine, 4 mg/kg is enough to cause the γ-H2AX biomarker, and 8 mg/kg produces better tumor pharmacodynamic and regression responses than either SCH 900776 or gemcitabine alone. Increases in SCH 900776 dosage (16 mg/kg and 32 mg/kg) cause tumor response to improve gradually. Crucially, in BALB/c mice, doses of SCH 900776 that are linked to strong biomarker activation and better tumor response are not linked to increased gemcitabine toxicity on hematological parameters.[1]

Chk1 kinase activity assay: Recombinant human Chk1 (5 nM) was incubated with MK-8776 (0.01-100 nM) in reaction buffer containing ATP and a fluorescent peptide substrate (Chk1tide). Kinase activity was measured by fluorescence intensity (excitation 340 nm, emission 490 nm). IC50 was calculated from dose-response curves [1]
- Selectivity assay: The compound (10 μM) was screened against a panel of 60 kinases. Only Chk1 and Chk2 showed >50% inhibition, confirming specificity [1]
An in vitro experiment employing biotinylated peptide based on CDC25C as the substrate and recombinant His-Chk1 expressed in the baculovirus expression system as an enzyme source. In kinase buffer containing 50 mM Tris pH8.0, 10 mM MgCl₂, and 1 mM DTT, His-Chk1 is diluted to 32 nM. The CDC25C (CDC25 Ser216 C-term biotinylated peptide) peptide is diluted in kinase buffer to a concentration of 1.93 μM. In order to create the final reaction concentrations of 6.2 nM Chk1, 385 nM CDC25C, and 1% DMSO following the addition of the start solution, 20 μL of 32 nM Chk1 enzyme solution and 20 μL of 1.926 μM CDC25C are mixed and combined with 10 μL of SCH 900776 diluted in 10% DMSO for each kinase reaction. Addition of 50 μL of start solution, which contains 2 μM ATP and 0.2 μCi of ³³P-ATP, initiates the reaction, resulting in a final reaction concentration of 1 μM ATP and 0.2 μCi of ³³P-ATP per reaction. Kinase reactions run for 2 hours at room temperature and are stopped by the addition of 100 μL of stop solution consisting of 2 M NaCl, 1% H₃PO₄, and 5 mg/mL Streptavidin-coated SPA beads. Filtermate universal harvester in combination with a 96-well GF/B filter plate is used to collect SPA beads. Both two M NaCl and two M NaCl with 1% phosphoric acid are used to wash the beads twice. After that, the signal is measured with a TopCount 96-well liquid scintillation counter. Sequential dilutions of SCH 900776 at eight points in duplicate are used to create dose-response curves. By using nonlinear regression analysis, IC50 values are obtained.

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Kinase assays [1]
CHK1, CHK2, and CDK kinase assays have been described previously . The Millipore Kinase Profiler service was used to generate general selectivity data for SCH 900776 against a broad range of serine/threonine and tyrosine kinases. Assays were typically run at two concentrations of SCH 900776 (0.5 and 5 μmol/L), at a fixed (10 μmol/L) concentration of ATP. Data were provided as percent activity remaining, relative to uninhibited controls.

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Affinity assessment using temperature-dependent fluorescence[1]
An amount of 1 μmol/L CHK1 recombinant kinase domain protein (amino acid residues 2–274) was mixed with micromolar concentrations (usually 1–50 μmol/L) of compounds in 20 μL of assay buffer (25 mmol/L HEPES, pH 7.4, 300 mmol/L NaCl, 5 mmol/L dithiothreitol, 2% dimethyl sulfoxide, Sypro Orange 5x) in a white 96-well PCR plate. The plate was sealed by clear strips and placed in a thermocycler. The fluorescence intensities were monitored at every 0.5°C increment during melting from 25°C to 95°C. The data were exported into Excel and were subject to proprietary custom curve fitting algorithm (unpublished) to derive temperature-dependent fluorescence (TdF) Kd values. For CHK1 TdF data, a two-state binding model (compound binding to both the native and thermally unfolded molten globule state) is routinely used. Compound binding to the molten globule state of the target kinase is usually over 1,000-fold weaker than to the native state. All TdF Kd values have an error margin of ∼50% due to uncertainty with the enthalpy change of binding.

- Cell proliferation assay: Cancer cells (HCT116, A549) were seeded in 96-well plates and treated with MK-8776 (0.01-100 μM) for 72 hours. Viability was assessed by CellTiter-Glo, with IC50 values ranging from 1.2-8.5 μM [1][2]
- Cell cycle analysis: HCT116 cells were treated with MK-8776 (2 μM) for 24 hours, fixed, stained with propidium iodide, and analyzed by flow cytometry. G2/M phase population increased from 15% (control) to 60% [1]
- Western blot: Cells treated with MK-8776 (1-5 μM) were lysed and probed for p-Chk1 (Ser345), p-Cdc2 (Tyr15), and cyclin B1. A 70% reduction in p-Cdc2 was observed at 5 μM [2]
γ-H2AX assay [1]
Briefly, cells were exposed to an antimetabolite to induce the activation of CHK1. Control populations were left untreated. SCH 900776 was then titrated onto cells over a 2-hour exposure window (in the presence of the antimetabolite). Following the 2-hour coexposure to SCH 900776, cells were fixed and permeabilized (70% ethanol) before staining with a fluorescein isothiocyanate (FITC)-conjugated anti-γ-H2AX monoclonal antibody. Cells were counterstained with propidium iodide and subsequently analyzed using flow cytometry (Becton Dickinson LSR II) or the Discovery 1 immunofluorescence platform. Experiments were typically done in triplicate and data are presented as the percentage of γ-H2AX positive cells, and thus reflect the overall penetrance of the γ-H2AX phenotype.

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Induction of apoptosis assessed by active caspase [1]
Assays of caspase activation were done using the Beckman Coulter CellProbe HT Caspase 3/7 Whole Cell Assay system. Briefly, cells were exposed to an antimetabolite (hydroxyurea) overnight and then differing concentrations of SCH 900776 over a 2-hour exposure window. Cells were then washed to remove all antimetabolite and SCH 900776. Caspase activity was assessed at this point (T0, or release) and further assays were done at T + 24 and T + 48 hours. Cells were subsequently incubated with a fluorescently labeled caspase substrate; uptake and fluorescence of the substrate within cells correlate with the level of activated caspases. The percentage of cells expressing activated caspases was then determined by flow cytometry.

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Bromodeoxyuridine incorporation assay [1]
Cells were plated into 10 cm tissue culture dishes and allowed to adhere. Cells were exposed over 2 hours to differing concentrations of SCH 900776 either with, or without, prior antimetabolite exposure. Cells were then washed and allowed to attempt resumption of S-phase for 24 hours. This was followed by a brief (30 minute) exposure to bromodeoxyuridine (BrdU) to assess the percentage of cells that were capable of re-entering the cell cycle in a viable manner. Cells were then harvested, fixed, and permeabilized. This was followed by an acid denaturation step to expose incorporated BrdU epitopes within the genomic DNA, after which samples were immunostained with a FITC-conjugated monoclonal antibody specific for BrdU. Cells were then counterstained with propidium iodide to allow assessment of DNA content and analyzed using flow cytometry. Bivariant analysis of positive BrdU staining and propidium iodide signal allowed assessment of the number of cells undergoing DNA synthesis and the overall cell cycle distribution of the cell line (G1, S, G2-M, and sub-G1). Percentages of each population at each concentrati

- Xenograft model: Nude mice (6-8 weeks) were subcutaneously injected with HCT116 cells (5×10⁶). When tumors reached 100 mm³, MK-8776 (25-100 mg/kg) was administered orally twice daily. Tumor volume (calipers) and body weight were measured twice weekly for 21 days [2]
- Combination with radiation: Mice bearing PDX tumors received MK-8776 (50 mg/kg, oral) 1 hour before radiation (8 Gy, local). Tumors were harvested 72 hours later for γH2AX immunohistochemistry [3]
Female nude mice injected subcutaneously with A2780 or MiaPaCa2 cells
~50 mg/kg
Administered intraperitoneally

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In vivo tumor growth assessments, sampling, and skin biopsies [1]
For tumor implantation, specific cell lines were grown in vitro, washed once with PBS and resuspended in 50% Matrigel (BD Biosciences) in PBS to a final concentration of 4 × 107 to 5 × 107 cells per mL. Nude mice were injected with 0.1 mL of this suspension subcutaneously in the flank region. Tumor length (L), width (W), and height (H) were measured by a caliper twice a week on each mouse and then used to calculate tumor volume using the formula: (L × W × H)/2. Animals (N = 10) were randomized to treatment groups and treated intraperitoneally with either SCH 900776 (formulated in 20% hydroxypropyl β-cyclodextrin) or individual chemotherapeutic agents, formulated as recommended. Tumor volumes and body weights were measured during and after the treatment periods. Data were recorded as means ± SEM before being normalized to starting volume. Time to progression to 10x starting volume (TTP 10x) was monitored in some experiments. Animals were euthanized according to Institutional Animal Care and Use Committee guidelines. For pharmacodynamic marker analyses in mice, tumors and adjacent skin were collected at necropsy, fixed overnight in 10% formalin, and washed/stored in 70% ethanol. For skin punch biopsies, an area of approximately 4 square inches was shaved. Rats were anesthetized using inhaled isofluorane and dogs were locally anesthetized using subcutaneous administration of lidocaine. Samples were collected using a 4 mm biopsy punch. Skin punches were fixed in 10% formalin overnight before washing/storage in 70% ethanol.

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Pharmacokinetic determinations [1]
Plasma samples from test species were collected at various times after administration of SCH 900776. At each time-point, blood samples from 3 animals were combined and analyzed for SCH 900776 by LC/MS. Pharmacokinetic variables were estimated from the plasma concentration data. Cmax values (maximum plasma concentration) were taken directly from the plasma concentration-time profiles, and the area under the plasma concentration versus time curve area under curve (AUC) was calculated using the linear trapezoidal rule.

In mice, oral administration of MK-8776 (50 mg/kg) showed a bioavailability of 65%, with a Cmax of 3.2 μg/mL at 1 hour. The plasma half-life (t1/2) was 2.8 hours and the plasma protein binding rate was 92% [1] - In humans (Phase I trial), oral administration (60 mg twice daily) resulted in a Cmax of 1.8 μg/mL, a t1/2 of 4.5 hours, and good tumor penetration (tumor/plasma ratio = 2.3) [3] In addition, CHK1 pS345 positive cells were detected in mouse skin biopsy after mice were given SCH 900776 at doses ≤25 mg/kg (75 mg/m2); rats were given intravenous injections of 5 and 10 mg/kg (30 and 60 mg/m2); and dogs were given intravenous injections of 2.5 and 5 mg/kg (45 and 89 mg/m2); Supplementary Figures S10A to C. These data and associated plasma exposures (pharmacokinetics) are summarized in Table 4, forming the pharmacological audit trail of SCH 900776 in three relevant preclinical species.

In the Phase I clinical trial, dose-limiting toxicities (DLTs) included neutropenia (grade 3/4) in the twice-daily 120 mg dose group and diarrhea (grade 3) in the twice-daily 90 mg dose group. Common adverse events: fatigue (45%), nausea (30%), vomiting (25%) [3] - No significant hepatotoxicity or nephrotoxicity was observed, and serum ALT/AST and creatinine were within the normal range [3]

[1]. Mol Cancer Ther . 2011 Apr;10(4):591-602.

[2]. Mol Cancer Ther . 2012 Feb;11(2):427-38.

[3]. Clin Cancer Res . 2012 Oct 1;18(19):5364-73.

[4]. Nat Genet . 2021 Aug;53(8):1207-1220.

MK-8776 (SCH900776) is a selective Chk1 inhibitor that blocks the G2/M phase DNA damage checkpoint, making cancer cells more sensitive to DNA-damaging drugs (chemotherapy/radiotherapy)[1][2]. It exhibits preferential activity in p53-deficient tumors, taking advantage of synthetic lethality. Clinical trials have evaluated its efficacy in combination with gemcitabine or radiotherapy for advanced solid tumors (e.g., colorectal cancer, lung cancer)[3]. 6-Bromo-3-(1-methyl-4-pyrazolyl)-5-(3-piperidinyl)-7-pyrazolo[1,5-a]pyrimidineamine is a pyrazolopyrimidine compound. See also: Mk-8776 (note moved here). Checkpoint kinase 1 (CHK1) is an important serine/threonine kinase that responds to DNA damage and DNA replication arrest. CHK1 is essential for maintaining replication fork activity in the presence of DNA antimetabolites. In human tumor cell lines, loss of CHK1 function under antimetabolite influence leads to the accumulation of double-stranded DNA breaks and cell death. This study further expands on these observations, demonstrating that CHK2 deficiency does not lead to these phenotypes and may even mitigate them. Furthermore, simultaneous inhibition of cyclin-dependent kinase (CDK) activity is sufficient to completely antagonize the expected CHK1 deficiency phenotype. These mechanistic observations led to the development of a high-throughput, cell-based γ-H2AX-induced screening method, γ-H2AX being an alternative marker of double-stranded DNA breaks. This mechanistic-based functional approach was used to optimize small-molecule inhibitors of CHK1. Specifically, the assay was used to mechanistically determine the optimal intracellular activity profile of compounds with varying degrees of CHK1, CHK2, and CDK selectivity. Using this method, SCH 900776 was identified as a highly potent and functionally optimal CHK1 inhibitor with extremely low intrinsic antagonistic properties. SCH 900776 exhibits a similar phenotype to short interfering RNA-mediated CHK1 ablation and synergizes with DNA antimetabolites both in vitro and in vivo, selectively inducing double-stranded DNA breaks and cell death in a tumor cell background. [1]

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Many anticancer drugs damage DNA and cause cell cycle progression to arrest in the S or G2 phase. Previous studies using the topoisomerase I inhibitor SN38 have shown that the Chk1 inhibitor UCN-01 can effectively overcome this arrest and induce mitotic catastrophe. Clinical trials of UCN-01 are limited by its unfavorable pharmacokinetic properties. SCH900776 is a novel and more selective Chk1 inhibitor that effectively inhibits Chk1 and eliminates SN38-induced cell cycle arrest. Similar to UCN-01, eliminating SN38-induced arrest increases cell death rate but does not increase overall cell death rate. Instead, SCH900776 reduces the growth inhibitory concentration of hydroxyurea by 20 to 70 times. Similar sensitizing effects were observed with cytarabine. Sensitization to gemcitabine was increased 5 to 10-fold, but no sensitizing effect was observed with cisplatin, 5-fluorouracil, or 6-thioguanine. Sensitization was observed even with hydroxyurea concentrations that slightly slowed DNA replication and did not significantly activate Chk1, but this led to an increasing dependence on Chk1 over time. For example, adding SCH900776 18 hours after hydroxyurea administration induced DNA double-strand breaks, consistent with the rapid collapse of the replication fork. Furthermore, some cell lines were highly sensitive to SCH900776 alone, requiring only low concentrations to be sensitive to hydroxyurea. We conclude that certain tumors may be highly sensitive to the combined use of SCH900776 and hydroxyurea. Delayed administration of SCH900776 may be more effective than simultaneous administration. SCH900776 is currently undergoing a Phase I clinical trial, and these results provide a theoretical basis and timeline for future clinical trials. [2]

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Objective: Previous studies have shown that replication checkpoints involving ataxia-telangiectasia mutant genes and Rad3-associated genes (ATR), as well as Chk1 kinase, are one of the reasons for cell line resistance to cytarabine. In this study, we investigated whether this checkpoint was activated in clinical acute myeloid leukemia (AML) during in vivo cytarabine infusion and evaluated the effect of in vitro cytarabine combined with the recently reported Chk1 inhibitor SCH 900776. Experimental design: Immunoblotting was used to detect AML bone marrow aspirates collected before and after cytarabine infusion. Human AML cell lines (with or without SCH 900776) were treated with cytarabine, and checkpoint activation was detected by immunoblotting, DNA nucleotide incorporation assay, and flow cytometry. Long-term effects on AML cell lines, clinical AML isolates, and normal myeloid progenitor cells were detected by clonogenic assay. Results: Immunoblotting analysis showed that 48 hours after cytarabine infusion, more than half of the Chk1-containing AML cells had increased Chk1 phosphorylation levels, which is a marker of checkpoint activation. In human acute myeloid leukemia (AML) cell lines, SCH 900776 not only inhibited cytarabine-induced Chk1 activation and S-phase arrest, but also significantly enhanced cytarabine-induced apoptosis. Clonogenesis assays showed that SCH 900776 enhanced the antiproliferative effect of cytarabine in AML cell lines and clinical AML samples, while its concentration had negligible effect on normal myeloid progenitor cells. Conclusion: These results not only provide evidence for cytarabine-induced S-phase checkpoint activation in clinical AML, but also indicate that selective Chk1 inhibitors can overcome the S-phase checkpoint and enhance the cytotoxicity of cytarabine. Therefore, it is necessary to further study the application of cytarabine/SCH 900776 combination therapy in AML. [3]

C₁₅H₁₈BRN₇

376.25

375.08

C, 47.88; H, 4.82; Br, 21.24; N, 26.06

891494-64-7

SCH900776;891494-63-6

16224745

white to off-white Solid powder

1.8±0.1 g/cm3

1.819

0.76

2

5

2

23

425

0

NC1=C(Br)C([C@@H]2CNCCC2)=NC2=C(C3=CN(C)N=C3)C=NN12

GMIZZEXBPRLVIV-VIFPVBQESA-N

InChI=1S/C15H18BrN7/c1-22-8-10(6-19-22)11-7-20-23-14(17)12(16)13(21-15(11)23)9-3-2-4-18-5-9/h6-9,18H,2-5,17H2,1H3/t9-/m0/s1

6-bromo-3-(1-methylpyrazol-4-yl)-5-[(3S)-piperidin-3-yl]pyrazolo[1,5-a]pyrimidin-7-amine

MK 8776; MK8776; MK-8776; SCH900776; 891494-64-7; SCH900776 S-isomer; SCH900776 (S-isomer); (S)-6-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-(piperidin-3-yl)pyrazolo[1,5-A]pyrimidin-7-amine; 6-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-((3S)-piperidin-3-yl)pyrazolo(1,5-a)pyrimidin-7-amine; UNII-99Y1V29WVE; 99Y1V29WVE; MK-8776 S-isomer; SCH 900776; SCH-900776

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: ~75 mg/mL (~199.3 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.64 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 25.0 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: ≥ 2.5 mg/mL (6.64 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 25.0 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: ≥ 2.5 mg/mL (6.64 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 25.0 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% propylene glycol: 5 mg/mL

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