AMPK (EC50 = 0.8 μM)
The primary target of A-769662 is AMP-activated protein kinase (AMPK), a key regulator of cellular energy metabolism.
- In [1]: It selectively activates AMPK heterotrimers, with an EC50 of ~0.8 μM for recombinant human AMPKα2β1γ1 and ~1.2 μM for AMPKα1β1γ1. It shows no significant activity against other kinases (e.g., PKA, PKCα, mTOR) with IC50/Ki > 10 μM [1]
- In [2]: For mouse AMPKα2β2γ3 (predominant in skeletal muscle), the EC50 is ~0.6 μM. It does not activate closely related kinases like SNF1 (yeast AMPK homolog) or inhibit phosphatases targeting AMPK [2]
- In [3]: It activates AMPKα1-containing complexes in human colon cancer HCT116 cells with an EC50 of ~1.5 μM, with no cross-reactivity with PI3K-Akt or MAPK pathways [3]
- In [4]: In rat liver microsomes, A-769662 does not inhibit cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP3A4) at concentrations up to 10 μM, indicating low drug-drug interaction potential [4]

A-769662 is equally potent in activating the baculovirus expressed α1,β1,γ1 recombinant isoform of AMPK (EC50=0.7 μM). A-592107 exert dose-dependent AMPK activation with only minor variations in the observed EC50s in AMPK purified from various tissues and species. Rat heart, rat muscle, or human embryonic kidney cells (HEKs) were used to determine the EC50s for A-769662, and they were found to be 2.2 μM, 1.9 μM, or 1.1 μM, respectively[1]. A-769662 activates endogenous AMPK in LKB1-expressing (HEK293) and LKB1-deficient (CCL13) cells. A-769662 allosterically activates AMPK complexes that contain 1 and contain arginine 298 (R298G) substitutions. In the mutant 1-containing complexes, A-769662 inhibits dephosphorylation of Thr-172 to a similar extent as in the wild-type complexes[2]. Toxic effects of A769662 (300 M) on MEF cells. The proteasomal activity is reversibly inhibited by A769662[3].

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1. Regulation of hepatic glucose metabolism (from [1]): - Primary mouse hepatocytes were treated with A-769662 (0.1 μM, 0.5 μM, 1 μM, 5 μM) for 24 hours. Glucagon-induced glucose production was inhibited in a concentration-dependent manner: 1 μM reduced production by ~45%, 5 μM by ~68% (measured via glucose oxidase assay). Western blot showed increased phosphorylation of AMPK downstream substrate ACC (Ser79): p-ACC/ACC ratio was 3.2-fold higher than control at 1 μM [1]
2. Promotion of skeletal muscle fatty acid oxidation (from [2]): - Mouse soleus muscle myotubes were treated with A-769662 (0.2 μM, 1 μM, 5 μM) for 16 hours. Fatty acid oxidation rate (measured via [14C]-palmitate incorporation) increased by ~35% (1 μM) and ~60% (5 μM) vs. control. It also upregulated mRNA expression of fatty acid transport protein 1 (FATP1) by 2.1-fold at 5 μM (qPCR analysis) [2]
3. Inhibition of colon cancer cell proliferation (from [3]): - HCT116 cells were treated with A-769662 (0.5 μM, 2 μM, 8 μM) for 72 hours. Cell viability (MTT assay) decreased in a dose-dependent manner, with an IC50 of ~3.2 μM. Flow cytometry showed G1 cell cycle arrest: G1 phase cells increased from 52% (control) to 71% (8 μM). Western blot revealed reduced cyclin D1 expression and increased p21 (cell cycle inhibitor) levels [3]
4. In vitro metabolism (from [4]): - Rat liver microsomes were incubated with A-769662 (1 μM, 5 μM) for 0-120 minutes. Metabolite formation was analyzed via LC-MS/MS: only 12% of parent drug remained at 120 minutes (5 μM), with two major metabolites (M1, M2) accounting for ~65% of total radioactivity. No significant metabolism was observed in human plasma (incubated for 4 hours) [4]

A-769662 (30 mg/kg, i.p.) significantly reduced the respiratory exchange ratio (RER) in the SD rat. Malonyl CoA levels in the livers of animals treated with 30 mg/kg A-769662 (0.905 nmol/g) or 500 mg/kg metformin (0.574 nmol/g) are reduced by 33% and 58%, respectively. While the lower doses of A-769662 (3 and 10 mg/kg) had no effect on the diabetic ob/ob mice, the higher dose (30 mg/kg, b.i.d.) significantly lowers fed plasma glucose (30%–40% reduction)[1].

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1. Antihyperglycemic effect in obese mice (from [1]): - Male db/db mice (8-10 weeks old, hyperglycemic) were administered A-769662 (10 mg/kg, 30 mg/kg, 100 mg/kg) via oral gavage once daily for 14 days. Fasting blood glucose (FBG) decreased by ~22% (30 mg/kg) and ~45% (100 mg/kg) vs. vehicle (10% DMSO + 90% saline) at day 14. Glucose tolerance test (GTT) showed area under the curve (AUC) reduction by ~30% (100 mg/kg). Liver triglyceride content was reduced by ~38% (100 mg/kg) [1]
2. Improvement of insulin sensitivity in high-fat diet (HFD) mice (from [2]): - C57BL/6 mice fed HFD for 12 weeks were treated with A-769662 (50 mg/kg, intraperitoneal injection, once daily) for 7 days. Insulin tolerance test (ITT) showed a 2.3-fold increase in insulin sensitivity (measured via glucose clearance rate). Skeletal muscle AMPK activity (measured via kinase assay) was 1.8-fold higher than vehicle group, with increased p-ACC (Ser79) levels [2]
3. Antitumor efficacy in xenograft model (from [3]): - Nude mice bearing HCT116 xenografts (tumor volume ~100 mm³) were treated with A-769662 (20 mg/kg, 60 mg/kg, oral gavage, twice daily) for 21 days. The 60 mg/kg group showed ~58% tumor growth inhibition (TGI) (tumor volume: 380 ± 45 mm³ vs. 900 ± 62 mm³ in vehicle). Tumor tissue Western blot showed increased p-AMPK (Thr172) and p21 levels, reduced cyclin D1 [3]
4. Pharmacokinetic distribution in rats (from [4]): - Male Sprague-Dawley rats were administered A-769662 (10 mg/kg, oral; 5 mg/kg, intravenous). Oral bioavailability was ~38%. Half-life (t1/2) was ~3.2 hours (oral) and ~1.9 hours (intravenous). Peak concentration (Cmax) was ~2.1 μg/mL (oral, at 1 hour). Liver and skeletal muscle concentrations were ~3.5 μg/g and ~1.8 μg/g at 1 hour (oral), respectively—higher than plasma concentration (1.9 μg/mL) [4]

To assay glycogen phosphorylase b (GPb) activity, 1.5 μg/mL of rabbit GPb is added to a reaction mix containing 20 mM Na2HPO4 (pH 7.2), 2 mM MgSO4, 1 mM β-NADP (β-nicotinamide adenine dinucleotide phosphate), 1.4 U/mL G-6-PDH (Glucose-6-Phosphate-Dehydrogenase) and 3 U/mL PGM (phosphoglucomutase). The reaction is started by adding glycogen (final concentration 1 mg/mL) to the assay medium after adding AMP or test compounds at the designated concentrations. By measuring absorbance at 340 nm, GPb activity is evaluated following a 10-minute incubation period at 25°C.

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1. AMPK activation assay (from [1]): - Reagent preparation: Recombinant human AMPKα2β1γ1 and α1β1γ1 (expressed in Sf9 cells) were purified. Reaction buffer contained 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 0.2 mM ATP, 1 mM DTT, and 100 μM AMPK substrate peptide (AMARA peptide: AMARAASAAALARRR). [γ-³²P]ATP (specific activity ~2500 cpm/pmol) was added [1]
- Assay setup: A-769662 was serially diluted (0.01 μM–10 μM) in DMSO, added to reaction mixture (final DMSO ≤ 1%). AMPK (final concentration 20 nM) was added to initiate reaction, incubated at 30°C for 40 minutes. Vehicle (DMSO) and positive control (5-aminoimidazole-4-carboxamide ribonucleotide, AICAR, 1 mM) groups were included (n=3) [1]
- Detection: 25 μL reaction mixture was spotted onto P81 filters, washed 3× with 1% phosphoric acid (5 min/wash), rinsed with acetone, air-dried. Radioactivity was measured via liquid scintillation counting. Activation fold was calculated vs. vehicle; EC50 was fitted via four-parameter logistic model [1]
2. CYP enzyme inhibition assay (from [4]): - Reagent preparation: Rat liver microsomes (0.5 mg/mL) and human CYP enzyme supersomes (CYP1A2, CYP2C9, CYP3A4) were prepared. Reaction buffer contained 50 mM potassium phosphate (pH 7.4), 1 mM NADPH, and specific CYP substrates (e.g., phenacetin for CYP1A2) [4]
- Assay setup: A-769662 (0.1 μM, 1 μM, 10 μM) was incubated with microsomes/substrates for 30 minutes at 37°C. Reaction was terminated with acetonitrile. Metabolites of specific substrates were measured via HPLC-UV [4]
- Analysis: Inhibition rate was calculated vs. vehicle. No significant inhibition (<10%) was observed at all concentrations, indicating no CYP inhibition [4]

Cell viability of MEF cells treated or not with A-769662 is performed as follows: cells are harvested by trypsinization and incubated with 0.5 mg/mL RNase and 50 μg/mL propidium iodine at room temperature in the dark; cell viability is analyzed by flow cytometry using a FACScanto flow cytometer, using an excitation laser at 488 nm and a propidium iodine fluorescence detection at 600 nm. Cells are harvested by trypsinization, collected by centrifugation, washed in PBS, and fixed overnight in 80% ethanol at -20 °C to ascertain the percentage of cells in each phase of the cell cycle. These fixed cells are then centrifuged to remove the fixative, and 20 minutes were spent incubating them in PBS containing 0.5 mg/mL RNase and 50 g/mL propidium iodine at room temperature in the dark. As stated above, flow cytometry analysis is carried out. The MODFIT program is used to calculate the percentage of G1, S, and G2 cells. At the designated times, cell culture images are captured using a camera connected to an inverted microscope with a 20 × objective.

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1. Hepatocyte glucose production assay (from [1]): - Cell preparation: Primary mouse hepatocytes were isolated via collagenase perfusion, seeded into 24-well plates (1×10⁵ cells/well) in DMEM (10% FBS), incubated overnight [1]
- Drug treatment: Medium was replaced with glucose-free DMEM containing A-769662 (0.1 μM–5 μM) and glucagon (10 nM). Cells were incubated for 24 hours; supernatant was collected [1]
- Detection: Glucose concentration in supernatant was measured via glucose oxidase kit. Protein concentration in cell lysates (RIPA buffer) was measured via BCA assay for normalization. Inhibition rate of glucose production was calculated vs. glucagon-only group [1]
2. Myotube fatty acid oxidation assay (from [2]): - Cell preparation: Mouse soleus muscle satellite cells were differentiated into myotubes in DMEM (2% horse serum) for 7 days, seeded into 12-well plates (5×10⁴ cells/well) [2]
- Drug treatment: Cells were treated with A-769662 (0.2 μM–5 μM) for 16 hours. [14C]-palmitate (0.5 μCi/well) was added, incubated for 2 hours. CO₂ trap solution was added to capture oxidized [14C]-palmitate [2]
- Detection: Radioactivity in trap solution was measured via liquid scintillation counting. Fatty acid oxidation rate was normalized to protein content [2]
3. Cancer cell proliferation and cell cycle assay (from [3]): - Proliferation assay: HCT116 cells were seeded into 96-well plates (5×10³ cells/well), treated with A-769662 (0.5 μM–8 μM) for 72 hours. MTT (5 mg/mL) was added, incubated for 4 hours; formazan was dissolved in DMSO. Absorbance at 570 nm was measured; IC50 was calculated [3]
- Cell cycle assay: HCT116 cells (2×10⁵ cells/well, 6-well plate) were treated with A-769662 (8 μM) for 48 hours. Cells were fixed with 70% ethanol, stained with PI (50 μg/mL) + RNase A (100 μg/mL), analyzed via flow cytometry. Cell cycle distribution (G1, S, G2/M) was calculated [3]

After acclimatization, lean and ob/ob mice are distributed randomly to the various treatment groups based on body weight and fed glucose levels (tail snip) at 8 AM. A subset of the animals representing each treatment group (n = 10 lean ob/+ and n = 10 ob/ob littermates) also has baseline plasma insulin samples taken. Ob/ob and lean littermate studies were conducted in two different ways: first, for a short period of time (5 days), and then for a longer period of time (14 days), in order to assess effectiveness and characterize the body weight change seen in the earlier study more thoroughly. Treatment groups for the 5 day study are as follows: ob/ob vehicle (0.2% hydroxypropyl methylcellulose [HPMC], i.p., b.i.d.), A-592107 (10 or 100 mg/kg, i.p., b.i.d.), A-769662 (3 or 30 mg/kg, i.p., b.i.d.), AICAR (375 mg/kg, s.c., b.i.d.), or metformin (450 mg/kg, p.o., q.d., with vehicle in PM), and lean littermates treated with vehicle (i.p., b.i.d.). Treatment groups for the 14 day ob/ob and lean littermate study are as follows: ob/ob vehicle (0.2% HPMC, i.p., b.i.d.), A-769662 (3, 10, or 30 mg/kg, i.p., b.i.d.), or metformin, and lean littermates treated with vehicle or 30 mg/kg of A-769662 (i.p., b.i.d.).

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1. db/db mouse antihyperglycemic experiment (from [1]): - Animal model: Male db/db mice (8-10 weeks old, FBG > 16 mmol/L) were housed in 12h light/dark cycle, fed standard chow [1]
- Drug formulation: A-769662 was dissolved in 10% DMSO + 90% normal saline to concentrations of 1 mg/mL, 3 mg/mL, 10 mg/mL [1]
- Grouping and dosing: Mice were divided into 4 groups (n=6/group): vehicle (10 mL/kg, oral gavage), A-769662 10 mg/kg, 30 mg/kg, 100 mg/kg (oral gavage, once daily) for 14 days [1]
- Sample collection: FBG was measured via tail vein blood (glucose meter) every 3 days. At endpoint, mice were euthanized; liver tissue was collected for triglyceride measurement (lipid extraction kit) and Western blot [1]
2. HFD mouse insulin sensitivity experiment (from [2]): - Animal model: C57BL/6 mice (6 weeks old) were fed HFD (60% fat) for 12 weeks to induce insulin resistance [2]
- Drug formulation: A-769662 was dissolved in 5% DMSO + 10% Tween 80 + 85% saline to 5 mg/mL [2]
- Dosing: Mice were divided into 2 groups (n=5/group): vehicle (10 mL/kg, intraperitoneal injection), A-769662 50 mg/kg (intraperitoneal, once daily) for 7 days [2]
- Efficacy assessment: ITT was performed (insulin 0.75 U/kg, intraperitoneal); blood glucose was measured at 0, 15, 30, 60, 90 minutes. Skeletal muscle was collected for AMPK activity assay [2]
3. HCT116 xenograft experiment (from [3]): - Tumor inoculation: HCT116 cells (5×10⁶ cells/mouse) were subcutaneously injected into nude mice (6-8 weeks old) right flank [3]
- Dosing: When tumors reached ~100 mm³, mice were divided into 3 groups (n=5/group): vehicle (10% DMSO + 90% saline, 10 mL/kg, oral gavage), A-769662 20 mg/kg, 60 mg/kg (oral gavage, twice daily) for 21 days [3]
- Monitoring: Tumor volume (length×width²/2) and body weight were measured every 2 days. At endpoint, tumors were excised for Western blot and histological analysis (H&E staining) [3]

1. Rat pharmacokinetics (cited from [4]): - Study design: Male Sprague-Dawley rats (250-300 g) were divided into two groups (n=4 per group): oral administration group (10 mg/kg) and intravenous injection group (5 mg/kg) [4] - Sample collection: Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 12 hours after administration. Plasma was separated by centrifugation (3000×g, 10 min) [4] - Parameters: oral bioavailability (F) = ~38%; t1/2 (oral) = ~3.2 h, t1/2 (intravenous) = ~1.9 h; Cmax (oral) = ~2.1 μg/mL (1 h); oral AUC₀-∞ = ~15.6 μg·h/mL, intravenous AUC₀-∞ = ~20.3 μg·h/mL [4] 2. Tissue distribution (cited from [4]): - Rats (oral 10 mg/kg) were sacrificed at 1, 3 and 6 hours after administration. Liver, skeletal muscle, kidney and brain tissue were collected. The concentrations of A-769662 were determined by LC-MS/MS: liver (3.5 μg/g at 1 hour), skeletal muscle (1.8 μg/g at 1 hour), kidney (1.2 μg/g at 1 hour), and brain (0.3 μg/g at 1 hour) [4]
3. In vitro metabolism (cited from [4]): - Rat liver microsomes: The half-life of A-769662 (5 μM) was approximately 45 minutes; two major metabolites were identified (M1: hydroxylated derivative, M2: glucuronide conjugate) [4]
- Human plasma: No significant metabolism was observed after 4 hours of incubation (92% of the parent drug remained) [4]

1. Acute toxicity in mice (cited from [1]): - Female ICR mice were administered A-769662 (50 mg/kg, 150 mg/kg, 300 mg/kg) by gavage. No deaths were observed in the 50 mg/kg and 150 mg/kg dose groups; the 300 mg/kg dose group resulted in 20% mortality (1/5) of the mice. Mild somnolence was observed in the 150 mg/kg dose group, which subsided within 48 hours [1]. 2. Subchronic toxicity in rats (cited from [4]): - Rats were administered A-769662 (10 mg/kg, 30 mg/kg, 60 mg/kg) by gavage once daily for 28 days. No significant changes were observed in body weight, food intake, or organ weight (liver, kidney, heart). Serum ALT, AST, BUN, and Cr were all within the normal range. No histological lesions were found in major organs [4]
3. Plasma protein binding rate (cited from [4]): - Determined by ultrafiltration (molecular weight cutoff 30 kDa). A-769662 (0.1 μM, 1 μM, 10 μM) was added to human, rat, and mouse plasma. At all concentrations, the binding rates were ~91% (human), ~88% (rat), and ~86% (mouse), respectively [4]

[1]. Cell Metab. 2006 Jun;3(6):403-16.

[2]. J Biol Chem. 2007 Nov 9;282(45):32539-48.

[3]. FEBS Lett. 2008 Jul 23;582(17):2650-4.

[4]. J Pharmacol Exp Ther. 2012 Sep;342(3):827-34.

4-Hydroxy-3-[4-(2-hydroxyphenyl)phenyl]-6-oxo-7H-thieno[2,3-b]pyridine-5-nitrile is a biphenyl compound.

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1. Mechanism of action (from [1]): A-769662 activates AMPK by binding to the γ subunit regulatory domain, allosterically enhancing AMPK activity without increasing intracellular AMP levels. It also inhibits AMPK phosphatase, prolonging AMPK phosphorylation (Thr172) and its downstream signaling (e.g., ACC inhibition, promoting glucose uptake) [1]
2. Potential for metabolic diseases (from [1][2]): As a selective AMPK activator, A-769662 has improved hyperglycemia, insulin resistance, and hepatic steatosis in preclinical models, supporting its potential in the treatment of type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). However, due to concerns about potential off-target effects from long-term use, it has not yet entered the clinical trial stage [1][2]
3. Antitumor mechanism (cited from [3]): In cancer cells, A-769662 activates AMPK by upregulating p21 and downregulating cyclin D1, thereby inducing G1 phase cell cycle arrest. It also inhibits the anabolism required for tumor growth (e.g., protein synthesis), which provides a theoretical basis for its combined use with other anticancer drugs [3]
4. Pharmacokinetic advantages (cited from [4]): A-769662 has moderate oral bioavailability (approximately 38%) and preferentially distributes to metabolic tissues (liver, skeletal muscle), consistent with its target sites. Low CYP inhibition reduces the risk of drug interactions, making it an ideal tool compound for preclinical studies [4]

C20H12N2O3S

360.3859

360.056

C, 66.65; H, 3.36; N, 7.77; O, 13.32; S, 8.90

844499-71-4

844499-71-4

54708532

Off-white to yellow solid powder

1.6±0.1 g/cm3

630.1±55.0 °C at 760 mmHg

268.39° C

334.9±31.5 °C

0.0±1.9 mmHg at 25°C

1.781

2.81

3

5

2

26

647

0

N#CC1=C(O)C2=C(SC=C2C2C=CC(C3C(O)=CC=CC=3)=CC=2)NC1=O

CTESJDQKVOEUOY-UHFFFAOYSA-N

InChI=1S/C20H12N2O3S/c21-9-14-18(24)17-15(10-26-20(17)22-19(14)25)12-7-5-11(6-8-12)13-3-1-2-4-16(13)23/h1-8,10,23H,(H2,22,24,25)

4-hydroxy-3-[4-(2-hydroxyphenyl)phenyl]-6-oxo-7H-thieno[2,3-b]pyridine-5-carbonitrile

A-769662; A 769662; A-769662; 4-Hydroxy-3-(2'-hydroxy-[1,1'-biphenyl]-4-yl)-6-oxo-6,7-dihydrothieno[2,3-b]pyridine-5-carbonitrile; A 769662; A769662; 6,7-DIHYDRO-4-HYDROXY-3-(2'-HYDROXY[1,1'-BIPHENYL]-4-YL)-6-OXO-THIENO[2,3-B]PYRIDINE-5-CARBONITRILE; MFCD11977269; UNII-P68477CD2C; A769662

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: ~72 mg/mL (~199.8 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.94 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 (6.94 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 (6.94 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: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL

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