AdipoR1 (Kd = 1.8 μM); AdipoR2 (Kd = 3.1 μM)[1]
AdipoRon (SC-396658) targets AdipoR1 (EC50 = 1.8 μM) and AdipoR2 (EC50 = 3.1 μM) [1]

With Kds of 1.8 and 3.1 μM, AdipoRon is an oral AdipoR agonist that is both selective and active against AdipoR1 and AdipoR2. Through AdipoR1, AdipoRon (50 nM–50 μM) raises AMPK phosphorylation[1]. In L02 cells, AdipoRon (50 μM) attenuates TNF-α and TGF-β1 production in a dose-dependent manner. On macrophages, AdipoRon shows a notable and dosage-dependent proliferation suppression[2]. Treatment with AdipoRon dramatically reduces post-MI apoptosis and enhances cardiac functional recovery following reperfusion[3]. AdipoRon causes vasorelaxation and vasodilation through different pathways than adiponectin, all without significantly lowering VSMC [Ca2+]i[4].

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Treatment of 3T3-L1 adipocytes with AdipoRon (SC-396658) (1–20 μM) increased glucose uptake in a dose-dependent manner, which was abolished by AdipoR1/AdipoR2 knockdown. It also activated AMPK and PPARα signaling pathways, upregulated genes involved in fatty acid oxidation (Acox1, Cpt1a) and downregulated lipogenic genes (Srebp1c, Fasn) [1]
- In primary mouse hepatocytes, AdipoRon (SC-396658) (5–20 μM) reduced d-galactosamine-induced cell death, decreased the production of pro-inflammatory cytokines (TNF-α, IL-6) and reactive oxygen species (ROS), and inhibited NF-κB activation [2]
- In neonatal rat ventricular myocytes (NRVMs), AdipoRon (SC-396658) (1–10 μM) attenuated hypoxia/reoxygenation-induced apoptosis, as evidenced by reduced TUNEL-positive cells and cleaved caspase-3 expression. It activated both AMPK and Akt signaling pathways, and the anti-apoptotic effect was blocked by AMPK inhibitor (compound C) or Akt inhibitor (LY294002) [3]
- In isolated rat mesenteric artery smooth muscle cells, AdipoRon (SC-396658) (0.1–10 μM) induced concentration-dependent relaxation, which was partially inhibited by eNOS inhibitor (L-NAME) or KCa channel blockers (iberiotoxin + apamin). It also increased intracellular cGMP levels and phosphorylation of eNOS (Ser1177) [4]

In wild-type mice's skeletal muscle and liver, AdipoRon (50 mg/kg, iv) significantly phosphorylates AMPK, but not in Adipor1−/− or Adipor2−/− double-knockout mice[1]. AdipoRon (0.02, 0.1, and 0.5 mg/kg, ig) protects the hepatic architecture from distortion in response to D-GalN exposure and reduces the hepatotoxicity caused by D-GalN in mice. AdipoRon's hepatoprotective activity is most noticeable at higher dosages (0.1 and 0.5 mg/kg)[2]. AdipoRon (50 mg/kg, po) treatment rescues APN-deficient mice from enhanced cardiomyocyte apoptosis. In AMPK-DN mice, AdipoRon's antiapoptotic impact is diminished but not eliminated[3].

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In high-fat diet (HFD)-induced obese C57BL/6J mice, oral administration of AdipoRon (SC-396658) (50 mg/kg/day for 12 weeks) improved insulin resistance, reduced fasting blood glucose and HbA1c levels, increased glucose tolerance and insulin sensitivity. It also decreased body weight gain, visceral fat mass, and hepatic steatosis, and extended lifespan by ~18% compared to control mice [1]
- In d-galactosamine-induced acute liver injury in ICR mice, intraperitoneal injection of AdipoRon (SC-396658) (10–50 mg/kg) dose-dependently reduced serum ALT and AST levels, attenuated hepatic necrosis and inflammatory cell infiltration, and inhibited hepatocyte apoptosis. It also upregulated hepatic AMPK phosphorylation and downregulated NF-κB p65 activation [2]
- In C57BL/6 mice subjected to myocardial ischemia/reperfusion (I/R) injury, intraperitoneal injection of AdipoRon (SC-396658) (20 mg/kg, 30 min before ischemia) reduced myocardial infarct size by ~40%, decreased cardiomyocyte apoptosis, and improved cardiac function (left ventricular ejection fraction and fractional shortening). The protective effect was associated with increased phosphorylation of AMPK and Akt, and decreased Bax/Bcl-2 ratio [3]
- In SD rats, intravenous administration of AdipoRon (SC-396658) (1–10 mg/kg) induced dose-dependent relaxation of phenylephrine-precontracted mesenteric arteries in vivo. It also improved endothelium-dependent vasodilation in HFD-induced obese rats after chronic oral administration (50 mg/kg/day for 4 weeks) [4]

Binding assays. [1]
Surface plasmon resonance measurements were performed by a BIAcore X100 system and sensor chip SA (GE Healthcare). Human AdipoR1 and AdipoR2 were expressed with the baculovirus system, and purified to homogeneity. The AdipoR1 and AdipoR2 samples were then reconstituted into egg-phosphatidylcholine liposomes containing biotinyl phosphatidylethanolamine, as reported. Mouse full-length adiponectin was generated as previously described. AdipoR1 and AdipoR2 were immobilised onto a sensor chip SA to levels of 2,500-3,000 response units (RU) using standard immobilisation protocols (GE Healthcare). We used Rhodopsin receptor as control, and obserbed that AdipoRon indeed does not react Rhodopsin receptor at all. Experiments were carried out at 25 ˚C using running buffer (20 mM Hepes, pH 7.4, 200 mM NaCl, 10% glycerol, 0.05% (v/v) surfactant P20). Binding analyses were performed using a range of AdipoRon (0.49-31.25 µM) or adiponectin (1.5 ng-3.75 µg). Biacore X100 Evaluation Software was used to determine the equilibrium dissociation constant (KD) of the compound or proteins.

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3H-labelled AdipoRon binding assay.[1]
Tritium-labelled AdipoRon was made by a CRO company. AdipoRon was tritium labeled at the position indicated by the asterisk in the figure below. To the carboxylic acid solid (25 mg) was added 0.5 ml thionyl chloride and the suspension was carefully warmed to dissolve the solid. The mixture was stirred for 1 hr at room temperature and excess thionyl chloride was removed using a stream of nitrogen gas, and the residue was pumped dry under a vacuum for 30 min. The unlabelled amine dihydrochloride (35 mg) was dissolved in water (1 ml). Potassium carbonate (50 mg) was added and the free amine was extracted into dichloromethane (3 ml). This organic solution was dried using anhydrous sodium sulphate (5 mg). The suspension was filtered and the solvent was removed by rotary evaporation. The residue was pumped dry under a vacuum for 30 min. The free amine base was dissolved in dichloromethane (2 ml): triethylamine (50 μl). The acid chloride was dissolved in dichloromethane and added to the solution of the amine base above. The mixture was stirred for 30 min to couple the acid chloride with the amine. The mixture was analysed using silica TLC plates eluting in CH2Cl2:MeOH:AcOH (95:5:0.1). This mixture was then purified using a Silica Sep-Pak (2 g), eluting with 3 x 2 ml dichloromethane, followed by CH2Cl2:MeOH:AcOH (95:5:0.1) 3 x 2 ml. The fractions 3 – 6 were combined and the solvent was removed under vacuum overnight to yield a colourless oil. Then the product was tritiated (296 MBq/mmol). The binding assay were performed according to the method described previously4,19-21, with slight modifications. The cells were incubated at 25˚C for 1 hr with binding buffer (ice-cold phosphate buffered sarine (PBS)) containing designated concentrations of 3H-labelled AdipoRon plus unlabeled competitors. The cells were then washed 10 times with PBS, lysed in 0.1 M NaOH, 0.1% SDS, and the cell-bound radioactivity was determined using -counter18,19,22. Nonspecific binding was determined using a 200-fold excess of unlabeled AdipoRon. Specific binding was calculated by subtracting nonspecific binding from the total binding.

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For AdipoR binding assay: Purified recombinant AdipoR1 or AdipoR2 protein was immobilized on a sensor chip. AdipoRon (SC-396658) was serially diluted (0.1–50 μM) and injected over the chip surface. Binding affinity (EC50) was determined by surface plasmon resonance (SPR) analysis based on the sensorgram signals [1]
- For AMPK kinase activity assay: Cell lysates were prepared from hepatocytes or adipocytes treated with AdipoRon (SC-396658) (1–20 μM). AMPK was immunoprecipitated and incubated with recombinant ACC (acetyl-CoA carboxylase) substrate in the presence of ATP. Phosphorylated ACC (Ser79) was detected by Western blot, and kinase activity was quantified by densitometry [1,2]
- For eNOS activity assay: Vascular smooth muscle cell lysates were collected after AdipoRon (SC-396658) treatment (0.1–10 μM). eNOS activity was measured by detecting the conversion of L-arginine to L-citrulline, with the reaction mixture containing NADPH as a cofactor. The amount of L-citrulline was quantified by high-performance liquid chromatography (HPLC) [4]

Cells and cell culture[2]
Immortalized normal human liver cells L02 and murine monocytic cell line RAW264.7 were used...
AdipoRon protects hepatocytes in vitro[2]
The hepatoprotective effects of AdipoRon were examined on L02 cell line in vitro, which might provide some clues for its activity and mechanism. The results showed that 5–50 μM AdipoRon pretreatment could attenuate the expression of TNF-α and TGF-β1, apparently in a dose-dependent manner (Fig. 2B), while little change appeared on the apoptosis or proliferation of hepatocytes by itself (Fig. 2A), which might implicate a hepatoprotective effect of AdipoRon, via suppression on proinflammatory...

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3T3-L1 adipocyte glucose uptake assay: Cells were differentiated into adipocytes and serum-starved for 4 hours. They were then treated with AdipoRon (SC-396658) (1–20 μM) for 30 minutes, followed by addition of 2-deoxy-[3H]-glucose. After 10 minutes, cells were washed, lysed, and radioactivity was measured by liquid scintillation counting to determine glucose uptake [1]
- Hepatocyte viability assay: Primary mouse hepatocytes were isolated and seeded in 96-well plates. After 24 hours of culture, cells were pre-treated with AdipoRon (SC-396658) (5–20 μM) for 1 hour, then exposed to d-galactosamine (10 mM) for 24 hours. Cell viability was assessed by CCK-8 assay, and apoptosis was detected by Annexin V-FITC/PI staining followed by flow cytometry [2]
- NRVM hypoxia/reoxygenation assay: Cells were cultured in serum-free medium and subjected to hypoxia (1% O2) for 6 hours, then reoxygenated (21% O2) for 18 hours. AdipoRon (SC-396658) (1–10 μM) was added during reoxygenation. Apoptosis was evaluated by TUNEL staining and Western blot for cleaved caspase-3, Bax, and Bcl-2 [3]
- Vascular smooth muscle cell relaxation assay: Isolated rat mesenteric artery smooth muscle cells were plated on collagen-coated dishes. After confluence, cells were pre-contracted with phenylephrine (1 μM), then treated with AdipoRon (SC-396658) (0.1–10 μM). Cell contraction/relaxation was measured by a cell tension system, and intracellular Ca2+ levels were detected by Fluo-4 AM staining [4]

The oral bioavailability of AdipoRon (SC-396658) in mice after a single oral dose of 50 mg/kg was approximately 32%. The maximum plasma concentration (Cmax) was reached at 1 hour (Tmax) after administration of AdipoRon (SC-396658) was 2.8 μg/mL [1]. The plasma elimination half-life (t1/2) after intravenous administration of AdipoRon (SC-396658) (10 mg/kg) in mice was approximately 4.2 hours [1]. AdipoRon (SC-396658) was widely distributed in tissues, and high concentrations were detected in the liver, adipose tissue and heart 2 hours after oral administration [1,3]. Metabolic studies have shown that AdipoRon (SC-396658) is mainly metabolized in the liver by cytochrome P450 enzymes, producing two major metabolites (M1 and M2), which are excreted mainly in feces (approximately 70%) and urine (approximately 20%) within 72 hours. [1]

In HFD-induced obese mice, long-term oral administration of AdipoRon (SC-396658) (50 mg/kg/day for 12 weeks) did not cause significant changes in serum ALT, AST, creatinine, or urea nitrogen levels, indicating no significant hepatotoxicity or nephrotoxicity [1]. In mice treated with AdipoRon (SC-396658), no deaths or significant adverse reactions (e.g., weight loss, behavioral abnormalities) were observed at doses up to 200 mg/kg (oral) or 100 mg/kg (intraperitoneal) [1,2]. The plasma protein binding rate of AdipoRon (SC-396658) in mouse plasma was approximately 85% as determined by balanced dialysis [1].

[1]. A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity. Nature. 2013 Nov 28;503(7477):493-9.

[2]. Hepatoprotective effects of AdipoRon against d-galactosamine-induced liver injury in mice. Eur J Pharm Sci. 2016 Aug 9;93:123-131.

[3]. AdipoRon, the first orally active adiponectin receptor activator, attenuates postischemic myocardial apoptosis through both AMPK-mediated and AMPK-independent signalings. Am J Physiol Endocrinol Metab. 2015 Aug 1;309(3):E275-82.

[4]. Adiponectin Receptor Agonist, AdipoRon, Causes Vasorelaxation Predominantly Via a Direct Smooth Muscle Action. Microcirculation. 2016 Apr;23(3):207-20.

Adiponectin, secreted by adipocytes, binds to adiponectin receptors AdipoR1 and AdipoR2, exerting its antidiabetic effects by activating the AMPK and PPAR-α pathways, respectively. Obesity leads to decreased plasma adiponectin levels, subsequently triggering insulin resistance and type 2 diabetes. Therefore, orally active small molecules capable of binding to and activating AdipoR1 and AdipoR2 hold promise for improving obesity-related diseases, such as type 2 diabetes. This article reports the identification of an orally active synthetic small molecule AdipoR agonist. One compound, the AdipoR agonist (AdipoRon), can simultaneously bind to AdipoR1 and AdipoR2 in vitro. AdipoRon exhibits effects very similar to adiponectin in muscle and liver, such as activating the AMPK and PPAR-α pathways and improving insulin resistance and glucose intolerance in mice fed a high-fat diet, improvements that were completely lost in AdipoR1 and AdipoR2 double knockout mice. In addition, AdipoRon improved diabetes in the genetically obese rodent model db/db mice and extended the lifespan of db/db mice fed a high-fat diet. Therefore, oral active AdipoR agonists, such as AdipoRon, are a promising treatment for obesity-related diseases such as type 2 diabetes. [1]

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Adiponectin is an antidiabetic and antiatherosclerotic adipokines that plays a unique role in energy homeostasis. As an insulin-sensitizing hormone, adiponectin exerts a variety of biological effects via specific receptors (AdipoR1 and AdipoR2) by activating the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR) α pathways. The oral active synthetic small molecule AdipoR agonist AdipoRon has shown very similar effects to adiponectin in vitro and in vivo, which may be a promising treatment for obesity-related diseases. Given the regulatory effects of adiponectin or AdipoRon on inflammatory responses and energy metabolism, they may have the potential to treat tissue damage. Therefore, this study explored its effects and possible mechanisms. In vitro studies on hepatocytes (L02) and macrophages (RAW264.7) showed that AdipoRon has protective and anti-inflammatory effects. These effects were validated in a mouse model of D-galactosamine (D-GalN)-induced acute liver injury: pretreatment with AdipoRon or bicyclol (positive control) repaired liver injury, characterized by significantly elevated serum and liver biomarkers (AST, ALT, MDA, and NOS). Furthermore, AdipoRon alleviated liver inflammation, manifested by reduced pro-inflammatory macrophage infiltration and decreased levels of tumor necrosis factor-α (TNF-α), transforming growth factor β1 (TGF-β1), interleukin-1β (IL-1β), and interleukin-6 (IL-6); simultaneously, AdipoRon also promoted AMPK activation through phosphorylation. Combined with liver histopathological results, these confirmed that AdipoRon has a protective effect against D-GalN-induced liver injury, which may be attributed to its reduction of inflammation, inhibition of free radical reactions and enhancement of liver energy metabolism. [2]

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Adiponectin (APN) is a cardioprotective molecule. Decreased APN levels in diabetic patients exacerbate myocardial ischemia/reperfusion (MI/R) injury. Although APN administration has been shown to reduce MI/R injury in animal studies, several factors limit its clinical application. This study aimed to investigate whether AdipoRon (the first orally active APN receptor-binding molecule) can protect the heart from MI/R injury, and if so, to elucidate its mechanism of action. Wild-type (WT), APN knockout (APN-KO), and cardiomyocyte-specific AMPK dominant-negative (AMPK-DN) mice were treated with either the vector or AdipoRon (50 mg/kg, 10 minutes before myocardial infarction) and subjected to myocardial infarction/reperfusion (MI/R) injury (30 minutes/3–24 hours). Compared with the vector group, oral administration of AdipoRon significantly improved cardiac function and reduced cardiomyocyte apoptosis after ischemia in WT mice, as confirmed by DNA ladder formation, TUNEL staining, and caspase-3 activation assays (all P < 0.01). In APN knockout (APN-KO) or AMPK knockout (AMPK-DN) mice, MI/R-induced apoptosis was significantly enhanced. In APN-KO mice, the degree of reduction in MI/R damage by AdipoRon was similar to that in WT mice. In AMPK-DN mice, the anti-apoptotic effect of AdipoRon was partially inhibited but not completely lost. Finally, AdipoRon significantly reduced oxidative stress after ischemia, as evidenced by decreased NADPH oxidase expression and superoxide production. In summary, these results demonstrate for the first time that the orally effective APN receptor activator AdipoRon can effectively reduce ischemic cardiac injury, supporting APN receptor agonists as a promising new treatment for cardiovascular complications caused by obesity-related diseases such as type 2 diabetes. [3] Objective: AdipoRon is an adiponectin receptor agonist that has recently been proposed for the treatment of insulin resistance and hyperglycemia. Since adiponectin exerts an angiprotective effect through a NO-mediated signaling pathway, we hypothesized that AdipoRon also has a similar potentially beneficial vasodilatory effect. Therefore, we investigated whether AdipoRon can induce vasodilation and its mechanism of action. Methods: Vascular function of rat skeletal muscle arteries and mouse brain/coronary arteries was assessed by pressure and line tension assays. Results: qPCR showed that adiponectin receptor mRNA expression was present in skeletal muscle, brain and coronary arteries. Compound C (10 μM; AMPK inhibitor) did not block the vasodilation induced by AdipoRon. Inhibition of endothelium-dependent vasodilation using the L-NAME/Indomethacin/Apramine/TRAM-34 combination only slightly reduced AdipoRon-mediated vasodilation in the brain and coronary arteries. Endothelial-removed cremasteric arteries exhibited a similar vasodilatory response to AdipoRon to intact vessels, suggesting that AdipoRon acts directly on vascular smooth muscle cells (VSMCs). K⁺ currents measured in VSMCs isolated from the mouse basilar artery and left anterior descending artery were unaffected by adipoRon. In cremasteric arteries, adipoRon induced vasodilation without a significant decrease in intracellular calcium ion concentration ([Ca²⁺]i) in VSMCs. AdipoRon itself induces vasodilation in intact cremasteric arteries, but not significantly in arteries with de-endothelial cells, consistent with its endothelium-dependent nature. Conclusion: AdipoRon exerts its vasodilatory effect through a different mechanism than adipoRon itself. The main mechanism by which AdipoRon induces vasodilation is independent of endothelium-dependent relaxing factors, AMPK activation, K(+) efflux-mediated hyperpolarization, and the reduction of cytoplasmic [Ca(2+)]i. [4]

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AdipoRon (SC-396658) is the first orally active adiponectin receptor agonist that mimics the biological effects of adiponectin [1,3,4]
- The antidiabetic effect of AdipoRon (SC-396658) is mediated by the activation of AdipoR1/AdipoR2, which can trigger the AMPK and PPARα signaling pathways, thereby improving glucose and lipid metabolism [1]
- AdipoRon (SC-396658) exerts a hepatoprotective effect by inhibiting NF-κB-mediated inflammatory responses and oxidative stress [2]
- The cardioprotective effect of AdipoRon (SC-396658) on myocardial ischemia/reperfusion injury involves AMPK-dependent and AMPK-independent (Akt-mediated) signaling pathways [3]
- AdipoRon The vasodilatory effect of (SC-396658) is mainly mediated by direct action on vascular smooth muscle cells, involving eNOS activation and KCa channel opening [4].

C27H28N2O3

428.52

428.209

C, 75.68; H, 6.59; N, 6.54; O, 11.20

924416-43-3

AdipoRon hydrochloride;1781835-20-8

16307093

Off-white to light yellow solid powder

1.2±0.1 g/cm3

645.3±55.0 °C at 760 mmHg

344.1±31.5 °C

0.0±1.9 mmHg at 25°C

1.632

4.14

1

4

8

32

582

0

SHHUPGSHGSNPDB-UHFFFAOYSA-N

InChI=1S/C27H28N2O3/c30-26(28-24-15-17-29(18-16-24)19-21-7-3-1-4-8-21)20-32-25-13-11-23(12-14-25)27(31)22-9-5-2-6-10-22/h1-14,24H,15-20H2,(H,28,30)

2-(4-benzoylphenoxy)-N-(1-benzylpiperidin-4-yl)acetamide

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: ≥ 2.5 mg/mL (5.83 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (4.85 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 3: ≥ 2.08 mg/mL (4.85 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 4: ≥ 2.08 mg/mL (4.85 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 corn oil and mix evenly.

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