Comparison of the tissue distribution and metabolism of AN1284, a potent anti-inflammatory agent, after subcutaneous and oral administration in mice.

This study is to compare the tissue distribution and metabolism of AN1284 after subcutaneous and oral administration at doses causing maximal reductions in IL-6 in plasma and tissues of mice. Anti-inflammatory activity of AN1284 and its metabolites was detected in lipopolysaccharide (LPS) activated RAW 264.7 macrophages. Mice were given AN1284 by injection or gavage, 15 min before LPS. IL-6 protein levels were measured after 4 h. Using a liquid chromatography/mass spectrometry method we developed, we showed that AN1284 is rapidly metabolized to the indole (AN1422), a 7-OH derivative (AN1280) and its glucuronide. AN1422 has weaker anti-inflammatory activity than AN1284 in LPS-activated macrophages and in mice. AN1284 (0.5 mg/kg) caused maximal reductions in IL-6 in the plasma, brain, and liver when injected subcutaneously and after gavage only in the liver. Similar reductions in the plasma and brain required a dose of 2.5 mg/kg, which resulted in 5.5-fold higher hepatic levels than after injection of 0.5 mg/kg, but 7, 11, and 19-fold lower ones in the plasma, brain, and kidneys, respectively. Hepatic concentrations produced by AN1284 were 2.5 mg/kg/day given by subcutaneously implanted mini-pumps that were only 12% of the peak levels seen after acute injection of 0.5 mg/kg. Similar hepatic concentrations were obtained by (1 mg/kg/day), administered in the drinking fluid. These were sufficient to decrease hepatocellular damage and liver triglycerides in previous experiments in diabetic mice. AN1284 can be given orally by a method of continuous release to treat chronic liver disease, and its preferential concentration in the liver should limit any adverse effects.

Synthesis and Biological Evaluation of Derivatives of Indoline as Highly Potent Antioxidant and Anti-inflammatory Agents.

We describe the preparation and evaluation of novel indoline derivatives with potent antioxidant and anti-inflammatory activities for the treatment of pathological conditions associated with chronic inflammation. The indolines are substituted at position 1 with chains carrying amino, ester, amide, or alcohol groups, and some have additional substituents, Cl, MeO, Me, F, HO, or BnO, on the benzo ring. Concentrations of 1 pM to 1 nM of several compounds protected RAW264.7 macrophages against H2O2 induced cytotoxicity and LPS induced elevation of NO, TNF-α, and IL-6. Several derivatives had anti-inflammatory activity at 1/100th of the concentration of unsubstituted indoline. Four compounds with ester, amine, amide, or alcohol side chains injected subcutaneously in mice at a dose of 1 µmol/kg or less, like dexamethasone (5.6 µmol/kg) prevented LPS-induced cytokine elevation in the brain and peripheral tissues. Subcutaneous injection of 100 µmol/kg of these compounds caused no noticeable adverse effects in mice during 3 days of observation.

Indoline derivatives mitigate liver damage in a mouse model of acute liver injury.

BACKGROUND: Exposure of mice to D-galactosamine (GalN) and lipopolysaccharide (LPS) induces acute liver failure through elevation of TNF-α, which causes liver damage resembling that in humans. The current study evaluated in this model the effect of two indoline derivatives, which have anti-inflammatory activity in macrophages. METHODS: AN1297 and AN1284 (0.025-0.75mg/kg) or dexamethasone (3mg/kg), were injected subcutaneously, 15min before intraperitoneal injection of GalN (800mg) plus LPS (50µg) in male Balb/C mice. After 6h, their livers were evaluated histologically by staining with hematoxylin and eosin for tissue damage and by cleaved caspase 3 for apoptosis. Activity of liver enzymes, alanine transaminase (ALT) and aspartate aminotransferase (AST) and levels of TNF-α and IL-6 were measured in plasma, and those of TNF-α and IL-6, in the liver. RESULTS: AN1297 (0.075-0.75mg/kg) and AN1284 (0.25-0.75mg/kg) maximally reduced ALT by 51% and 80%, respectively. Only AN1284 (0.25 and 0.75mg/kg) reduced AST by 41% and 48%. AN1297 and AN1284 (0.25mg/kg) decreased activation of caspase 3 (a sign of apoptosis) by 80% and plasma TNF-α by 75%. AN1297 and AN1284 (0.075mg/kg) prevented the rise in TNF-α and IL-6 in the liver. AN1284 (0.25mg/kg) reduced mortality from 90% to 20% (p<0.01) and AN1297, to 60% (p=0.121). Both indoline derivatives inhibited the phosphorylation of MAPK p38 and DNA binding of the transcription factor, AP-1. CONCLUSION: While both compounds are highly potent anti-inflammatory agents, AN1284 is more effective in mitigating the underlying causes of GalN/LPS-induced acute liver failure in mice.

Synthesis and in vitro evaluation of anti-inflammatory activity of ester and amine derivatives of indoline in RAW 264.7 and peritoneal macrophages.

UNLABELLED: A prolonged increase in pro-inflammatory cytokines, TNF-α and IL-6 occurs in inflammatory diseases. Although existing therapies like steroids and TNF-α antagonists are effective they may cause serious adverse effects. We describe the preparation and evaluation for anti-inflammatory activity of 11 novel derivatives of indoline carbamates with a propionic ester, 2-aminoethyl, 3-aminopropyl 2-(dimethylamino)ethyl or 3-(dimethylamino)propyl group in positions 3 or 1. Compounds 25, 26 and 29 were previously shown to inhibit acetylcholinesterase with IC50s ranging from 0.4 to 55µM and to prevent cytotoxicity induced by reactive oxygen species in a concentration range of 100pM-1µM. Compounds 25, 26, 29, 9, 10, 17 and 18, reduced NO, TNF-α and IL-6 at concentrations of 1-10pM in LPS-activated RAW-264.7 and mouse peritoneal macrophages. The reduction in cytokines by compound 25 was associated with an increase in IκBα degradation and a decrease in the phosphorylation of p38 but not that of ERK. CONCLUSION: Indoline derivatives substituted at position 3 with chains carrying ester or amino groups may have potential for the treatment of chronic inflammatory and neurodegenerative diseases.

Carbamate derivatives of indolines as cholinesterase inhibitors and antioxidants for the treatment of Alzheimer's disease.

The cascade of events that occurs in Alzheimer's disease involving oxidative stress and the reduction in cholinergic transmission can be better addressed by multifunctional drugs than cholinesterase inhibitors alone. For this purpose, we prepared a large number of derivatives of indoline-3-propionic acids and esters. They showed scavenging activity against different radicals in solution and significant protection against cytotoxicity in cardiomyocytes and primary cultures of neuronal cells exposed to H2O2 species and serum deprivation at concentrations ranging from 1 nM to 10 µM depending on the compound. For most of the indoline-3-propionic acid derivatives, introduction of N-methyl-N-ethyl or N-methyl-N-(4-methoxyphenyl) carbamate moieties at positions 4, 6, or 7 conferred both acetyl (AChE) and butyryl (BuChE) cholinesterase inhibitory activities at similar concentrations to those that showed antioxidant activity. The most potent AChE inhibitors were 120 (3-(2-aminoethyl) indolin-4-yl ethyl(methyl)carbamate dihydrochloride) and 94 (3-(3-methoxy-3-oxopropyl)-4-(((4-methoxyphenyl)(methyl) carbamoyl)oxy)indolin-1-ium hydrochloride) with IC50s of 0.4 and 1.2 µM, respectively.