Anti-Amyloid Effects of Small Molecule Aß-Binding Agents in PS1/APP Mice.

AIMS: One promising approach for treatment of Alzheimer's disease (AD) is use of anti-amyloid therapies, based on the hypothesis that increases in amyloid-beta (Aß) deposits in brain are a major cause of AD. Several groups have focused on Aß immunotherapy with some success. Small molecules derivatives of Congo red have been shown to inhibit Aß aggregation and protect against Aß neurotoxicity in vitro. The agents described here are all small molecule Aß-binding agents (SMAßBA's) derivatives of Congo red. MAIN METHODS: Here, we have explored the anti-amyloid properties of these SMAßBA's in mice doubly transgenic for human prensenilin-1 (PS1) and APP gene mutations that cause early-onset AD. Mice were treated with either methoxy-X04, X:EE:B34 and X:034-3-OMe1. After treatment, brains were examined for Aß-deposition, using histochemistry, and soluble and insoluble Aß levels were determined using ELISA. KEY FINDINGS: A range of anti-amyloid activity was observed with these three compounds. PS1/APP mice treated with methoxy-X04 and X:EE:B34 showed decrease in total Aß load, a decrease in Aß fibril load, and a decrease in average plaque size. Treatment with methoxy-X04 also resulted in a decrease in insoluble Aß levels. The structurally similar compound, X:034:3-OMe1, showed no significant effect on any of these measures. The effectiveness of the SMAßBA's may be related to a combination of binding affinity for Aß and entry into brain, but other factors appear to apply as well. SIGNIFICANCE: These data suggest that SMAßBA's may significantly decrease amyloid burden in brain during the pathogenesis of AD and could be useful therapeutics alone, or in combination with immunotherapy.

Chrysamine-G binding to Alzheimer and control brain: autopsy study of a new amyloid probe.

Chrysamine-G (CG) is a carboxylic acid analogue of Congo red, a histologic dye which stains amyloid. CG binds to the beta-amyloid protein of Alzheimer's disease (AD) in vitro and partitions into the brain of normal mice. In this study, we demonstrate increased binding of [14C]CG to homogenates of several regions of AD brain as compared to control. The total binding of CG to AD brain was approximately two- to three-fold that of control brain. The cerebellum could be used as an internal standard for each brain as CG binding to cerebellum did not differ between AD and control. The binding of [14C]CG correlated with numbers of senile plaques and neurofibrillary tangles. In addition, CG could be used to stain cerebrovascular amyloid in tissue sections. These results suggest that CG may prove useful as an in vivo probe of amyloid deposition in AD.

Development of small molecule probes for the beta-amyloid protein of Alzheimer's disease.

This study describes the synthesis and in vitro testing of small molecule probes that may eventually prove useful as markers of amyloid deposition in living patients. The prototype agent, Chrysamine G (CG), is a derivative of Congo red. CG binds synthetic beta-amyloid well in vitro, as does a fluorinated derivative. The mechanism of binding appears to be the same as Congo red--through a bidentate attachment spanning several amyloid peptide chains. CG is much more lipophilic than Congo red and crosses the blood-brain barrier in normal mice, achieving a brain/blood ratio over 10/1. There was no acute toxicity in mice at doses 10 times those used in the distribution studies. CG appears to be a relatively high affinity probe for beta-amyloid that appears to have low toxicity and can cross the blood-brain barrier. These characteristics are promising for development of in vivo amyloid probes similar to CG.

Chrysamine-G, a lipophilic analogue of Congo red, inhibits A beta-induced toxicity in PC12 cells.

Increasing evidence suggests that deposition of amyloid-beta (A beta) peptide leads to neurodegeneration in Alzheimer's disease. Congo red, a histologic dye that binds to amyloid has previously been shown to diminish the toxic effects of A beta in cell culture. Since Congo red is too highly charged to enter the brain in significant quantities, a lipophilic derivative, Chrysamine-G, was tested for the ability to attenuate A beta[25-35]-induced toxicity in PC12 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Chrysamine-G showed a concentration-dependent inhibition of A beta[25-35]-induced toxicity. This protective effect became significant at 0.2 microM, a concentration very close to the Ki for Chrysamine-G binding to synthetic A beta (0.37 microM). A decarboxy derivative of Chrysamine-G, which does not bind to A beta, also did not protect against A beta-induced toxicity. The protective effects of Chrysamine-G may relate to its ability to bind directly to A beta and may involve other post-binding effects as well.

Inactivity of phosphoethanolamine, an endogenous GABA analog decreased in Alzheimer's disease, at GABA binding sites.

Phosphoethanolamine (PE) is a metabolite of the phospholipid metabolism which is decreased in Alzheimer's disease brain. PE shows a strong structural similarity to the inhibitory neurotransmitter, GABA, and the GABAB receptor partial agonist, 3-amino-propylphosphonic acid. The ability of PE to compete for binding to GABAA and GABAB binding sites was investigated. GABAA sites were studied using [3H]SR-95531 and [3H]muscimol. GABAB sites were studied using [3H]GABA in the presence of isoguvacine to saturate GABAA sites. Total [3H]GABA binding was also examined. PE showed little activity at any of the GABA binding sites investigated. PE was most potent at GABAB sites, but the IC50 of 7.5 +/- 0.75 mM was considerably higher than its maximal physiologic concentration of approximately 1.5 mM. The efficient exclusion of PE from GABA binding sites may be an important physiologic mechanism in the control of inhibitory neurotransmission. The structural basis for this exclusion is discussed in reference to the GABAB partial agonist 3-amino-propylphosphonic acid.

Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity and readily enter the brain.

In vivo assessment of the beta-sheet proteins deposited in amyloid plaques (A beta peptide) or neurofibrillary tangles (tau protein) presents a target for the development of biological markers for Alzheimer's disease (AD). In an effort to develop in vivo beta-sheet imaging probes, derivatives of thioflavin-T (ThT) were synthesized and evaluated. These compounds lack the positively charged quaternary heterocyclic nitrogen of ThT and are therefore uncharged at physiological pH. They are 600-fold more lipophilic than ThT. These ThT derivatives bind to A beta(1-40) fibrils with higher affinity (Ki = 20.2 nM) than ThT (Ki = 890 nM). The uncharged ThT derivatives stained both plaques and neurofibrillary tangles in post-mortem AD brain, showing some preference for plaque staining. A carbon-11 labeled compound, [N-methyl-11C]6-Me-BTA-1, was prepared, and its brain entry and clearance were studied in Swiss-Webster mice. This compound entered the brain at levels comparable to commonly used neuroreceptor imaging agents (0.223 %ID-kg/g or 7.61 %ID/g at 2 min post-injection) and showed good clearance of free and non-specifically bound radioactivity in normal rodent brain tissue (brain clearance t(1,2) = 20 min). The combination of relatively high affinity for amyloid, specificity for staining plaques and neurofibrillary tangles in post-mortem AD brain, and good brain entry and clearance makes [N-methyl-11C]6-Me-BTA-1 a promising candidate as an in vivo positron emission tomography (PET) beta-sheet imaging agent.

Hypolipidemic activity of benzohydroxamic acids and dibenzohydroxamic acids in rodents.

A series of benzo- and dibenzohydroxamic acids were shown to have potent hypolipidemic activity in mice and rats lowering both serum cholesterol and triglyceride levels at 20 mg/kg/day. Selected derivatives lowered tissue lipids, i.e. liver, small intestine and aorta, and accelerated fecal lipid excretion in rats. The VLDL and LDL cholesterol content was reduced and HDL cholesterol was significantly elevated after 14 days administration, orally. The agents were not HMG CoA reductase inhibitors; however, other lipid regulator enzyme activities were inhibited, e.g. acyl CoA cholesterol acyl transferase, ATP-dependent citrate lyase and acetyl CoA synthetase. The triglyceride levels were probably reduced due to the derivatives inhibiting the enzymatic activities of sn-glycerol-3-phosphate acyl transferase and phosphatidylate phosphohydrolase.

Synthesis and cytotoxic action of 3,5-isoxazolidinediones and 2-isoxazolin-5-ones in murine and human tumors.

The 3,5-isoxazolidinediones and 2-isoxazolin-5-ones demonstrated potent cytotoxicity against the growth of human Tmolt3 T cell leukemia, murine P388 and L1210 leukemias, as well as human HeLa-S3 uterine carcinoma and glioma tumor cell growth. The specificity of the 3,5-isoxazolidinedione and 2-isoxazoline-5-one derivatives as cytotoxic agents varied with the histological type of tumor cell. Selected compounds were active against solid HeLa uterine. KB nasopharynx, skin A431, SW-480 adenocarcinoma, osteosarcoma and glioma growth. Selected compounds demonstrated in vivo antineoplastic activity against Ehrlich ascites carcinoma growth. In L-1210 leukemia cells, the agents blocked DNA and protein synthesis at 25, 50 and 100 microM over 60 min. The agents were effective in reducing rate limiting enzymes in the de novo purine and pyrimidine pathways. In addition they suppressed dihydrofolate reductase and ribonucleoside reductase activities with moderate inhibition of DNA and RNA polymerase activities. DNA itself was not a target of the agents.

The antineoplastic and cytotoxicity of benzohydroxamic acids and related derivatives in murine and human tumor cells.

Benzohydroxamic acids proved to be potent cytotoxic agents suppressing the growth of a number of murine and human cell lines grown in tissue culture, e.g. leukemia, colon, uterine and glioma. Selected compounds demonstrated activity against the growth KB nasopharynx, bronchogenic lung, osteosarcoma and skin cancer. In vivo activity against Ehrlich ascites carcinoma growth was shown with certain compounds. In L1210 cells compound 2 inhibited DNA synthesis significantly within 60 min. the site of action of the agent appears to involve the purine de novo synthesis pathway at PRPP amido transferase and IMP dehydrogenase. Dihydrofolate reductase and nucleoside kinase activities were inhibited by the agent. The levels of d(NTP)s in L1210 cells were reduced after drug treatment. The drug did not appear to affect the DNA template directly causing any damage which might alter transcription and replication nor was there any inhibition of HeLa topoisomerase activity by the drug. Thus the drug appears to be a metabolic inhibitor of nucleoside metabolism.

Investigation of 3,5-isoxazolidinediones as hypolipidemic agents in rodents.

A series of 2-benzoyl-4,4-dialkyl-3,5-isoxazolidinediones proved to have potent hypolipidemic activity, lowering both serum cholesterol and triglyceride levels at 10 or 20 mg/kg/day, IP and orally in rodents. 2-(3,4,5-Trimethoxybenzoyl)-4,4-diethyl-3,5-isoxazolidinedione+ ++ (4) afforded the best hypolipidemic activity lowering normolipidemic CF1 mouse serum cholesterol levels 49% and serum triglyceride levels 34% at 20 mg/kg/day, IP. Compound 4 was selected as a typical derivative of the chemical class for further detailed studies. Serum cholesterol levels in normolipidemic Sprague Dawley male rats were reduced 45% after 8 weeks at 10 and 20 mg/kg/day of compound, orally. Serum triglyceride levels were reduced 38-49% at 10 and 20 mg/kg/day, orally. In vitro liver enzyme activities studies in normolipidemic CF1 mice showed the compound inhibited mitochondrial citrate exchange, acetyl CoA synthetase, HMG CoA reductase, acyl CoA cholesterol acyl transferase, acetyl CoA carboxylase, sn-glycerol-3-phosphate acyl transferase, phosphatidylate phosphohydrolase and heparin-induced lipoprotein lipase activities with increases in the activities of cholesterol ester hydrolase and ATP-dependent citrate lyase. Similar enzyme activities were inhibited in vivo except HMG CoA reductase activity was not inhibited in rat liver or small intestinal mucosa after 8 weeks drug administration. Cholesterol levels were reduced in tissues after 8 weeks administration of compound 4 in normolipidemic rats. Bile cholesterol and triglyceride levels were elevated after two weeks administration to rats at 20 mg/kg/day. Serum lipoprotein levels in normolipidemic and hyperlipidemic rats showed the cholesterol levels in VLDL and LDL fractions after 4, 6 and 8 weeks at 10 and 20 mg/kg/day were reduced whereas HDL-cholesterol levels were significantly elevated. Studies demonstrated that 3H-cholesterol and 14C-palmitic acid incorporation into lipids of the lipoprotein fraction was reduced by the drug but 32P-incorporation was generally elevated. The agent demonstrated no observable toxicity in rats after 8 weeks administration, orally. The acute toxicity study in normolipidemic mice at 20, 40 and 100 mg/kg/day, IP, demonstrated no observable harmful effects of the drug.