In vivo characterization of Abeta(40) changes in brain and cerebrospinal fluid using the novel gamma-secretase inhibitor N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) in the rat.

Plaques in the parenchyma of the brain containing Abeta peptides are one of the hallmarks of Alzheimer's disease. These Abeta peptides are produced by the final proteolytic cleavage of the amyloid precursor protein by the intramembraneous aspartyl protease gamma-secretase. Thus, one approach to lowering levels of Abeta has been via the inhibition of the gamma-secretase enzyme. Here, we report a novel, bioavailable gamma-secretase inhibitor, N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) that displayed oral pharmacokinetics suitable for once-a-day dosing. It was able to markedly reduce Abeta in the brain and cerebrospinal fluid (CSF) in the rat, with ED(50) values of 6 and 10 mg/kg, respectively. Time-course experiments using MRK-560 demonstrated these reductions in Abeta could be maintained for 24 h, and comparable temporal reductions in rat brain and CSF Abeta(40) further suggested that these two pools of Abeta are related. This relationship between the brain and CSF Abeta was maintained when MRK-560 was dosed once a day for 2 weeks, and accordingly, when all the data for the dose-response curve and time courses were correlated, a strong association was observed between the brain and CSF Abeta levels. These results demonstrate that MRK-560 is an orally bioavailable gamma-secretase inhibitor with the ability to markedly reduce Abeta peptide in the brain and CSF of the rat and confirm the utility of the rat for assessing the effects of gamma-secretase inhibitors on central nervous system Abeta(40) levels in vivo.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in viral meningitis: upregulation of MMP-9 and TIMP-1 in cerebrospinal fluid.

A hallmark of viral meningitis is the invasion of monocytes, lymphocytes and, in the initial phase of the disease, neutrophils into the subarachnoidal space. By their degradation of different macromolecular components in the extracellular connective tissue, matrix metalloproteinases (MMPs) may be essential for the breakdown of the vessel wall in the meninges and the choroid plexus. In this study, the occurrence of MMP-1, MMP-2, MMP-3 and MMP-9 and the two tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, was monitored in the cerebrospinal fluid (CSF) from patients with viral meningitis. Of the proteinases, MMP-9 was found in 13 of 39 (33%) patients, but not in controls; the levels being correlated with the neutrophil cell number in CSF. The CSF concentration of TIMP-1 was increased three-fold compared to the control group (median 233 ng/ml; range 9.4-1252.5 ng/ml) and was correlated to the levels of total protein in CSF. Of the other MMPs and TIMPs assayed, MMP-2 and TIMP-2 were constitutively expressed and not upregulated in viral meningitis. High levels of MMP-9 and MMP-2, as measured by ELISA, was associated with high proteolytic activity detected in CSF by zymography. In conclusion, invasion of the leukocytes into the CSF compartment in viral meningitis may involve MMP-9, its proteolytic effect likely being controlled by expression of TIMP-1.

Attenuation of experimental subarachnoid hemorrhage-induced cerebral vasospasm by CGS 26303, an endothelin-converting enzyme inhibitor.

The effect of CGS 26303, an endothelin-converting enzyme inhibitor, on the prevention and reversal of cerebral vasospasm was investigated in a rabbit model of subarachnoid hemorrhage (SAH). In the prevention study, rabbits were injected with 3 ml of autologous blood in the cisterna magna and treatment with CGS 26303 i.v. was initiated 1 h later. The compound was subsequently administered at 12, 24, and 36 h post SAH and animals were sacrificed at 48 h post SAH. Treatment with CGS 26303 at 3, 10, and 30 mg/kg resulted in dose-dependent increases in the concentrations of the compound in cerebrospinal fluid samples, and the arterial narrowing after SAH was significantly attenuated in all three groups. Morphologically, corrugation of the internal elastic lamina of vessels was often observed in the vehicle-treated group, but it was not prominent in the CGS 26303-treated groups and the healthy controls. In the reversal study, treatment with CGS 26303 was initiated 24 h post SAH and a second injection was given 12 h later. Arterial narrowing was significantly attenuated in rabbits treated with CGS 26303 at 30 mg/kg. These results demonstrate that CGS 26303 may be an effective agent in prevention and reversal of cerebral vasospasm after aneurysmal SAH.

Investigation of inhibition angiotensin-converting enzyme (ACE) activity and opioid activity of two hemorphins, LVV-hemorphin-5 and VV-hemorphin-5, isolated from a defined peptic hydrolysate of bovine hemoglobin.

Two peptides, LVV-hemorphin-5 and VV-hemorphin-5, were isolated from a defined peptic bovine hemoglobin hydrolysate by reversed-phase HPLC. These peptides were identified as 31-38 and 32-38 fragments of beta chain of bovine hemoglobin. Their inhibitory activity towards angiotensin-converting enzyme and opioid potency were determined. Since their amino acid sequences show close homology with spinorphin, which is found in human cerebrospinal fluid and in the bovine spinal cord, the possible physiological role in vivo of these peptides was discussed.

Laboratory examinations correlated with severity of dementia.

Acetylcholine esterase, alpha 1-antichymotrypsin, homovanillic acid (HVA), 3-methoxy-4-hydroxy-phenylenglycol (MHPG), norepinephrine, dopamine, 5-hydroxy-indolacetic acid (5-HIAA), and gamma-aminobutyric acid (GABA) in the serum and cerebrospinal fluid (CSF) were quantified. Positive wave with the latency about 300 msec (P300) and electroencephalography (EEG) were examined in 10 patients with Alzheimer's disease, 10 patients with vascular dementia, and 10 age-matched healthy controls. Serum alpha 1-antichymotrypsin concentrations were significantly higher in the Alzheimer's disease group than in the vascular dementia and healthy control groups. Homovanillic acid concentrations in CSF were significantly lower in the vascular dementia group than in the Alzheimer's disease and the healthy control groups. A significant positive correlation was present between the mini-mental state examination (MMSE) score (normal range of 24 to 30) and the acetylcholine esterase concentration in the CSF. Significant negative correlations were present between the MMSE score and the P300 latency, between the MMSE score and the MHPG concentration in the CSF, between the MMSE and the norepinephrine concentration in the CSF, and between the MMSE score and the dopamine concentration in the CSF.

Proteinase-antiproteinase imbalance in meningitis: determination of alpha 1 proteinase inhibitor (alpha 1PI), elastase-alpha 1PI complex, and elastase inhibition capacity in cerebrospinal fluid.

Mortality and long-term neurologic sequelae are still frequent complications of meningitis despite effective antibiotic treatment. This suggests that pathogen-independent inflammatory mechanisms may play an important role in the course of this illness. Neutrophil granulocytes form the primary immune defense in meningitis. Once activated, these cells release elastase into the cerebrospinal fluid (CSF). Elastase may induce tissue damage if local antiproteinase capacity is low as under normal conditions. To define the relevance of this mechanism we studied 22 patients with meningitis. Concentrations of elastase in complex with the main antiproteinase alpha 1-proteinase inhibitor (elastase-alpha 1 PI), alpha 1-proteinase inhibitor (alpha 1PI), and elastase inhibition capacity (EIC) were measured in CSF of 9 patients with bacterial meningitis (BM), aged 1 month-14 years; 13 patients with non-bacterial meningitis (NBM), aged 1 month-15 years; and 20 patients in whom meningitis was excluded after spinal tap (control group), aged 6 months-15 years. The concentration of elastase-alpha 1PI in the BM group (median 552 micrograms/l) was significantly higher than in either the NBM group (median 30 micrograms/l, p less than 0.01) or the control group (median 30 micrograms/l, p less than 0.01). Similarly, the alpha 1PI-concentration in the BM group was significantly higher (median 113 mg/l) than either the NBM group (median 13.7 mg/l, p less than 0.025) or the control group (median 6.3 mg/l, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble derivatives of the beta amyloid protein precursor in cerebrospinal fluid: alterations in normal aging and in Alzheimer's disease.

We isolated and sequenced a soluble approximately 25 kDa amino-terminal derivative of the beta amyloid protein precursor (beta APP) that is readily detected in human cerebrospinal fluid (CSF). In CSF samples from 24 Alzheimer's disease (AD) patients and 12 controls, we then quantitated this approximately 25 kDa form as well as the approximately 125 and approximately 105 kDa derivatives that we previously identified. Our analysis shows (1) that, in AD, there is a significant decrease in the relative amount of the approximately 105 kDa form and a corresponding significant increase in the relative amount of the approximately 25 kDa form; (2) that these changes correlate with the mental status of the AD patients; and (3) that the same changes occur to a lesser extent in elderly as compared with young control patients. These observations indicate that processing of the beta APP changes in normal individuals as they age and to a greater extent in those who develop AD. The changes in beta APP derivatives that we have observed in CSF could have major implications because they may reflect fundamental mechanisms responsible for amyloid deposition and can be measured in living patients.

Determination of amyloid beta protein precursors harboring active form of proteinase inhibitor domains in cerebrospinal fluid of Alzheimer's disease patients by trypsin-antibody sandwich ELISA.

beta-Amyloid protein precursors (APP) having proteinase inhibitor domains (APPI) were quantified by a new sandwich enzyme linked immunosorbent assay for detection of active (free) form of proteinase inhibitors by using trypsin in place of the first antibody and by denaturation of APPI-trypsin complex in the microtiterplate. The concentration of APPs having APPI in cerebrospinal fluid of Alzheimer's disease patients was found, by this method, to be significantly elevated compared with those of multi-infarct dementia.

Mutation in cystatin C gene causes hereditary brain haemorrhage.

Hereditary cystatin C amyloid angiopathy (HCCAA) is an autosomal dominant disorder in which a cysteine proteinase inhibitor, cystatin C, is deposited as amyloid fibrils in the cerebral arteries of patients and leads to massive brain haemorrhage and death in young adults. A full length cystatin C cDNA probe revealed a mutation in the codon for leucine at position 68 which abolishes an Alu I restriction site in the cystatin C gene of HCCAA patients. The Alu I marker has been used to show that this mutation is transmitted only in affected members of all eight families investigated, and that the mutated cystatin C gene causes HCCAA.

Elastase-alpha 1-proteinase inhibitor: an early indicator of septicemia and bacterial meningitis in children.

In 26 infants and children with septicemia or bacterial meningitis, significantly elevated plasma levels of elastase-alpha 1-proteinase inhibitor (E-alpha 1-PI) were present at time of recognition of infection, even in those patients with neutropenia (range of reference values: 25 to 190 micrograms/L, n = 142; patients: 444 to 2049 micrograms/L, n = 26). After initiation of therapy, normalization of E-alpha 1-PI levels was observed in all patients who recovered from infection. In addition, 18 of 19 children with bacterial meningitis had increased cerebrospinal fluid concentrations of E-alpha 1-PI above the range of normal (range of reference values: 0 to 39 micrograms/L, n = 62; patients: 30 to 3490 micrograms/L, n = 19); concentrations of E-alpha 1-PI in bacterial meningitis were significantly increased when compared with those in aseptic meningitis (range 25 to 194 micrograms/L; n = 15). In 30 patients with local bacterial infections (pneumonia, urinary tract infections, etc.), E-alpha 1-PI was also elevated. These data suggest that E-alpha 1-PI is a sensitive indicator of systemic and local bacterial infection in childhood.

Proteinase inhibitors in cerebrospinal fluid in multiple sclerosis.

Levels of the proteinase inhibitors alpha 2-macroglobulin (alpha 2-m) and alpha 1-antitrypsin (alpha 1-at), and total protein, IgG and transferrin were measured in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and other neurological diseases. All groups except that termed "meningitis" had similar alpha 2-m levels, but alpha 1-at and transferrin were significantly depressed in MS. Total protein levels were normal and IgG levels were elevated in MS. Serum levels of alpha 1-at were normal so the decreases observed in the CSF in MS were not due to impaired systemic production. In view of previous reports that proteinase activity is high in MS plaques and CSF, the inhibitory capacity of alpha 2-m and alpha 1-at in CSF was measured. As any decreases in inhibitory capacity noted in MS were slight, they could only be important in the local environment of a plaque where enzyme levels may be critically high.

Proteolytic enzyme activity in patients with severe head injury and the effect of a proteinase inhibitor.

A study was performed to detect the inhibitory effect of intravenously administered aprotinin (Trasylol) on brain and CSF protease activity in 25 patients with severe head injury. The data presented include measurements of CSF protease activity, alpha-1-antitrypsin, alpha-2-macroglobulin, haptoglobulin, polyacrylamidgel-electrophoresis pattern, total protein and hemoglobin content. The results indicate that increased protease activity is present and that this induces autolytic processes which can be inhibited by aprotinin treatment. The survival rate was higher after aprotinin treatment. Total CSF protein content was significantly higher in nonsurvivors than in survivors.