- Email: [email protected]
Neuropathologic correlates of trial-related clinical assessments
S142
Oral O3-07: Developing Therapeutics
Ab plaque burden. Catalytically incompetent 2E6 treated with a protease inhibitor and a non-proteolytic control IgVL2 did not express these activities. The major 2E6-cleavage site in Ab was the His14-Gln15 peptide bond. Epitope mapping indicated competitive inhibition of 2E6-catalyzed 125I-Ab degradation by the remote Ab29-40 peptide, identifying this region as the noncovalent recognition epitope. 2E6-Ab immune complexes were undetectable by ELISA, consistent with rapid progress of the reaction to the catalysis step. 2E6 did not cleave His-Gln containing proteins, indicating that specificity for Ab derives from the noncovalent binding step. Conclusions: The Ab fragment targets monomer and aggregate forms of Ab and clears brain Ab plaques by initial noncovalent recognition of the amyloidogenic C terminal region followed by cleavage at remote peptide bonds. Catalysis is rapid and no immune complexes are detectable. This catalytic antibody fragment can be developed for more effective and safe AD immunotherapy. O3-07-02
NOVEL, SMALL MOLECULE INHIBITORS OF PROTEIN AGGREGATION FOR TREATMENT OF AMYLOID-RELATED DISEASES
Gal Bitan1, Sharmistha Sinha1, Aida Attar1, Panchanan Maiti1, Miao Tan1, Shubhangi Prabhudesai1, Peter Talbiersky2, Reena Bakshi1, Pei-Yi Kuo1, Fusheng Yang1, Dana Gant1, Mychica R. Jones1, Cui-Wei Xie1, Jeff M. Bronstein1, Sally A. Frautschy1, Frank-Gerrit Kla¨rner2, Thomas Schrader2, 1UCLA, Los Angeles, CA, USA; 2University of Duisburg-Essen, Essen, Germany. Contact e-mail: [email protected] Background: Aberrant protein folding and assembly causes >30 diseases, termed amyloidoses, for none of which there is a cure. Alzheimer’s and Parkinson’s diseases are two prominent examples. In each disease, particular proteins self-assemble into toxic oligomers and polymers that disrupt cellular function and lead to cell death. Developing inhibitors of aberrant assembly is difficult. In particular, the oligomers, which are the most toxic species, are highly challenging targets because they do not have a stable structure. Methods: Using a rational approach, we have developed small molecules that inhibit aberrant protein assembly in a process-specific, rather than a protein-specific manner. The new inhibitors bind to specific amino acid residues and block a combination of electrostatic and hydrophobic interactions that mediate assembly and toxicity. We used an array of biophysical and biological methods, including in vitro and in vivo assays to test the efficacy of the inhibitors. Results: Our lead compound, CLR01, inhibits the aggregation and toxicity of at least seven distinct, disease-related, amyloid proteins in vitro. In vivo, CLR01 effectively and significantly reduces Ab load in transgenic mouse brain and rescues zebrafish from a-synuclein-induced toxicity. Intracellular a-synuclein is maintained soluble enabling its degradation by the proteasome. Several in vitro assays show that CLR01 has low toxicity and a good therapeutic index. In vivo, using multiple doses no side effects have been observed to date. Conclusions: Our data demonstrate that inhibition of aberrant protein aggregation in vivo enables clearance of the offending proteins simply by keeping them soluble. In the soluble form in the presence of CLR01, the proteins are not toxic. Taken together, the data suggest that CLR01 is a promising candidate for development of effective prevention and threatment for amyloidoses in general, and for Alzheimer’s and Parkinson’s diseases in particular. O3-07-03
A NOVEL MULTIVALENT ABETA PEPTIDE VACCINE WITH PRECLINICAL EVIDENCE OF A CENTRAL IMMUNE RESPONSE THAT GENERATES ANTISERA RECOGNIZING A WIDE RANGE OF ABETA PEPTIDE SPECIES
Mary J. Savage, Guoxin Wu, Alexander McCampbell, Keith R. Wessner, Michael Citron, Xiaoping Liang, Sidney Hsieh, Abigail L. Wolfe, Gene G. Kinney, Laura B. Rosen, John J. Renger, Merck & Company, West Point, PA, USA. Contact e-mail: [email protected] Background: Preclinical and clinical data support a vaccination approach to reduce levels of amyloid (Ab) peptide in the brains of Alzheimer’s Dis-
ease (AD) patients. Many Ab regions could drive an immune response, however, vaccine antigens should target the most toxic Ab species, without a T-cell response. In addition, the degree to which antibodies generated in response to Ab vaccines enter the central nervous system (CNS) following peripheral immunization is not fully understood. We sought to define the Ab species recognized by antisera following immunization with a multivalent Ab peptide vaccine and the degree of antibody response detected in the CNS compared to the blood. Methods: Guinea pigs and Rhesus monkeys were immunized with vaccine, followed by several boosts. Animals were immunized ranging from 7 to 33 weeks. Plasma and cerebrospinal fluid (CSF) were analyzed for Ab peptide concentration. Serum and CSF were analyzed for levels of amyloid IgG. Serum from both immunized species were tested in a one-site ELISA for reactivity to a panel of full length and truncated Ab peptides; versions of which have been isolated from AD brain. Results: After excluding CSF samples contaminated by serum, the levels of CSF Ab IgG that were measured in both species ranged between 0.07-0.1 % of the level present in serum, based on relative titer measurements. These relative titer ratios were independent of the absolute level of serum titers, which ranged between 104 and 106. Total Ab levels in the plasma were elevated many-fold after V950 immunization, while CSF total Ab levels were not significantly different compared to baseline. The antisera from both species recognized many forms of N-terminally truncated Ab, including species that are modified to include pyroglutamate. Conclusions: Following immunization of Guinea pig and Rhesus monkey with a multivalent Ab vaccine, Ab antibodies were present in the CSF as well as the serum. Antibodies present locally in the CNS may allow additional pathways for Ab clearance beyond the proposed peripheral sink. Several vaccines now in clinical trial target an extreme N-terminal epitope of Ab. A vaccine which targets multiple Ab epitopes may bind and clear toxic Ab species more effectively.
O3-07-04
NEUROPATHOLOGIC CORRELATES OF TRIAL-RELATED CLINICAL ASSESSMENTS
Jeffrey L. Cummings, Cathy Lee, UCLA, Los Angeles, CA, USA. Contact e-mail: [email protected] Background: Disease-modifying agents are being developed to treat Alzheimer’s disease (AD). These agents target the neuropathology of AD such as amyloid and tau. These pathologies are reflected at autopsy in plaques and tangles. Clinical assessments reflect the success in altering these pathologies. We examined the relationship of the commonly used clinical trial assessments to neuropathological findings using data in the National Alzheimer Coordinating Center (NACC) database. Methods: We analyzed data from 169 patients in the NACC database who had been assessed with trial-related instruments and who had come to autopsy. We examined the relationships between the neuropathologic findings and the clinical measures. Results: Correlations between MMSE (last visit) and neuritic plaques, diffuse plaques, and Braak staging were highly significant (p ¼ 0.0001). The Uniform Data Set (UDS) memory score was highly correlated with neuritic plaques and Braak staging (p ¼ 0.0001). The Clinical Dementia Rating (CDR) was highly correlated with neuritic plaques and Braak staging (p ¼ 0.0001). The Functional Activity Questionnaire (FAQ) a measure of Instrumental Activities of Daily Living was correlated with neuritic plaques and Braak staging (p ¼ 0.0001). The Neuropsychiatric Inventory (NPI) was correlated with Braak staging (p ¼ 0.00). Conclusions: There are strong statistical correlations between clinical trial-related assessments captured at the last visit and the neuropathologic findings at autopsy. Cognitive and functional instruments correlate with plaques and tangles; behavioral measures correlate with tangles. The variance accounted for by the correlations is modest suggesting that other processes (e.g., cell death) are contributing importantly to the clinical outcomes.
Oral O3-07: Developing Therapeutics
Ab plaque burden. Catalytically incompetent 2E6 treated with a protease inhibitor and a non-proteolytic control IgVL2 did not express these activities. The major 2E6-cleavage site in Ab was the His14-Gln15 peptide bond. Epitope mapping indicated competitive inhibition of 2E6-catalyzed 125I-Ab degradation by the remote Ab29-40 peptide, identifying this region as the noncovalent recognition epitope. 2E6-Ab immune complexes were undetectable by ELISA, consistent with rapid progress of the reaction to the catalysis step. 2E6 did not cleave His-Gln containing proteins, indicating that specificity for Ab derives from the noncovalent binding step. Conclusions: The Ab fragment targets monomer and aggregate forms of Ab and clears brain Ab plaques by initial noncovalent recognition of the amyloidogenic C terminal region followed by cleavage at remote peptide bonds. Catalysis is rapid and no immune complexes are detectable. This catalytic antibody fragment can be developed for more effective and safe AD immunotherapy. O3-07-02
NOVEL, SMALL MOLECULE INHIBITORS OF PROTEIN AGGREGATION FOR TREATMENT OF AMYLOID-RELATED DISEASES
Gal Bitan1, Sharmistha Sinha1, Aida Attar1, Panchanan Maiti1, Miao Tan1, Shubhangi Prabhudesai1, Peter Talbiersky2, Reena Bakshi1, Pei-Yi Kuo1, Fusheng Yang1, Dana Gant1, Mychica R. Jones1, Cui-Wei Xie1, Jeff M. Bronstein1, Sally A. Frautschy1, Frank-Gerrit Kla¨rner2, Thomas Schrader2, 1UCLA, Los Angeles, CA, USA; 2University of Duisburg-Essen, Essen, Germany. Contact e-mail: [email protected] Background: Aberrant protein folding and assembly causes >30 diseases, termed amyloidoses, for none of which there is a cure. Alzheimer’s and Parkinson’s diseases are two prominent examples. In each disease, particular proteins self-assemble into toxic oligomers and polymers that disrupt cellular function and lead to cell death. Developing inhibitors of aberrant assembly is difficult. In particular, the oligomers, which are the most toxic species, are highly challenging targets because they do not have a stable structure. Methods: Using a rational approach, we have developed small molecules that inhibit aberrant protein assembly in a process-specific, rather than a protein-specific manner. The new inhibitors bind to specific amino acid residues and block a combination of electrostatic and hydrophobic interactions that mediate assembly and toxicity. We used an array of biophysical and biological methods, including in vitro and in vivo assays to test the efficacy of the inhibitors. Results: Our lead compound, CLR01, inhibits the aggregation and toxicity of at least seven distinct, disease-related, amyloid proteins in vitro. In vivo, CLR01 effectively and significantly reduces Ab load in transgenic mouse brain and rescues zebrafish from a-synuclein-induced toxicity. Intracellular a-synuclein is maintained soluble enabling its degradation by the proteasome. Several in vitro assays show that CLR01 has low toxicity and a good therapeutic index. In vivo, using multiple doses no side effects have been observed to date. Conclusions: Our data demonstrate that inhibition of aberrant protein aggregation in vivo enables clearance of the offending proteins simply by keeping them soluble. In the soluble form in the presence of CLR01, the proteins are not toxic. Taken together, the data suggest that CLR01 is a promising candidate for development of effective prevention and threatment for amyloidoses in general, and for Alzheimer’s and Parkinson’s diseases in particular. O3-07-03
A NOVEL MULTIVALENT ABETA PEPTIDE VACCINE WITH PRECLINICAL EVIDENCE OF A CENTRAL IMMUNE RESPONSE THAT GENERATES ANTISERA RECOGNIZING A WIDE RANGE OF ABETA PEPTIDE SPECIES
Mary J. Savage, Guoxin Wu, Alexander McCampbell, Keith R. Wessner, Michael Citron, Xiaoping Liang, Sidney Hsieh, Abigail L. Wolfe, Gene G. Kinney, Laura B. Rosen, John J. Renger, Merck & Company, West Point, PA, USA. Contact e-mail: [email protected] Background: Preclinical and clinical data support a vaccination approach to reduce levels of amyloid (Ab) peptide in the brains of Alzheimer’s Dis-
ease (AD) patients. Many Ab regions could drive an immune response, however, vaccine antigens should target the most toxic Ab species, without a T-cell response. In addition, the degree to which antibodies generated in response to Ab vaccines enter the central nervous system (CNS) following peripheral immunization is not fully understood. We sought to define the Ab species recognized by antisera following immunization with a multivalent Ab peptide vaccine and the degree of antibody response detected in the CNS compared to the blood. Methods: Guinea pigs and Rhesus monkeys were immunized with vaccine, followed by several boosts. Animals were immunized ranging from 7 to 33 weeks. Plasma and cerebrospinal fluid (CSF) were analyzed for Ab peptide concentration. Serum and CSF were analyzed for levels of amyloid IgG. Serum from both immunized species were tested in a one-site ELISA for reactivity to a panel of full length and truncated Ab peptides; versions of which have been isolated from AD brain. Results: After excluding CSF samples contaminated by serum, the levels of CSF Ab IgG that were measured in both species ranged between 0.07-0.1 % of the level present in serum, based on relative titer measurements. These relative titer ratios were independent of the absolute level of serum titers, which ranged between 104 and 106. Total Ab levels in the plasma were elevated many-fold after V950 immunization, while CSF total Ab levels were not significantly different compared to baseline. The antisera from both species recognized many forms of N-terminally truncated Ab, including species that are modified to include pyroglutamate. Conclusions: Following immunization of Guinea pig and Rhesus monkey with a multivalent Ab vaccine, Ab antibodies were present in the CSF as well as the serum. Antibodies present locally in the CNS may allow additional pathways for Ab clearance beyond the proposed peripheral sink. Several vaccines now in clinical trial target an extreme N-terminal epitope of Ab. A vaccine which targets multiple Ab epitopes may bind and clear toxic Ab species more effectively.
O3-07-04
NEUROPATHOLOGIC CORRELATES OF TRIAL-RELATED CLINICAL ASSESSMENTS
Jeffrey L. Cummings, Cathy Lee, UCLA, Los Angeles, CA, USA. Contact e-mail: [email protected] Background: Disease-modifying agents are being developed to treat Alzheimer’s disease (AD). These agents target the neuropathology of AD such as amyloid and tau. These pathologies are reflected at autopsy in plaques and tangles. Clinical assessments reflect the success in altering these pathologies. We examined the relationship of the commonly used clinical trial assessments to neuropathological findings using data in the National Alzheimer Coordinating Center (NACC) database. Methods: We analyzed data from 169 patients in the NACC database who had been assessed with trial-related instruments and who had come to autopsy. We examined the relationships between the neuropathologic findings and the clinical measures. Results: Correlations between MMSE (last visit) and neuritic plaques, diffuse plaques, and Braak staging were highly significant (p ¼ 0.0001). The Uniform Data Set (UDS) memory score was highly correlated with neuritic plaques and Braak staging (p ¼ 0.0001). The Clinical Dementia Rating (CDR) was highly correlated with neuritic plaques and Braak staging (p ¼ 0.0001). The Functional Activity Questionnaire (FAQ) a measure of Instrumental Activities of Daily Living was correlated with neuritic plaques and Braak staging (p ¼ 0.0001). The Neuropsychiatric Inventory (NPI) was correlated with Braak staging (p ¼ 0.00). Conclusions: There are strong statistical correlations between clinical trial-related assessments captured at the last visit and the neuropathologic findings at autopsy. Cognitive and functional instruments correlate with plaques and tangles; behavioral measures correlate with tangles. The variance accounted for by the correlations is modest suggesting that other processes (e.g., cell death) are contributing importantly to the clinical outcomes.