CTAD Task Force Paper: Neuropsychiatric Symptoms in AD: Clinical Trials Targeting Mild Behavioral Impairment: A Report from the International CTAD Task Force.

The International CTAD Task Force (TF) addressed challenges related to designing clinical trials for agitation in dementia, presenting accomplishments from the two previous TFs on neuropsychiatric symptoms (NPS). In addition, this TF proposed a paradigm shift in NPS assessment and management, presenting Mild Behavioral Impairment (MBI) as a clinical syndrome. MBI is marked by later-life emergent and persistent NPS in dementia-free older persons (ranging from cognitively unimpaired to subjective cognitive decline to mild cognitive impairment), which facilitates earlier detection and better prognostication of Alzheimer's disease (AD). The TF has made the following recommendations for incorporation of NPS into AD preventative trials: (1) clinical trials targeting improvement in MBI symptoms should be undertaken; (2) treatment trials for MBI should be disease specific and confirm the diagnosis of participants using biomarkers; trials should include measures sensitive to cognitive changes in preclinical AD, which can serve as outcome measures, in addition to changes in biomarker levels; (3) as a first step, pharmacotherapeutic trials should address the full MBI complex as well as the specific symptoms/domains that constitute MBI; (4) clinical trials using problem-adaptation psychotherapy to target affective MBI should be considered; and (5) MBI should be considered in AD trials of disease modifying therapies. The well-validated and widely-used MBI Checklist (MBI-C) is an appropriate symptom rating scale for these studies, as it was developed specifically to identify and measure MBI in dementia-free persons. Other scales such as the Neuropsychiatric Inventory (NPI) may be used, although administration at two timepoints may be necessary to operationalize the MBI criterion of symptom persistence.

Clinical Meaningfulness in Alzheimer's Disease Clinical Trials. A Report from the EU-US CTAD Task Force.

Recent positive results of three phase III anti-amyloid monoclonal antibody trials are transforming the landscape of disease-modifying therapeutics for Alzheimer's disease, following several decades of failures. Indeed, all three trials have met their primary endpoints. However, the absolute size of the benefit measured in these trials has generated a debate on whether the change scores observed on clinical outcome assessments represent a clinically meaningful benefit to patients. An evidence-based conclusion is urgently required to inform decision-making related to the approval, reimbursement, and ultimately, the management of emerging therapies in clinical practice. The EU-US CTAD Task Force met in Boston to address this important question. The current state-of-the-art knowledge for interpreting clinical meaningfulness of AD clinical trial results, including the point of view of patients and study partners on what is clinically meaningful, was discussed and is summarized here. A combination of methodologies to address the challenges emerged. There remain gaps in the understanding of clinical meaningfulness that only long-term longitudinal studies will be able to address.

Can We Use Blood Biomarkers as Entry Criteria and for Monitoring Drug Treatment Effects in Clinical Trials? A Report from the EU/US CTAD Task Force.

In randomized clinical trials (RCTs) for Alzheimer's Disease (AD), cerebrospinal fluid (CSF) and positron emission tomography (PET) biomarkers are currently used for the detection and monitoring of AD pathological features. The use of less resource-intensive plasma biomarkers could decrease the burden to study volunteers and limit costs and time for study enrollment. Blood-based markers (BBMs) could thus play an important role in improving the design and the conduct of RCTs on AD. It remains to be determined if the data available on BBMs are strong enough to replace CSF and PET biomarkers as entry criteria and monitoring tools in RCTs.

The Institute on Methods and Protocols for Advancement of Clinical Trials in ADRD (IMPACT-AD): A Novel Clinical Trials Training Program.

BACKGROUND: Alzheimer's Disease and Related Dementias (ADRD) clinical trials require multidisciplinary expertise in medicine, biostatistics, trial design, biomarkers, ethics, and informatics. OBJECTIVES: To provide focused interactive training in ADRD clinical trials to a diverse cadre of investigators. DESIGN: The Institute on Methods and Protocols for Advancement of Clinical Trials in ADRD (IMPACT-AD) is a novel multidisciplinary clinical trial training program funded by the National Institute on Aging and the Alzheimer's Association with two educational tracks. The Professionals track includes individuals who fill a broad variety of roles including clinicians, study coordinators, psychometricians, and other study professionals who wish to further their knowledge and advance their careers in ADRD trials. The Fellowship track includes current and future principal investigators and focuses on the design, conduct and analysis of ADRD clinical trials. SETTING: The 2020 inaugural iteration of IMPACT-AD was held via Zoom. PARTICIPANTS: Thirty-five trainees (15 Fellowship track; 20 Professionals track) were selected from 104 applications (34% acceptance rate). Most (n=25, 71%) identified as female. Fifteen (43%) were of a non-white race; six (18%) were of Hispanic ethnicity; eight (23%) indicated they were the first person in their family to attend college. MEASUREMENTS: Participants completed daily evaluations as well as pre- and post-course assessments of learning. RESULTS: Across topic areas, >90% of trainees evaluated their change in knowledge based on the lectures as "very much" or "somewhat increased." The mean proportion correct responses in pre- and post-course assessments increased from 55% to 75% for the Professionals track and from 54% to 78% for the Fellowship track. CONCLUSIONS: IMPACT-AD successfully launched a new training opportunity amid a global pandemic that preliminarily achieved the goals of attracting a diverse cohort and providing meaningful training. The course is funded through 2025.

What Have We Learned from Expedition III and EPOCH Trials? Perspective of the CTAD Task Force.

Although the results were disappointing from two recent clinical trials of amyloid-targeting drugs in mild-to-moderate AD, the trials provided information that will be important for future studies, according to the EU-US CTAD Task Force, which met in November 2017 to discuss the EXPEDITION3 and EPOCH trials. These trials tested two of the predominant drug development strategies for AD: amyloid immunotherapy and BACE inhibition in populations largely composed of mild AD dementia patients. The results of these trials support the emerging consensus that effective amyloid-targeted treatment will require intervention in early, even pre-symptomatic stages of the disease. Further, the Task Force suggested that a refinement of the amyloid hypothesis may be needed and that other hypotheses should be more fully explored. In addition, they called for improved biomarkers and other outcome assessments to detect the earliest changes in the development of AD.

Age-related alterations of enzyme activities and subunits of hepatic glutathione S-transferases in male and female Fischer-344 rats.

Enzyme activities of glutathione S-transferases (GSTs) toward five different substrates (benzalacetone (PBO), styrene oxide (STOX), sulfobromophthalein (BSP), 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB)) as well as concentrations of four subunits of GST isozymes (1, 2, 3 and 4) were determined using cytosol fractions obtained from livers of young (6 months) and old (26 months) Fischer-344 rats of both sexes. Values for enzyme activities for three substrates (DCNB, BSP and PBO) in young male rats were significantly higher than the corresponding values in female rats. In old male rats, values were generally lower than the corresponding values in young male rats, becoming close to corresponding values in young female rats. Old female rats, however, exhibited values close to those in young female rats, except for DCNB and STOX values, which were slightly lower in old female rats. GST subunits 3 and 4, as determined by high-performance liquid chromatography after purification by affinity chromatography using S-hexyl-glutathione, were predominant in young males, whereas concentrations of subunits 1 and 2 were higher in females than in males. In male rat livers, concentrations of subunits 3 and 4 decreased considerably with age while those of subunits 1 and 2 increased, so that the subunit pattern in old male rats tended to be similar to that of young female rats. In old females, a decrease in the concentration of subunits 3 and 4 and an increase in the concentration of subunit 1 were also observed as in old male rats, while the subunit 2 concentration tended to decline. Furthermore, the elution pattern of affinity chromatography changed with age, yielding an earlier elution of most subunits in old male rats and of subunit 1 in old female rats. The results suggest that age-related changes that occur with GSTs in livers of male rats are essentially a feminization of the isozyme pattern. However, despite rather unremarkable changes in enzyme activities with age in females, considerable changes of subunit pattern (a general decrease in concentration of subunits 2, 3 and 4 and an increase in the concentration of subunit 1) were also observed in female rats, and these were much greater than could be predicted from enzyme activity changes with age in this sex.

Short-term feeding of a diet enriched in phospholipids increases bile formation and the bile acid transport maximum in rats.

Earlier studies suggested that the secretory rate maximum (SRm) of bile acid and the cholestasis which occurs after the SRm is reached may be determined by the hepatic or extrahepatic biliary phospholipid pool. We therefore investigated whether bile formation and the bile acid SRm could be influenced by feeding a diet enriched in phospholipids. Male rats were fed phospholipid (PLD) or triacylglycerol (TgD)-enriched diet for 3 days, and bile formation as well as biliary lipid output were measured on the 4th day. In other similarly fed groups, cholic acid was infused in stepwise increasing doses to determine the effect of PLD on the SRm of cholic acid. The plasma lipid levels were significantly lower in PLD and TgD diets compared to basal diet. But, while the levels of total cholesterol (CH), HDL-CH, and phospholipid (PH) were not significantly altered by PLD compared to TgD, the triacylglycerol levels were markedly increased by PLD. In the liver of PLD fed rats, triacylglycerol and CH ester contents decreased by 39 and 62%, respectively, while free CH and PH contents were not significantly changed. The PLD significantly augmented spontaneous bile flow, bile acid, PH and CH secretion rates compared to TgD diet (65, 124, 164 and 654%, respectively). The enhanced biliary secretory function was associated with an increase in pericanalicular vacuoles and diverticuli in centrilobular hepatocytes. Compared to TgD fed rats, PLD rats showed a 2-fold decrease in the ratio of cholic acid/chenodeoxycholic acid in bile and a significant decrease in the % contribution of taurine conjugated BA. The PH fatty acids in bile were similar in both groups except that in PLD group the % contribution of C18:2 was higher than in TgD group. No differences were found in plasma membrane CH/PH content or total fatty acid composition. During bile acid infusion, the SRm and the total cholic acid secreted were significantly higher in the PLD than in the TgD rats. Moreover, the cholestatic response observed after high bile acid dose was markedly reduced by PLD. The results show that short-term feeding of PLD induces changes in CH and bile acid metabolism which result in enhanced biliary output of CH and PH. The enhanced pool of biliary lipid may protect plasma membranes from the deleterious effects of high bile acid concentrations.

Age-related changes in antioxidant enzyme activities are region and organ, as well as sex, selective in the rat.

Enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were determined in the liver as well as several specific brain regions of young and old Fischer-344 rats of both sexes. In the liver of male rats, activities of CAT as well as Mn-SOD were lower, while activities of Cu Zn-SOD were higher in old (30-month-old) rats than in young (7-month-old) ones. Activities of total SOD as well as GSH Px were comparable for young and old male rat livers. In contrast to male rats, in female rat livers, activities of CAT were significantly higher in old (28-months-old) rats, while activities of Mn-SOD were slightly (but significantly) higher in old rat livers. In old male rats, activities of Mn-SOD were significantly higher than in young males in several specific regions of the brain (the substantia nigra (s. nigra), striatum, hippocampus) but lower in the cerebellum. In particular, SOD activities in s. nigra, striatum and hippocampus in old male rats were several fold higher than corresponding values in young male rats. Activities of Cu Zn-SOD were generally unchanged with age. Activities of CAT as well as GSH-Px (both Se-dependent and non-Se-dependent forms) were also relatively unaffected by age. In female rat brains, activities of Mn-SOD as well as those of others all remained mostly unaffected by aging, although there was a general tendency of slightly higher activities in most cerebral regions for Mn-SOD in old female rats. Thus, age-related changes of these antioxidant enzymes in the liver and brain are markedly sex dependent and some enzyme activities (such as CAT in the liver) change in an opposite direction with age. Changes of Mn-SOD in the brain were markedly region-specific in male rats. Results suggest that the significance of the changes of these antioxidant enzyme activities during aging needs to be carefully interpreted, taking into consideration the fact that changes are markedly variable depending on sex as well as the organs and brain regions examined.

Difference in response of hepatic glutathione S-transferase activities to protein-free diet between young and old C57/BL male mice.

Responses of hepatic glutathione S-transferase (GST) activities to protein-free diet (PFD) and normal diet (ND) refeeding were compared for young (6-month-old) and old (22-month-old) C57/BL male mice. Enzyme activities toward 1-chloro-2,4-dinitrobenzene (CDNB) were not significantly different between young and old rat livers in the basal condition without diet manipulation. When animals were fed PFD for 1 week, GST activities toward CDNB significantly declined in both age groups in comparison to respective basal values, but there was no significant difference in activities between the two age groups after a 7-day PFD. When they were refed with ND for 2 days (on day 2 of ND), the activities in young mice rose to a level significantly higher than the corresponding basal value. In contrast, in old animal livers, the activity slightly but further tended to decline on day 2 of ND. Activities in old rat livers returned to the basal level on day 5 of ND, while activities in young animal livers that increased to levels higher than basal levels due to the overshoot returned to the basal level on day 7 of ND. Enzyme activities toward 1,2-dichloro-4-nitrobenzene (DCNB) were significantly higher in young rat livers than in old ones at the basal period. However, enzyme activities also overshot the basal level on day 2 of ND after 7-day PFD in young mouse livers, while in old mouse livers the activities were lowest on this day. Activities returned to the basal level on day 7 of ND in both age groups. Thus, the greatest difference in enzyme activities between young and old mouse livers for both substrates was observed on day 2 of ND after 7-day PFD, rather than at either the basal period or immediately after 7-day PFD. The results essentially agree with our previous findings on female C57/BL mice as well as female Fischer-344 rats, suggesting that the age-induced changes in the GST system become clearly manifest after diet manipulation of PFD followed by ND refeeding, rather than in values during a basal period without diet manipulation, regardless of sex or species of animal.

Pharmacological modifications of endogenous antioxidant enzymes with special reference to the effects of deprenyl: a possible antioxidant strategy.

Limited information is available on the upregulation of endogenous antioxidant enzymes by means of administering various pharmaceuticals and/or chemicals. It has been reported that ursodeoxycholic acid (UDCA), a bile acid originally identified from black bear bile (a Chinese medicine, Yutan) increased glutathione S-transferase (GST) activities in mouse livers, resulting in a decrease in systemic lethal toxicity of orally challenged 1-2-dichloro-4-nitrobenzene (DCNB). Also, ursolic acid found in herbal medicines (e.g. leaves of loquat) was reported to increase catalase (CAT) activities in mouse liver. Interestingly, the chemical structures of these two compounds are surprisingly similar to each other, despite the difference in their original sources. These results suggest that in the future, more and more compounds will be found to have effects on increasing endogenous antioxidant enzyme activities. Deprenyl is a monoamine oxidase B inhibitor but also possesses many other different pharmacological activities. Among these various pharmacological effects of deprenyl, a possible causal relationship between two effects of deprenyl, namely the prolongation of the survival of animals and upregulation of antioxidant enzymes in selective brain regions, has been postulated by the authors. In at least four different animal species (rats, mice, hamsters and dogs), a significant prolongation of survival by chronic administration of the drug has been reported by different groups including that of the authors. This group has reported that repeated administration of the drug for 2-3 weeks can significantly increase activities of both types of superoxide dismutase (SODs) (Cu, Zn-, and Mn-SODs) as well as of CAT selectively in brain dopaminergic regions. Both effects are dose dependent but excessive dosages become less effective and even cause an adverse effect (i.e. a decrease in enzyme activities and shortening of life span). The parallelism of the dose-effect relationship between the two phenomena suggests that modification of SOD and CAT levels is one possible mechanism for deprenyl's ability to prolong the life span of animals.

Hepatic protein synthesis and serum aminoacid levels during liver regeneration in young and old malnourished rats.

The aim of the present study was to measure protein synthesis in regenerating liver and to evaluate the impact of malnutrition in young and old rats. Two groups of male Wistar rats were used: young rats (4 months old) and old rats (18 months old). The rats were allocated to malnutrition or ordinary food intake for 1 week. Half of each group was sham-operated and the other was partially hepatectomized 2 days before the end of diet manipulation. Hepatic protein synthesis was significantly increased in all hepatectomized groups compared with their respective sham group: young well-nourished hepatectomized rats, 44%; young malnourished hepatectomized rats, 55%; old well-nourished hepatectomized rats, 47%; and old malnourished hepatectomized rats only 21%. Hepatic DNA content was unchanged in all groups and liver RNA content was higher in young malnourished hepatectomized rats (21%, P < 0.05). Serum total amino acid concentration did not change in young well-nourished hepatectomized and young malnourished hepatectomized rats. This value did not show significant changes between old well-nourished hepatectomized and old well-nourished sham, but it increased 14% (P < 0.05) in old malnourished hepatectomized. It was concluded that (a) regeneration is not impaired by malnutrition in young rats and may even be better than in rats eating a normal diet, and (b) the deleterious effect of aging is revealed once old animals are exposed to malnutrition. It is manifested in the decreased rate in hepatic protein synthesis observed in old malnourished hepatectomized rats and in the augmentation of total serum amino acid concentration, where the hypercatabolism induced by hepatectomy is significantly greater.

Differences in the influence of diet on hepatic glutathione S-transferase activity and glutathione content between young and old C57 black female mice.

The effect of a protein-free diet (PFD) on hepatic activity of glutathione S-transferase (GST) and hepatic content of total glutathione (GSH) was examined in young (9-month-old), middle-aged (17-month-old) and old (27-month-old) C57BL/6CrS1c female mice. There were no significant differences in the control values of GSH or of enzyme activity for four of five substrates among young, middle-aged, and old animals fed normal diet (ND) only. Both GSH and GST activity were significantly decreased by the 7-day PFD in both young and old groups but the decrement was generally greater in old mice. After a 2-3-day refeeding of ND, young mouse enzyme activities were significantly higher than control (basal) values for all five substrates, whereas old mouse values were still significantly lower than corresponding control values. There was no overshooting of GSH levels after refeeding of ND in either young or old animals. This study indicates that an age difference in this detoxification system can be clearly demonstrated in the hepatic response to PFD feeding and especially to ND refeeding, despite the enzymes' stable basal activities with aging.

Mechanism underlying anti-apoptotic activity of a (-)deprenyl-related propargylamine, rasagiline.

A potent inhibitor of type B monoamine oxidase, (-)deprenyl, is known to protect or rescue dying neurons, independent of inhibition of the enzyme activity. After long term administration to rodents, a propargylamine structurally related to (-)deprenyl, (R)(+)-N-propargyl-1-aminoindan (rasagiline) increased the activities of anti-oxidative enzymes, superoxide dismutase and catalase. Rasagiline protected in vitro dopamine cells from apoptosis induced by oxidative stress or neurotoxins. The mechanism of the anti-apoptotic effect was studied by in vitro experiments using human dopaminergic neuroblastoma, SH-SY5Y cells. Peroxynitrite-generating N-morpholino sydonimine (SIN-1) induced apoptosis in SH-SY5Y cells via disruption of mitochondrial membrane potential (DeltaPsim), followed by caspase 3 activation. Rasagiline prevented the loss of DeltaPsim, the initial step to apoptosis, and also following caspase 3-activation and DNA fragmentation. The results suggest that rasagiline may interact with the specific molecule in the mitochondria and suppress the death signal transduction. By the anti-apoptotic function, rasagiline may rescue or protect declining neurons in aging and neurodegenerative disorders, such as Parkinson's disease.

Effect of protein-free diet on activities and subunits of glutathione S-transferase in livers of young and aged female rats.

Female Fischer-344 rats of different ages (8 and 25 months old) were fed a protein-free diet (PFD) for 7 days and refed a normal diet (ND) (23% protein) thereafter. Rats were killed immediately after the PFD was stopped (day 0) and at different time intervals during refeeding of a ND. Four subunits (1,2,3 and 4) and activities of glutathione S-transferases (GSTs) toward five different substrates, [styrene oxide (STOX), 1,2-dichloro-4-nitrobenzene (DCNB), 1-chloro-2,4-dinitro benzene (CDNB), sulfobromophthalein (BSP) and benzalacetone (PBO)] were determined. There were no significant differences between young and old rats in the liver enzyme activities before the PFD. The PFD caused significant decreases in activities for three substrates (DCNB, BSP and STOX) in both age groups, with no significant differences between young and old rats a day 0. During recovery from the PFD, activities for the three substrates exceeded basal levels in young rats but at different time intervals (STOX, day 2; BSP, day 5; DCNB, day 9), while enzyme values in old rats tended to return slowly to basal values with no "overshoot." Concentrations of subunits 3 and 4 in young rat livers that were depressed by the PFD did not recover until day 9 of the ND, while subunits 2 and especially 1 increased during the ND refeeding, overshooting the basal levels. In contrast, in old rat livers the only change was a reduction of subunit 1 by the PFD and its gradual recovery during ND refeeding. These results demonstrate that our previous observation of overshooting of enzyme activities in mice is reproducible in rats but with certain substrate specificities. Furthermore, changes in subunit concentrations caused by aging and a PFD are more complex than what was predicted from changes in enzyme activities of GSTs.

Involvement of mu class glutathione S-transferase subunit M2 (rGST M2) levels in the initiation and promotion of hepatocellular carcinogenesis in old rats.

Age-associated differences in the response of the initiation and promotion of hepatocellular carcinogenesis in the rat were analyzed. Male Wistar rats 5 and 18 months-old were used throughout. They underwent an experimental design of multistage model of hepatocarcinogenesis: hepatic cells were initiated with the complete carcinogen Aflatoxin B1 (0.5mg/Kg b.w.) and the promotion was performed through a combined treatment of proliferation (partial hepatectomy, 65%) and administration of the tumorigenic promoter phenobarbital (0.1% in drinking water for 21 days). After the treatment, rats were sacrificed and the following parameters were determined: activity and subunit composition of the glutathione S-transferase enzyme system, the number of liver preneoplastic foci and the proliferation cell index. The combined treatment (initiation + promotion) lowered the expression of the mu class GST (rGST M1, rGST M2). The inhibition in rGST M2 in old animals (which in basal conditions had already been lower) was significant. On the other hand, the treatment increased the alpha class GST (rGST A, rGST A3). The number of preneoplastic foci was higher in old rats (number of foci/cm(2): 6.9+/-0.3 vs 3.9+/-0.3 in young rats, p< 0.05). The proliferation cell index did not show age-related differences. Because rGST M2 deficiency coexisted with induced expression of alpha class, the livers would be resistant to some toxic insults, being selectively sensitive to potentially genotoxic substances for which M2 is an essential detoxification pathway. The transition to a rGST M2-deficient phenotype during aging could induce higher responsiveness to genotoxic effects, and might favor the likelihood of further progression, indicating a higher susceptibility of aged animals to the development of carcinogenesis.

Inhibition by interferon alpha-2b of rat liver regeneration: effect on ornithine decarboxylase and total protein synthesis.

Polyamines are key factors in macromolecule synthesis during liver regeneration. It has been postulated that interferon-alpha (IFNalpha) decreases putrescine levels in regenerating liver by inhibiting ornithine decarboxylase (ODC) activity, the main enzyme in polyamine biosynthesis. In the present study, we analysed the effects of a pharmacological dose of IFNalpha on polyamine and ODC levels during the regenerative process following partial hepatectomy in rats. Synthesis of ODC by isolated hepatocytes from IFN-treated rats with regenerating livers was also assessed. Furthermore, we investigated the effect of IFNalpha-2b on DNA and total protein synthesis in 24-hr regenerating livers. No effect on DNA synthesis was observed at the dose of IFNalpha-2b used, but total protein synthesis decreased significantly in IFNalpha-2b-treated rats undergoing liver regeneration (7.0 +/- 2.0 and 12.1 +/- 1.7%. min(-1) in hepatectomized rats treated with IFNalpha-2b and saline, respectively). ODC levels were also reduced significantly (by 50%) in hepatectomized rats treated with IFNalpha-2b versus saline. In parallel with the ODC decrease, the concentrations of putrescine and spermidine (63 +/- 25 vs 101 +/- 15 nmol/g liver and 1.08 +/- 0.35 vs 2.14 +/- 0.22 micromol/g liver, respectively, in IFNalpha-2b- and saline-treated hepatectomized rats) showed similar, significant diminutions. Moreover, the incorporation of [35S]methionine into ODC was decreased dramatically in isolated hepatocytes from IFNalpha-2b-treated hepatectomized rats 12 hr after surgery. In conclusion, the protein synthesis rate in regenerating liver was impaired by therapeutic doses of IFNalpha-2b. In addition, the results presented in this study suggest that IFNalpha-2b negatively regulates ODC synthesis, causing a reduction in polyamine levels during liver regeneration.