281 ‒ Longevity drugs, aging biomarkers, and updated findings from the Interventions Testing Program

Intro (00:00:00)

• There is strong evidence for at least three mutants, calorie and methionine restriction diets, and some drugs, such as rapamycin, delaying aspects of aging.
• The host, Peter, introduces his guest for the podcast, expressing excitement for the discussion about longevity drugs and aging biomarkers.

An overview of the Interventions Testing Program (ITP) (00:00:50)

• The National Aging Institute created the Interventions Testing Program (ITP) about 20 years ago, now in its 20th year and applying for additional funding.
• ITP tests drugs for their potential to slow aging and extend the lifespan of mice across three labs: the University of Michigan, the University of Texas Health Science Center at San Antonio, and the Jackson Labs.
• Anyone in the world can nominate a drug for testing by the ITP, with an annual call for applications.
• Four significant drug hits have been published by ITP, with two more showing smaller significant effects, and others soon to be published.
• ITP goes beyond initial findings, testing varying doses, examining pathology, and sharing tissue samples with other researchers.
• Insights from ITP could impact human aging research though there are many steps between successful mouse treatments and human drugs.
• The number of drug candidate nominations varies each year; the record was 28 nominations, but typically it is 10-15, with about five to seven selected for testing.
• The National Institute on Aging (NIA) provides $1 million annually to each site involved in the ITP, with additional indirect costs for university maintenance and operations.
• The NIA's budget for the ITP doubled from half a million after the first ten years, reflecting the program's success.
• Although NIA's overall budget is significant due to its focus on Alzheimer's research, funding for aging biology as a general field is comparatively small.
• Potential for philanthropic contributions exists as foundations can directly support research labs or projects at universities involved with the ITP.

How the mice used by the ITP are superior for research relative to mouse models used in most research (00:08:51)

• The standard mouse model commonly used in research is the C57BL/6, known as the B6 mouse.
• B6 mice have a single genotype, leading to limitations as drug effects can vary significantly between different mouse models.
• Inbred B6 mice do not represent mice in general, despite widespread use in studies.
• The Intervention Testing Program (ITP) uses a genetically heterogeneous mouse model called UM-HET3, which consists of mice sharing half of their genes randomly like siblings.
• UM-HET3 mice can be consistently reproduced and have a diverse genetic variety, allowing for results less likely to be skewed by an unusual genotype.
• This diversity in the UM-HET3 model can provide more reliable identification of drug effects and has enabled gene mapping related to lifespan.
• The heterogeneity of UM-HET3 mice allows for a broader understanding of how inheritance modifies aging and drug response.
• In contrast, B6 mice are homozygous, meaning they have identical genes from both parents, resulting in severe health issues and not being representative of a healthy population.
• Inbred mouse models are mostly suitable for studying specific diseases they are prone to and for transplantation experiments where identical genotypes are necessary.
• The continued use of B6 mice in research is often due to a lack of awareness of alternative models, financial constraints, and adherence to preceding research practices.
• F1 mice, which are offspring of two different inbred strains, are healthier and live longer, serving as a better option for transplantation studies and as a foundation for creating models like the UM-HET3.

Design of ITP studies, outcomes tested, and metrics of interest (00:17:11)

• ITP studies test the effect of candidate molecules on aging after board review for biological plausibility.
• Primary outcomes are calculated using the proportional hazard or risk of death over a lifespan, approximating median lifespan.
• Median lifespan is the age by which half the population has died; increases are significant markers of longevity.
• Maximum lifespan is assessed differently, using the 90th percentile survival rather than last mouse death to account for statistical variance.
• Significant longevity effects are inferred when a higher percentage of surviving mice at the 90th percentile are drug-treated compared to controls.
• The studies avoid choosing metrics based on the best outcome and pre-specify the 90th percentile to prevent false positives.
• Power analysis is consistent across experiments with adequate planning to account for potential site-specific disasters.
• Sample sizes at each site typically involve 100 males and 100 females for controls and 50 of each for treated groups, with controls being oversampled for comparison across drugs.

Variability and consistency across sites (00:17:11)

• Control mice survival rates are usually consistent across sites for females but not for males.
• Males at the Michigan site consistently live 5-10% longer than those at other sites, with the cause still unknown.
• Weight differences are also observed, with Michigan mice being about 10% lighter for both males and females, suggesting potential site-specific environmental factors.
• The survival benefit of lighter weight is only evident in Michigan males, not females, and causality is unclear.
• Site-specific differences in survival for males are managed by site stratifying comparisons of controls and treated mice within individual sites.
• Results are then pooled, stratifying for both median and maximum lifespan metrics, but site-specific results are also reported separately in studies.

The process and challenges of drug formulation for mice (00:29:00)

• Ensuring proper drug formulation and dosing for mice involves significant planning and testing.
• Randy Strong and Brett Ginsburg at the University of Texas focus on drug measurement in mice food and tissue.
• Before starting lifespan experiments, food is tested with drugs to confirm proper dosage.
• Testing involves checking MRNAs in the liver to ensure the drug's biological effect.
• Issues, such as the degradation of drugs like rapamycin in the stomach, have been overcome with techniques like encapsulation.
• Drug efficacy is impacted by factors like gender, as blood concentration differences can lead to variance in results.
• Administration of drugs to mice is done through food, not intravenously or intramuscularly, due to practicality and mouse welfare.
• Monitoring food intake is not precise since individual consumption rates cannot be tracked.

Four drugs identified by the ITP that extends the lifespan of mice (00:36:11)

• Over 100 drugs have been tested over 20 years, with some tested multiple times leading to notable successes and failures.
• Rapamycin was the first molecule to prove lifespan extension in the ITP, with significant effects even when started late in life.
• Other successful drugs include 17-alpha-Estradiol, Acarbose, and Canagliflozin, all of which have varying effects based on the sex and age at which they are started.
• Not all drugs show equal efficacy in male and female mice, suggesting sex-specific metabolic processing of drugs.
• Differences in xenobiotic metabolism between sexes account for drug concentration variations, a phenomenon also present in humans.
• Despite known differences in male and female drug metabolism, human medicine does not typically use sex-specific dosing beyond weight differences.

The Success of Rapamycin and What It Tells Us About the Biology of Aging (00:43:32)

• Rapamycin (Rap) was nominated for ITP study by Dave Sharp, influenced by its FDA approval for organ transplants and emerging genetic data from invertebrates.
• The initial interest in Rap was not due to its immune-suppressing properties but its role in controlling growth and its potential to extend lifespan.
• The Immune functions of Rapamycin are dose and context-dependent, with both suppressive and boosting effects observed.
• Unexpected findings showed that elderly mice treated with Rapamycin exhibited improved influenza vaccine responses due to enhanced B cell production.
• The decision not to abort the study despite delays in formulating Rap for oral administration led to breakthrough findings.
• In the study, both old and young mice treated with Rapamycin showed significant lifespan extension, with no significant difference when initiated in middle age compared to younger ages.
• The effectiveness of Rapamycin in extending lifespan suggests a broader range of drug-sensitive aging processes still amenable to intervention even at middle age.
• General positive health effects of treatment were observed, not just on lifespan but also on physical capabilities like grip strength and balance.

Other Measures of Healthspan Evaluated by the ITP in Stage 2 Studies (00:51:49)

• The ITP's next focus includes evaluating cognitive functions in mice treated with longevity drugs, a topic of divided predictions among researchers.
• Healthspan measures being assessed in stage 2 studies include grip strength, motor tasks, stamina, muscle mass, and response to age-related diseases.
• Stage 1 studies focus solely on lifespan and body weight, while stage 2 studies explore a broader range of health markers.
• Specific interests in health markers drive the choice of additional measures at different labs.
• An existing collaborative interactions program (CIP) enables sharing of tissue samples with external researchers but may be replaced by a centralized NIA system, potentially slowing down access to these resources.
• Although the exact mechanisms and optimal tissue targets for studying drug effects remain unclear, tissues are actively being used for epigenetic and metabolomic studies, with the goal of eventually pinpointing precise cellular targets and pathways affected by anti-aging interventions.

Distinguishing aging rate indicators from biomarkers of aging (00:59:18)

• Biomarkers of aging are analogous to an odometer in a car, indicating the extent to which a body has "aged."
• Presently, there is skepticism about the existence or utility of robust biomarkers of aging.
• Functional tests like strength, vision, and cardiovascular fitness can suggest biological youth, but these are not the same as assay-based biomarkers.
• Aging rate indicators are compared to a speedometer, showing the rate of aging rather than the accumulated effects.

Aging rate indicators identified through the examination of slow-aging mice (01:01:16)

• Identified 13 biomarkers that change consistently across all examined slow-aging mouse models, including both genetically altered mice and those treated with specific diets or drugs.

• Identified biomarkers are useful early indicators of aging rate, even when the mice are young adults.

• One of the key biomarkers is UCP1, a mitochondrial protein that is associated with increased thermogenesis and has protective effects against obesity, diabetes, metabolic syndrome, and certain types of inflammation.

• All slow-aging mice showed increased UCP1 levels in their fat tissues, with the exception of responses to certain drugs that only extend lifespan in male mice, suggesting sex-specific effects.

• Discussed the variability in biological markers due to factors like exercise, diet, and time of day, but indicated that consistent differences between control and treated or mutant mice suggest a robust phenomenon.

• Utilized humane euthanasia methods for mice to minimize stress-related artifacts in biological measurements.

• Other key aging biomarkers identified in the slow-aging mice include a decrease in inflammatory macrophages (M1) and an increase in anti-inflammatory macrophages (M2), suggesting a reduction in inflammatory tone.

• Increases in brain-derived neurotrophic factor (BDNF) and doublecortin (DCX) in the brain were observed, implying enhanced neuroprotection and neurogenesis, respectively. These changes were also sex-specific.

• GPLD1 is another biomarker found to be elevated in slow-aging mice, which is involved in releasing proteins from cell surfaces and is associated with improvements in cognitive ability.

• Slow-aging mice exhibited increased cap-independent mRNA translation, specifically for GPLD1, linking protein translation mechanisms to physiological effects observed in aging.

Why proteomics are essential to understand changes in the cell (01:15:36)

• Proteomics, the study of proteins, is crucial for understanding cellular changes because RNA levels are poorly correlated with protein levels.
• Studies indicate only about 30% correlation between changes in RNA and proteins related to aging.
• Changes in proteins with age are not fully explained by transcriptional changes.
• The process of protein formation involves multiple steps beyond transcription, including translation and degradation, which can impact protein levels independent of mRNA.
• Differential RNA translation and autophagy are among mechanisms that modify the proteome.
• Recognizing the importance of these mechanisms will lead to greater emphasis on studying proteins and their nuances.

Unraveling aging rate indicators: dose-effect, duration, and future frontiers (01:25:30)

• Indicators of aging rate can potentially be influenced by genetic mutations, diet, and drugs.
• Questions remain about how quickly aging indicators respond to interventions and how long effects persist.
• Rapid changes in indicators would allow for screening large numbers of drugs to predict lifespan effects.
• Past research shows that transient early-life treatment with growth hormone can have long-term effects on aging rate indicators.
• Future research aims to understand whether positive interventions can have similarly persistent effects.
• These aging indicators may also apply to human aging research, offering a potentially valuable tool for testing interventions in people.
• Experiments with mice suggest that timing is crucial for interventions, with differing results depending on when growth hormone treatment is applied.

A closer look at aging rate indicators: bridging the gap from mice to humans (01:31:54)

• Biomarkers like BDNF and DCX are measured in mouse brains directly or in CSF.
• For human application, less invasive methods than CSF or biopsies are desired, like plasma testing.
• A UCSF project gathered data including muscle and fat biopsies from healthy 70-year-olds, which will be used to look for aging rate indicators.
• Two muscle and fat biomarkers relevant to fitness and aging, fndc5 and ucp1, are also measurable in human plasma.
• Aim to find mouse and human plasma markers that correlate with tissue-specific aging rate indicators.
• Study planned with Katherine Karowski and metabolic expert Costus Latas, to correlate blood metabolites with tissue aging markers in mice.
• Hamilton at Stanford has developed a way to correlate plasma signals to specific organ changes; the methodology is not fully disclosed.
• Irisin, a product from the muscle protein FNDC5, correlates with slow aging in mice.
• Previous irisin research had inaccuracies, but new assays have clarified its concentration and potential promise.
• While irisin and gPLD1 seem promising, they would likely be injectable drugs due to their peptide nature.
• Anti-aging drugs like rapamycin have demonstrated aging-delaying effects, but broader aging intervention requires strong evidence across various functions, not just anti-cancer properties.
• There's increasing evidence that certain interventions act on aging broadly, not just one disease, challenging traditional medical perspectives.

Treatment efficacy and the holistic approach to slowing aging

• Drugs must not only extend life but improve multiple age-sensitive functions to be considered true anti-aging drugs.
• Evidence for a drug's anti-aging properties requires comprehensive testing, as seen with Snell dwarf mice and various diets.
• Molecules like rapamycin are starting to build a strong case for genuinely slowing the aging process.
• Anti-aging research is gradually shifting understanding from targeting specific diseases to a more holistic approach to health and longevity.

What do laboratory mice die from? (01:44:32)

• Laboratory mice predominantly die from cancer; approximately 80% succumb to various neoplasms.
• Lymphoid or leukemia cancers are common in males, whereas females often die from breast or liver cancer.
• Other kinds of cancers spread across the remaining 20% cause mortality in both sexes.
• Necropsy data is usually too limited to statistically confirm if treatment-induced survival extension changes specific cancer proportions.
• A notable exception was a drug that extended lifespan but didn't alter the age at death from breast cancer in females, hinting at complex drug-aging interactions.

Distinguishing between a drug that improves an age-sensitive outcome and a drug that improves all aspects of aging (01:48:09)

• No drugs have been found that improve health metrics like grip strength or treadmill time without also extending lifespan in the Interventions Testing Program (ITP).

The ITP study of 17α-estradiol: mechanisms of life extension and surprising sex differences (01:48:48)

• The ITP had success with 17α-estradiol, a stereoisomer of 17β-estradiol (common estrogen), which does not bind well to typical estrogen receptors.
• It was hypothesized that since estrogens are beneficial, 17α-estradiol, without causing feminization, might improve male longevity.
• Unexpectedly, 17α-estradiol extended male mouse lifespan beyond females, with no positive effect on female longevity, suggesting a different mechanism than typical estrogen.
• Its physiological targets and actions remain largely unknown.
• Research is underway to understand 17α-estradiol's effect, especially on the brain, with only a few labs currently investigating.
• When combined with another drug, rapamycin, male mouse lifespan increased by 29%, but 17α-estradiol has not achieved more than a 19% increase alone.
• Experiments are testing if combining 17α-estradiol with rapamycin will have additive effects.
• A related compound, hydroxy estriol, has shown efficacy in males equivalent to or better than 17α-estradiol, while being the first drug to reduce female lifespan.
• Males, but not females, showed an increased production of estriol when treated with 17α-estradiol, dependent on testosterone or another testicular hormone.
• The reasons for choosing hydroxy estriol for study and the specific impact on mortality in females are under investigation.

Unsuccessful drugs studied by the ITP: resveratrol, metformin, and nicotinamide riboside (01:58:34)

• The Interventions Testing Program (ITP) revealed that resveratrol, metformin, and nicotinamide riboside (NR), popularly believed to be effective anti-aging drugs, did not show the expected results.
• These drugs garnered significant attention in the media and at conventions, with resveratrol particularly being associated with anti-aging properties supposedly found in red wine.
• However, the effective concentration of resveratrol in red wine for anti-aging is impractically high, and its status as an activator of certain longevity pathways is disputed by biochemists.
• Metformin, while safe for human use, did not extend lifespan in mice according to ITP studies and follow-up research.
• NR generated a lot of hype but ultimately failed to extend mouse lifespan in ITP testing. It has been proposed that NR or its metabolite, NMN, may still hold promise if combined with other substances or tested in different formulations.
• Even though these substances were unsuccessful in mice, there's a possibility they may have different effects in humans, but robust testing in humans would be required to determine their efficacy.

Over-the-counter successes in the ITP: meclizine and astaxanthin (02:09:13)

• Meclizine, an over-the-counter seasickness drug, has shown a 10% increase in lifespan in male mice but not in females.
• The intervention's effectiveness may not be directly linked to its properties as a TOR inhibitor but could be related to other CNS effects.
• Astaxanthin, known for giving salmon their pink color and available over the counter, also improved longevity in male mice, but had no impact on maximum lifespan.
• These findings are significant as they suggest over-the-counter drugs could affect aging, presenting new possibilities for mechanistic exploration.

A senolytic drug, fisetin, fails to extend lifespan (02:16:00)

• Fisetin, a drug undergoing human trials as a potential senolytic, did not extend lifespan in male or female mice nor did it remove senescent cells.
• Different dosage regimes suggested by experts were tested without effect, questioning the notion that fisetin can remove senescent cells.
• The definition of what constitutes a senescent cell is unclear and debated, which complicates understanding the effectiveness of senolytics.