272 ‒ Rapamycin: potential longevity benefits, surge in popularity, unanswered questions, and more

Intro (00:00:00)

• Rapamycin consistently demonstrates longevity and health span benefits across various species, including yeast, worms, fruit flies, mice, and pet dogs.

David & Matt’s expertise in mTOR & rapamycin (00:01:00)

• David has extensively researched rapamycin and its effects on the mTOR pathway, focusing on nutrient sensing and its relations to anabolism and catabolism.
• Matt's contributions to the field include discovering mTOR's impacts on aging through unbiased genetic research, leading to further exploration into rapamycin's potential as a longevity therapeutic.
• Both are recognized as leading authorities on mTOR and rapamycin, with David's work being more mechanistically focused and Matt's trending towards translational research.
• Their complementary expertise spans the continuum of scientific benchwork to potential clinical applications, respecting the complexity of the mTOR signaling network.
• The field's credibility is upheld by their scientific rigor and cautious approach to commercialization, setting a standard for research into potential aging therapeutics.

The discovery of rapamycin & first use in humans as an immunosuppressant (00:12:00)

• Rapamycin was discovered in a soil sample from Easter Island (Rapa Nui) by pharmaceutical company Wyeth Ayerst.
• The compound came from a bacterium called Streptomyces hygroscopicus and was named after Easter Island where it was found.
• Initially, rapamycin showed antifungal properties and later was used as an immunosuppressant.
• Its clinical development path was long, with approval for human use coming decades after its discovery.
• The long clinical path and its initial use as an immunosuppressant may have negatively impacted the reputation and development for other uses.

Emergence of rapamycin as a molecule with the potential to prolong lifespan (00:19:05)

• The connection between rapamycin and nutrients led scientists to consider its potential impact on lifespan, building on existing knowledge about caloric restriction and aging.
• The Interventions Testing Program's (ITP) study in 2009 showed that rapamycin extended lifespan in mice, a significant finding linking yeast and mammalian aging.
• No other small molecules except for caloric restriction and potentially Alpha-ketoglutarate have shown consistent lifespan extension across a broad evolutionary spectrum of organisms.
• Genetic inhibition of the mTOR pathway, which rapamycin affects, similarly extends lifespan and health span in various model organisms.
• There is substantial evolutionary conservation in genetic control of longevity, which supports the study of fundamental biological aging processes and their translation to human aging.

Groundbreaking rapamycin study on mouse lifespan extension & the open questions about the timing & frequency of dosing (00:25:53)

• Geroscience focuses on fundamental aging interventions unlike disease-specific treatments.
• The 2009 NIA study showed rapamycin could extend the lifespan when started in middle-aged mice.
• This finding was serendipitous due to delays in formulating rapamycin in mouse food.
• Treating middle-aged organisms is more practical for potential human applications.
• Starting rapamycin in middle age is beneficial, but the ideal initiation time and dosing are unknown.
• There is a lack of comprehensive dose-response studies due to high costs and funding challenges.

Research funding challenges and the importance of high-risk studies [Discussion throughout]

• Current biomedical research funding is risk-averse, often hindering high-impact studies.
• The accidental discovery with rapamycin suggests a need for funding high-risk, high-reward research.
• Intermittent dosing studies in ITP mice could inform treatments in larger animals and humans.
• Such studies, while costly, are considered valuable and could potentially find funding outside NIH.
• Exploring other mTOR inhibitors and dosing strategies is critical but underfunded.
• The current NIH funding structure does not prioritize these types of pragmatic, translational studies.

Explaining mTOR & the biology behind rapamycin’s effects (00:36:26)

• Rapamycin is a unique drug that binds to a cellular protein FKBP and then attaches it to mTOR, affecting its function.
• mTOR is a kinase involved in anabolism (building up) and catabolism (breaking down), including autophagy, the self-eating process of the cell.
• The discovery that mTOR needs to be bound to other proteins to be active was significant. Detergents used in experiments initially disrupted these complexes.
• Proteins like Raptor and Richter are components of the mTOR complexes known as mTORC1 and mTORC2, respectively, and they play roles in cellular and organismal lifespan.
• mTORC1 is connected to many upstream signals and downstream targets, with thousands of mTOR complexes present in a typical cell.
• mTOR resides at the lysosome, which is critical for nutrient sensing and cellular metabolism.
• mTOR is evenly distributed across various tissues and is considered integral to cellular health.
• Rapamycin is an allosteric inhibitor that blocks the entrance where substrates would normally bind to mTORC1, affecting its function by steric hindrance while allowing smaller substrates to pass.
• Both mTORC1 and mTORC2 are affected differently by various types of inhibitors, with rapamycin primarily affecting mTORC1.

Differences in how rapamycin inhibits mTOR complex 1 (MTORC1) versus mTOR complex 2 (MTORC2) (00:47:13)

• Rapamycin partially inhibits MTORC1 and can, over time, partially inhibit MTORC2.
• MTORC1 and MTORC2 have different canonical substrates; for MTORC1, it's S6 kinase, and for MTORC2, it's a protein called AKT.
• FKBP-rapamycin can bind to "naked" MTOR and prevent the formation of MTORC2 by blocking the necessary interaction between MTOR and Richter.
• Prolonged rapamycin exposure leads to the binding of FKBP-rapamycin to MTOR, thus inhibiting the formation of new MTORC2 complexes.
• The inhibition mechanisms for MTORC1 and MTORC2 through rapamycin are distinct, with MTORC1 being directly inhibited, while MTORC2 is prevented from forming when rapamycin binds to MTOR prior to MTORC2 assembly.

Reconciling the biochemical mechanism of rapamycin with its longevity benefit (00:51:20)

• Longevity benefits of rapamycin are seen with both chronic and intermittent dosing, despite the biochemical mechanism's complexity.
• Rapamycin impacts life and health span potentially due to MTORC1 inhibition, but the complete mechanisms are not yet fully understood.
• Genetic data from various organisms suggest that benefits are associated with MTORC1 inhibition, but the evidence is not comprehensive.
• The idea that longevity benefits come only from MTORC1 inhibition and side effects from MTORC2 inhibition is still under investigation.
• Low levels of rapamycin are used in longevity studies, which might not inhibit MTORC2 as much, allowing some cells to escape inhibition.
• The rapamycin dosing is less constant in a living organism as compared to controlled experiments, suggesting transient exposure might be sufficient for benefits.
• Determining the specific contributions of MTORC1 and MTORC2 inhibition to rapamycin’s benefits is complex due to shared components and a lack of clean experimental tools.
• The network of signaling regulated by MTORC is dynamic and adaptive, continuously seeking homeostasis despite pharmacological interventions.

Important discoveries about the interplay of amino acids (leucine in particular) & mTOR (00:56:42)

• David Sabatini discovered mTOR (mechanistic target of rapamycin) in Saul Snyder's lab and initially overlooked the importance of cell biology in understanding mTOR's function.
• Using an antibody, he observed mTOR's unique punctate pattern in cells, but the specific cellular location was unclear until years later.
• After many years, Tim Peterson in Sabatini's lab discovered that mTOR resides on lysosomes, which are cellular compartments responsible for breaking down materials into basic components like amino acids.
• Peterson's experiment showed that amino acids' presence or absence could move mTOR on and off lysosomes, indicating nutrients' role in mTOR localization.
• A complex network of about 20 proteins communicates with nutrients to anchor mTOR to the surface of lysosomes via a docking station.
• Joe Avruch's research identified leucine and arginine as key amino acids regulating mTOR, prior to the discovery of mTOR's association with lysosomes.
• David Sabatini's lab pursued how leucine, known for increasing satiety and muscle mass, was detected by cells.
• They identified sestrin as the protein receptor for leucine, which was a significant breakthrough, and observed leucine binding in the structure of sestrin.
• Rachel Wolfson and Lynn Chantranupong discovered sestrin as the leucine sensor; Bobby Saxton and Thomas Schwartz elucidated the crystal structure of leucine bound to sestrin.
• The structure showed leucine's snug fit within sestrin, explaining why it is challenging to mimic leucine's anabolic effects with other compounds.
• Leucine's binding and release from sestrin may involve a lid that closes over it and possibly requires phosphorylation to reopen, suggesting an active release mechanism.

Reconciling rapamycin-mediated mTOR inhibition with mTOR's significance in building & maintaining muscle (01:04:43)

• mTOR is essential for nutrient sensing and muscle mass, and its activation promotes muscle building.
• Sarcopenia, or low muscle mass, is a significant risk for both lifespan and health span.
• Rapamycin inhibits mTOR, expected to lead to muscle loss, but has been observed to preserve muscle mass in rodents.
• The dose of rapamycin likely impacts whether it leads to muscle loss or preservation.
• At lifespan-extending doses, rapamycin may preserve muscle mass in the context of aging.
• It's unclear whether lower doses of rapamycin would impede muscle building in humans during resistance training.
• Rapamycin's effect on chronic inflammation might preserve muscle by reducing inflammation-related muscle synthesis impairment.
• There are differences between rodent models and humans regarding sarcopenia, urging caution when extrapolating findings from mouse studies to human health.

Unanswered questions around the tissue specificity of rapamycin (01:12:00)

• High doses of rapamycin can inhibit mTOR in all examined tissues, but CNS penetration is more challenging, requiring multiple doses.
• At lower doses, as used in lifespan and healthspan studies, the effect on different tissues may vary.
• There is a need to understand which tissues are most affected by low-dose rapamycin related to lifespan impact.
• Studies have not been done systematically to determine mTOR's role in individual tissues regarding aging.
• No biological system is immune to aging, implying a need for tissue-specific information under low-dose rapamycin treatment.
• Rapamycin does not uniformly affect all mTOR substrates, and its inhibition varies across tissues and doses, raising more questions about its tissue-specific effects during aging.

Rapamycin’s ability to cross the blood-brain barrier & its potential impacts on brain health & neurodegeneration (01:18:09)

• Efficacy of rapamycin in crossing the blood-brain barrier (BBB) is debated; effective inhibition of mTOR complex 1 in the brain occurs at higher doses.
• The BBB may weaken with age, potentially allowing better penetration of rapamycin in older individuals; however, direct data on this is lacking.
• Rapamycin has a relatively large size for a small molecule (around 1000 Daltons) which could impact its ability to traverse the BBB.
• Its lipophilic nature is likely more relevant to its ability to cross into the brain, potentially becoming trapped in lipid-rich areas.
• Biomarker C2N, used to monitor amyloid levels in humans, suggests that intermittent rapamycin dosing lowered amyloid in plasma for high-risk patients, but causality with brain amyloid levels is unclear.
• The hypothesis posits that peripheral actions of rapamycin may affect the brain, including possible effects on immune dysregulation and inflammation.
• Systemic immune dysregulation with age could lead to more immune cells penetrating the BBB and contributing to brain inflammation.
• Effects on brain disorders could be achieved by targeting mTOR inhibition in peripheral tissues such as the liver.
• Clinical practice includes using rapamycin for patients with mild cognitive impairment and amyloid beta elevations, as there is no existing effective treatment, and it may improve inflammation and autophagy.
• Alzheimer's disease complexity could mean various subtypes respond differently to rapamycin, emphasizing the need for more clinical research.

Rapamycin's Transition to Human Use and its Patent History (01:26:40)

• Rapamycin's transition to humans was slow, resulting in minimal profitability and diminished research interest.
• Everolimus, a derivative of rapamycin, is part of the Novartis portfolio.
• Original patents on rapamycin did not adequately cover derivatives, leading to multiple rapalogs, such as Everolimus.
• Rapalogs have similar biochemical mechanisms, binding FKBP12 to inhibit mTOR complex 1, but differ in bioavailability, tissue distribution, and metabolism.

Rejuvenation of the Immune System Study and Rapamycin Derivatives

• Impacts derivatives of rapamycin on the immune system were initially tested in other mammals before humans.
• The study by Joan Mannick used drug everolimus, showing potential for immune rejuvenation.
• Rapalogs demonstrated almost identical actions in cell cultures, with subtle variations mostly serving patent strategies.
• Rapamycin became a generic, off-patent drug early in its lifecycle, yet remained expensive due to a lack of alternatives.

The Immune Modulating Effects of Rapamycin and its Derivatives

• Everolimus proved it could enhance immune response to the flu vaccine in older adults without significant side effects.
• The concept changed from perceiving rapamycin as an immune suppressant to an immune modulator.
• While initially used as an immunosuppressant for transplants, data in cells and mice did not strongly indicate rapamycin’s effectiveness in this role.
• No evidence suggests rapamycin increases infections contrary to its immunosuppressive branding.

Clinical Trial Endpoints for Gero-Therapeutics

• Clinical studies like Mannick’s are pivotal for finding functional endpoints for FDA approval of gero-therapeutic drugs.
• Trials have shown that older mice and humans can have improved immune response through treatments like rapamycin derivatives.
• Human trials confirmed that lower doses of rapamycin derivatives are tolerable and may have potential as gero-therapeutics for immune rejuvenation.
• Future studies of rapamycin and its derivatives might focus on clarifying the distinction between immune modulation and suppression.

Might rapamycin induce changes in T cell methylation patterns, potentially reversing biological aging? (01:40:41)

• There is agreement that rapamycin would change the epigenome in T cells, but uncertainty if this equates to reversing biological aging.
• The current understanding and measurement of epigenetic age may not accurately reflect biological aging, raising skepticism around whether rapamycin could reverse epigenetic aging.
• Research on canonical age-related epigenetic changes in T cells and their link to functional declines is still in early stages.
• Although impressive immune system changes were observed in mice with mtor inhibition, it remains unclear if this is due to cell cycle delay or a fundamental rewriting of the genetic code.
• Rapamycin's impact could be on the state of the cells, not necessarily on inducing a specific epigenetic change through a defined signal transduction pathway.
• While rapamycin affects all 12 Hallmarks of Aging, its link to epigenetic changes is weaker compared to other areas like mitochondrial functionality.
• It's speculated that rapamycin may chiefly reduce hyperactivation of the immune system, therefore resetting its function to respond more like a young immune system to a vaccine.
• There's evidence that transient treatment with rapamycin can rejuvenate function in other tissues and organs such as the heart, brain, and ovaries, with persistent effects post-treatment.

(As requested, the summary contains only key points from the specific sections of the YouTube video transcript structured into bullet points under the relevant heading.)

Rapamycin side effects & impacts on mental health: fascinating results of Matt’s survey on off-label rapamycin use (01:49:00)

• Survey compared over 300 off-label rapamycin users with nearly 200 non-users who were equally health-conscious.
• Both groups had similar demographics and lifestyle habits, likely biased towards health-conscious individuals.
• Key takeaway: no significant side effects associated with off-label rapamycin use except for mouth sores.
• Frequency of mouth sores among users was approximately 15%, which aligns with the prevalence in organ transplant patients.
• The cause of mouth sores might be linked to rapamycin's effect on fast-proliferating epithelial cells and potential disruption of barrier function.
• No noticeable side effects on hair and nails; however, pre-treatment with rapamycin can mitigate hair loss from chemotherapy in mice.
• Suggested potential remedies for mouth sores include FK 506 mouthwashes or using inert analogs that bind to FKBP without targeting mTOR.
• Side effects that were lower in rapamycin users included abdominal cramps, depression, and anxiety.
• Growing evidence suggests rapamycin could have beneficial neurocognitive effects.
• Discussions around rapamycin's role combined with ketamine for depression and chronic pain, with anecdotal reports indicating enhanced effects or reduced frequency and dosage requirements.
• Contradictory studies highlight the need for clarity on the interaction between rapamycin, ketamine, and mTOR, especially regarding dosing and administration methods.

Unanswered questions & future research directions [Expansion on mental health impacts]

• The precise mechanisms by which rapamycin might relieve depressive and anxiety symptoms are still unclear.
• The combination of rapamycin with ketamine in clinical settings points to potential therapeutic approaches that warrant further exploration.
• Discrepancies in existing studies necessitate reexamination to ensure a thorough understanding among researchers and clinicians.
• The relationship between rapamycin's side effects, dosage, and administration methods remains an area of active inquiry.

Impact of taking rapamycin in people who contracted COVID-19: more insights from Matt’s survey (01:59:32)

• People taking rapamycin did not show a significant difference in the likelihood of a positive COVID-19 test.
• Among rapamycin users, there were no observable differences in COVID-19 severity or post-infection symptoms for those who started taking it after their infection.
• Those who took rapamycin throughout their infection had statistically lower severity rates and a decreased likelihood of long COVID symptoms.
• Continuous rapamycin use might be beneficial against chronic inflammatory responses linked to severe or long-term COVID-19.
• The properties of rapamycin could potentially apply to other viral infections, not just COVID-19.

What David would like to study with mTOR inhibitors (02:05:20)

• David is interested in studying rapamycin and other mTOR inhibitors to induce a metabolic state that can’t be achieved through dietary means.
• Allosteric inhibitors like rapamycin partially inhibit mTORC1 and mTORC2 but do not completely stop their activity.
• Catalytic inhibitors fully suppress mTOR activity but are highly toxic to cells and animals and could potentially cause death.
• There's interest in the potential for catalytic inhibitors to massively activate autophagy and rewire the system temporarily, possibly having epigenetic impacts.
• Dietary restriction impacts the mTOR pathway but differs from what can be achieved with rapamycin.
• The broader effects of dietary restriction, both positive and negative, are not often considered when comparing it to rapamycin use.
• Catalytic inhibitors often lack specificity for mTOR, affecting other kinases, which could underline why they are less ideal for clinical use due to side effects.

Joan Mannick’s studies of RTB101 & other ATP-competitive inhibitors of mTOR (02:09:50)

• RTB101 is an ATP-competitive mTOR inhibitor that also inhibits other kinases.

• Studies at Restore Bio dosed people with RTB101 without significant side effects, but efficacy was in question as the trial was shut down.

• In the RTB101 trial, it was combined with everolimus, with two arms in the study: combination and RTB101 alone.

• RTB101 was originally a Novartis molecule known as NVP-103 with dual mTOR and PI3 kinase inhibiting activities, considered a "dirty" molecule with multiple target interactions.

• Wyeth developed an mTOR inhibitor that was exquisitely specific, though Pfizer de-emphasized it upon acquisition.

• There is curiosity about the relative benefits and side effects profiles of various mTOR inhibitors in animal models, and whether anything is superior to rapamycin for longevity.

• Catalytic inhibitors, like RTB101, are hydrophobic and challenging to use in dosing for animal experiments.

• RTB101, in cell culture models, showed antiviral gene expression, providing a rationale for using it in clinical trials related to vaccine response and infection resistance.

• A decision was made to proceed to a pivotal trial with RTB101 alone, potentially influenced by patent life and a clear path to market.

• The pivotal trial of RTB101 was halted mid-way as it failed to meet its FDA-mandated endpoint based on patient-reported infections.

• Post-trial analysis revealed that RTB101 recipients had a significantly lower risk of subsequent infection from influenza viruses and coronaviruses, leaving the drug's failure and its impact on immune function as an open topic.

• The effectiveness, mechanism of action, and potential issues with the original study of RTB101 remain unclear due to its broad targeting profile.

Impact of mTOR inhibition on autophagy & inflammation & a discussion of biomarkers (02:20:10)

• mTOR inhibition impacts protein synthesis, which in turn affects various processes, including autophagy, inflammation, and cellular senescence.
• Autophagy is believed to be a key mechanism through which mTOR inhibitors like rapamycin confer their longevity benefits.
• Inhibition of mTOR can also reduce the production of soluble factors by senescent cells and influence proteomics significantly.
• Autophagy can be linked to the recycling of cellular components and is directly regulated by mTOR through pathways such as those involving the transcription factor TFEB, which promotes lysosome production.
• Impact on aging is also due to mTOR's role as a general regulator that modifies many different processes, which is important because aging affects nearly all cellular components.
• Different downstream effects of mTOR may be significant in various contexts or diseases, e.g., cell size in hypertrophy or cell cycle in cancer.
• In C. elegans models, the longevity benefits of mTOR inhibition seem to be mostly attributed to autophagy activation, but in yeast, effects on mRNA translation are predominant.
• In mammals, including humans, anti-inflammatory effects of mTOR inhibitors like rapamycin might account for many functional benefits seen with age, particularly through the reduction of sterile inflammation.
• There is an identified need for better biomarkers to capture the impact of drugs like rapamycin on processes such as inflammation and autophagy, but current markers for autophagy are notably insufficient compared to those available for inflammation.

The Dog Aging Project: What We’ve Learned From Testing Rapamycin in Dogs (02:28:24)

• Companion animals, specifically dogs, are valuable for aging studies because they share our environment and are not genetically inbred like lab mice.
• Dogs age faster than humans, allowing for timely clinical trials to measure aging-related outcomes.
• More than half of the population views pets as family members, making their health span and longevity a worthy study focus.
• The Dog Aging Project assesses rapamycin's effects on dogs' lifespan and health metrics in a rigorous clinical trial that began 7-12 years ago.
• The study is a double-blind, randomized, placebo-controlled trial with a cohort of 580 dogs to detect a 9% change in overall lifespan.
• Candidates for the study are dogs that are at least seven years old, weigh between 40 and 110 pounds, and have no significant pre-existing age-related diseases.
• Lifespan is the primary endpoint, but health metrics such as cardiac, neurological, and cognitive functions are also evaluated.
• Results may translate to human biology and contribute to the understanding of rapamycin's potential as a longevity extension therapy.

Preliminary Results of Primate Studies With Rapamycin (02:36:20)

• Non-human primate studies, specifically with marmosets, provide important insights since they have a shorter lifespan than rhesus monkeys.
• Preliminary data indicates potential lifespan extension due to rapamycin treatment in marmosets.
• The primate study conducted in captivity may have limitations related to environmental factors compared to real-world scenarios.
• It is acknowledged that the effects of rapamycin in a controlled lab environment could differ from those in a non-lab, free-living setting.
• The potential benefits in marmosets are promising but must be considered with caution due to the controlled study conditions, which may not fully represent the daily challenges faced in more natural settings.

Dosing of rapamycin (02:39:31)

• Initial study tested rapamycin doses of 0.1 mg/kg thrice a week and 0.05 mg/kg thrice a week.
• Follow-up dosing protocol settled at 0.15 mg/kg once a week for a large clinical trial.
• Decision based on prior outcomes, desire to mimic the once-weekly dosing seen in everolimus studies, and practical considerations for pet owners.
• Concerns include the dose being too low to see significant effects, balancing risk aversion in pet studies.
• Mouse studies used a much higher dosage of rapamycin compared to what is extrapolated for human dosing.
• Most off-label human users take rapamycin once weekly, primarily 6 mg, due to historical precedent and community consensus.
• Some individuals take much higher doses up to 20 mg once a week, often adjusting over time to gauge side effects.
• Concurrent intake of grapefruit juice observed among some users to increase bioavailability of rapamycin.
• Warnings against using compounded formulations of rapamycin due to potential issues with Purity and bioavailability unless there are FDA certificates.
• Importance of enteric-coated capsules for rapamycin to ensure bioavailability given its instability at gastric pH levels.

Considerations and Warnings for Off-label Use and Dosing [9571.0 continued]

• Rapamycin is not a cheap drug, with prices around $5 per milligram.
• Caution advised against using compounded formulations from compounding pharmacies, due to variability in Purity and concentration.
• The branded drug rapamune and generic serulimus are recommended over compounded versions.
• A podcast is mentioned that covers the ins and outs of compounding pharmacies; not all are implausible, but due diligence is necessary.
• Emphasized importance of ensuring rapamycin is in an enteric-coated capsule for proper bioavailability.

Effect of rapamycin on fertility (02:49:33)

• Research into the effects of rapamycin on ovarian aging and spermatogenesis is lacking due to limited funding, largely because it lacks a direct profit motive.
• Studies have shown that in female mice, transient rapamycin treatment can delay or possibly reverse ovarian atrophy, restoring reproductive capacity to sterile mice.
• Conversely, in male mice, rapamycin appears to impair spermatogenesis and potentially induce sterility.
• The differential effects between male and female fertility might be due to the cell proliferation rates in spermatogenesis vs. oogenesis.
• Spermatogenesis is a rapid proliferation process and is possibly more impacted by rapamycin due to its effects on cell growth, while oogenesis is a slower process.
• Once male mice are taken off rapamycin, there might be a preservation of sperm quality, indicating that effects are dependent on dose and duration of treatment.
• The role of rapamycin in anti-aging could be attributed to either a slowing of the cell cycle or true rejuvenation of cells.
• There are indications from not yet published research that rapamycin may enable structural rejuvenation of atrophied ovaries in mice.
• The potential to improve fertility outcomes by preserving or restoring Anti-Müllerian Hormone (AMH) levels in women who have not reached the lowest AMH threshold is a topic of great interest.
• Human clinical trials funded by smaller grants, such as one led by Columbia's reproductive aging center, are exploring the safety and potential efficacy of rapamycin for ovarian function in women with premature ovarian failure in a double-blind, placebo-controlled randomized clinical trial.