Hello, I'm excited to be part of this symposium focusing on bone health. I'm sorry I'm not able to join you in person today. The purpose of this talk is to address whether there are any effects of bisphosphonate beyond the skeleton where we typically use these agents for. And really this is meant to open up our horizons or our minds into possibly effects of bisphosphonate beyond the skeleton. And so we'll start by talking about cardiovascular effects. We'll talk about mortality and cancer, and then we'll discuss some new interest in senolytic effects specifically of zoledronate and then impact on muscle function and safety measures. So we're starting with cardiovascular and focusing on atrial fibrillation. This is data from the FIT trial that led to the approval of alendronate in the 90s. 6,500 women, mean age 69 years. And there was really no increased risk in development of any atrial fibrillation in these women with 2.5% versus 2.2% and non-significant hazard ratio. A concern whether alendronate may increase the cumulative incidence of the risk was very minimal compared to placebo and the hazard ratio did not reach statistical significance. Looking beyond that at observational studies, this is a case control study from the United States that looked at about 800 cases of atrial fibrillation versus 900 controls. And the odds ratio of having used alendronate in patients with atrial fibrillation was slightly higher. So a higher risk of having been exposed to alendronate in the AFib group compared to controls. In Europe, which a much larger case converse is 68,000 controls. And then looking at current users of alendronate, the adjusted relative risk was not significant indicating possibly no effect of alendronate on atrial fibrillation. What about zoledronate and atrial fibrillation from the HORIZON trial that used zoledronate versus placebo and osteoporosis? Risk of any atrial fibrillation was slightly increased 2.4% versus 2%. However, did not reach statistical significance. But serious atrial fibrillation slightly increased the risk 1.3% versus half a percent in placebo and the risk was statistically significant. And this is more closer to what we've seen with alendronate that serious atrial fibrillation, the risk may slightly be higher with use of this phosphonase. The prevention trial, which used a lower dose of zoledronate and osteopenia, the risk of atrial fibrillation was not increased in these women. Beyond atrial fibrillation, however, if you were to look at cardiovascular events in general, this is again the HORIZON trial. And as a reminder, HORIZON was 8,000 women, mean age was 73. And here they were looking at the impact of any cardiovascular events. So, MI, stroke, death from stroke, death from cardiovascular causes, and all-cause mortality. And you can see that even though there was no difference in cardiovascular events between zoledronate and placebo and any of these, the cumulative incidence, if you were to put all of these together, there was a decreased incidence with zoledronate compared to placebo. This is raising the question whether actually zoledronate is cardioprotective in terms of reducing risk of mortality, although there was no difference specifically in cardiovascular mortality. Again, the PREVENTION trial, which is the trial that used zoledronate at a lower dose for osteopenia, not for osteoporosis and mortality. This is the data on mortality listed here. There was possibly a reduced risk of mortality, so survival benefit in women using zoledronate, but certainly a lower risk of myocardial infarction, possibly reaching statistical significance with zoledronate reducing the risk of MI compared to placebo in these women. Again, the data is very slim, and as you can see, all of these numbers are very close to each other, so there is a hint that could be reducing risk of cardiovascular events with bisphosphonate. But I showed you some data on mortality, and I wanted to look a little bit deeper into the mortality improvement with these medications. Going back to allandronate and mortality, again, a renewed interest in this came in the past maybe eight years to try to see if there is any cardiovascular protection with allandronate. When it looks at overall mortality, looking at all of the trials that used allandronate overall, there was no difference when it comes to mortality with allandronate. The story is a little bit different with zoledronate. As I showed you earlier, with the HORIZON and the PREVENTION trial, there may have been an improvement in mortality. This was shown much more in the two studies. The HORIZON recurrent fracture trial, so this is women who already had a fracture receiving zoledronate, and there was a significant reduction in mortality in these women, but this was not seen in the pooled analysis of those aged 75 or older, so it was younger women that benefited in terms of survival following a fracture. This is the study that looked at zoledronate every 18 months for four doses in women with osteopenia, so 65 years or older with osteopenia, not osteoporosis. There, there was a reduction in risk of total cancer incidence. This is mostly driven by a reduction in risk of breast cancer in these women with a significantly lower risk with the use of low-dose zoledronate. Going back to that graph I showed you earlier with zoledronate reducing mortality compared to placebo in these women, there was a reduction in mortality. So what did these women overall die from? And this is what this graph is showing us. This is the risk of deaths from different causes, and this is placebo in gray and zoledronate in black. And you can see that it is possibly what's driving an improvement, more survival with zoledronate, maybe a reduction in risk in arrhythmia, which was statistically significant, and lower risk of pneumonia with zoledronate. There was possibly an increased risk with stroke, death from stroke, but this did not reach statistical significance. So we still don't really understand why there is an improvement in mortality with zoledronate beyond the effect of the skeleton and in women who already had a fracture where you are reducing the risk of mortality following a fracture. And so looking at overall mortality with all the studies that use zoledronate, again, as I mentioned, maybe a hint that there may be an improvement in mortality with zoledronate, but it was more apparent in some of the studies, not in all of these studies. In older patients, there were a couple of studies that looked at frail older women, and these are called the ZEST studies, the ZEST and ZEST-2. Both of these studies were looking at frail women. The ZEST was frail women 65 years or older, but the mean age was 85. So they were older women living in nursing facilities that only received a single infusion of zoledronate, and there was no impact on functional status, but it was not powered for functional outcomes. The ZEST-2 study that looked at placebo versus zoledronate in the same population, so older frail women, but these are women who had osteoporosis and received annual zoledronate for three years, the typical dose that you use for osteoporosis. And although the primary outcome here was on bone density change, there was secondary outcome to look at mortality, and this is why I wanted to show you this here. There is prelim data from the ZEST-2 study that is now published on clinicaltrials.gov. I just accessed this earlier this month, and this is the data that they are putting. There was no full manuscript or publication yet from this data, but it's showing that in women, frail older women that received a reduction in mortality. So 8% death in the zoledronate arm versus 12% in the placebo arm. Now, these are small numbers, so I don't know if they reach statistical significance or not, but this is continuing the same story that in someone who has a fracture or someone who's frail, there may be a mortality benefit to zoledronate specifically, even if not an andronate in these women. This is the data from Taiwan that looked at 45,000 new cases of osteoporosis following a hip fracture that received different types of medications, and here it is showing us the survival for these different drugs. Obviously, someone who fractures and does not receive any medication following a hip fracture, these have the highest mortality. The lowest mortality was with the Nosimab, so a significant improvement in survival with the Nosimab, with zoledronate, but also with oral bisphosphonates in the gray and yellow here. So all these agents do have a survival benefit in someone who's had a hip fracture as compared to no treatment or treatment with a CERN. Looking at mortality and this cancer incidence that we mentioned earlier, we did go a study in Rochester using the Rochester Epidemiology Project. Just a quick note on this, this is a population-based database that gives us access to medical records of all community members in Olmstead County in Minnesota, and we are able to look at population-based analyses of their health data over time. We included men and women over the age of 50 who were prescribed a bisphosphonate between 2005 and 2021. We excluded anyone that received the prior medication before the bisphosphonate. We also excluded anyone who had a history of cancer. We did propensity score matching for referent population and did Cox adjustment for age, sex, and Elixhauser score. And this is the data for the bisphosphonate users, 3,800 individuals compared to 3,800 referents, age matched, mostly women, mostly white, and then they have an average BMI. Half of them were smokers and they had about three comorbidities in general. So they were prescribed alendronate, less so resedronate, zoledronate, or other bisphosphonates. And looking specifically at all bisphosphonate, there was no benefit with mortality. So in all population, men and women, no mortality benefit. With alendronate specifically, again, no mortality benefit in this population. With zoledronate, there was possibly an increased risk of mortality in men receiving zoledronate. Now keep in mind that we excluded men that had a history of cancer. So this is zoledronate for osteoporosis, not for prostate or other types of cancer, but it makes you wonder what type of men are we prescribing these zoledronates to. So the increased risk of mortality may be related to the reason for prescribing this medication in a more frail or higher risk population rather than zoledronate itself increasing mortality. What about cancer incidence? Because this was shown in one of the studies that these bisphosphonates may reduce the risk of cancer. The case here in turquoise is showing us the bisphosphonate users compared to the red, which is the referent population, females and males. There was possibly an increased incidence of cancer in those using bisphosphonates, again, different than the data that was seen in other population-based studies mentioned earlier. And so we still don't know in terms of the mortality or in terms of cancer incidence, whether there is any benefit for bisphosphonate. Data is all over the place, as I showed you, some showing benefits, some no benefit, and some in our data may be increased risk. But there was a proposed hypothesis, and this is data from a Canadian multicenter osteoporosis study that looked at the difference between different types of bisphosphonates. So nitrogen-containing versus non-nitrogen-containing bisphosphonate, and whether the difference is related to that. And you see survival probability in nitrogen bisphosphonate, but not in the other types of bisphosphonate, like, excuse me, ethidronate. And so, again, many hypotheses when it comes to that, including the type of bisphosphonate. In our study, we did not specify which bisphosphonate, we grouped them all together. But when we looked at alendronate and zaledronate separately, there was no difference compared to the overall group. But also brings up the question of what patients are benefiting the most, and we don't have good answers when it comes to that. Moving on to another possible benefit from bisphosphonate, or impact of bisphosphonate outside of the skeleton is on cellular senescence. Very briefly, what is cellular senescence? Cellular senescence is considered one of the core pillars of aging. So with aging, we do have a lot of changes that occur, DNA damage, oxidative stress, epigenetic changes, stem cell exhaustion, telomere attrition. But at the core of all of that is something called cellular senescence. And what cellular senescence is, and this is depicted in this scheme, this can happen at any tissue in the body, where you can have one of the cells that develop all of these changes over time that I mentioned at the DNA, but also genomic and protein level, where you have accumulation of errors in metabolism. The cell goes into a stage called the senescence stage, where the cell is no longer dividing, it goes into a cellular arrest G0 stage, but also the cell is not dying, it is resistant to apoptosis. And this is why it is called senescent. Loosely, some people call them the zombie cells, because they are resistant to apoptosis, but also are not multiplying anymore. These cells that determine senescent cells will start secreting a senescent profile called the SASP, senescence-associated secretory phenotype. And these are a number of chemokines and inflammatory markers that will impact the surrounding tissue. Even the healthy tissue around them will become dysfunctional, causing chronic disease. And so with the cellular senescence developing at multiple tissue level over time with age, we can see that this will be responsible for development of many of the chronic age-related conditions, including osteoporosis, but diabetes, other metabolism dysfunction, including immune dysfunction, eventually leading to frailty, sarcopenia, and a decrease in health span, and eventually leading to death. The important part of this is if you can find the targeting settlement of these chronic conditions and increasing the health span of the population with less frailty, less sarcopenia, even if we are not impacting life, but we are impacting their health span. And so with the hypothesis related to the bisphosphonate, we looked specifically at the senescence in human bone. This is bone biopsies in young women and older women. So young women were average age of 30 and older average age was 70. And in the bone biopsy, we looked at markers of the cellular senescence, expression of P16 and P21 and P53. All of these have been shown in animal models to be increased expression with aging and in cellular senescence. And you can see that older women in the bone, they have a higher expression of P16 and P21, but not P53, telling us that there is increase in cellular senescence in the bone microenvironment. And in the same biopsies, we looked at various markers, expression of various markers of these SAS that I mentioned earlier, the chemokines and inflammatory markers that are being produced during cellular senescence. And we showed a significant increase in multiple of these senescent markers and the inflammatory markers, I'm sorry, chemokines affecting the bone microenvironment in these human bone biopsies. And so using animal models, we went back and looked at zoledronate, both in humans and in animals. We took human senescent fibroblast, that is derived from peripheral tissue, and they were treated in vitro with increasing concentrations of zoledronate for 72 hours. And we showed that there was a decrease in the senescent cells that were dying more preferentially at increased doses of zoledronate compared to the total number of cells. And so at very high levels of zoledronate, toxic levels, everything will die, healthy and senescent cells are dying. But at lower doses of zoledronate, there was preferential loss of the senescent cells compared to the total number of cells, telling us that zoledronate was preferentially killing senescent cells. And these are the same data showing different concentrations of zoledronate with the senescent cells in green and then in blue, the total number of healthy cells. So there was a decrease in the senescence burden with zoledronate in vitro. And this is, again, showing us the same type of concentration in a different type of graph, showing us preferential loss of senescent cells with increasing concentration of zoledronate. We went back and looked at the same study, but in vivo, looking at zoledronate in mice, the 22-year-old, months-old, sorry, mice, which is the equivalent of about 80-year-old in human. Zoledronate, given intraperitoneally, compared to vehicle, twice weekly for eight weeks. And we looked at these SAS markers that I mentioned earlier, but we also did in mice, frailty profiling, as well as after the mice died, we looked at the bone samples to look at PCR analysis. These are the SAS markers that I mentioned earlier that we did in human bone biopsies. This is in these mice. And you can see that there was preferential change in some of these markers that were differently expressed with zoledronate compared to mice that were treated with vehicle, telling us that zoledronate is impacting the expression of some of these senescent markers in the tissue. And looking specifically at some of these markers, you can see a lower expression of many of these markers with zoledronate compared to placebo-treated mice. And again, the P21 and P16 that we talked about earlier, this is specifically looking at the bone PCR, bone tissue PCR, there was a reduction in P21, but not in P16 with zoledronate use. Looking at frailty markers, this is grip strength, and you can see that grip strength was higher in the zoledronate-treated mice compared to the placebo, and possibly higher hang endurance and maybe treadmill endurance. Only the grip strength was statistically significant. Other markers of frailty were not statistically significant, but there was a trend for possibly better muscle function with zoledronate. And so talking about frailty, I wanted simply to mention that there has been a couple of data looking at frailty in humans with zoledronate. I mentioned this study earlier, frail women receiving a single infusion of zoledronate, and it did not show any functional change in these women. However, I wanted to show you here, this is the data from the study showing us no significant functional changes. So activities of daily living, gait speed, no difference between zoledronate and placebo. But at baseline, these women with zoledronate were already falling higher. So there was a higher number of falls in the past year, and a higher number of recurrent falls in the past year. So it is possible that we're starting from a more frail group that went into the zoledronate compared to placebo, that may have confounded those results. There are currently two ongoing clinical trials, one of them is looking at zoledronate versus denosumab, with the primary outcome being frailty outcome measures. These are pilot studies looking at 20 older adults. So we look forward to seeing whether this will have any impact on these frailty markers. So to summarize, as I mentioned earlier, this is simply to open up our minds into maybe there are benefits from bisphosphonate beyond the skeleton. Evidence suggestive of cardiovascular benefit of bisphosphonate, but not sufficient. No significant signal for higher risk for atrial fibrillation. No clear mortality benefit with oral bisphosphonate, but zoledronate may reduce risk of MI, overall mortality, particularly following hip fracture. And there may be a senolytic effect of zoledronate, but we don't know what the clinical implication of these are. We did not talk about mechanisms today. I simply wanted to leave you with this. There is, we published some data about what are the possible mechanisms for bisphosphonates into reducing mortality and having some of these benefit. There is preclinical data showing bisphosphonate effect on lipid profile, arterial wall calcification and phosphate metabolism, including FGF23 that may reduce the atherosclerosis impact in patients receiving these bisphosphonate may be leading to that. You would also, I suggest that there may be also an immunomodulatory effect and senolytic effect of these bisphosphonate, particularly zoledronate that may also be impacting many of these pathways leading to a decrease in mortality. And with that, I'll be happy to take questions. Thank you for your attention. Thank you.

bisphosphonates

cardiovascular effects

zoledronate

senescence

atrial fibrillation

senolytic properties