Great. Thanks very much, Anthony, and thanks everyone for tuning in this evening or this afternoon if you happen to be in Colorado. Great to be here as always and, and, you know, I think that Anthony or one of his, his crew actually was the, with the people who suggested this particular topic.
And actually, I think it's quite timely, as I was just mentioning before the, the webinar started. I think that this is certainly something that's getting a lot of press recently and I have to say as an oncologist who does a lot of consultation with with general practise vets, I, I do think that this is a kind of therapy that, that is very attractive, but because of its attractiveness, it does have the potential to be, let's say overused or or, or abused, in circumstances where it might not be the most appropriate therapy. So I think having an opportunity to sort of take a step back and say, well, what, what do we really know about metronomic chemotherapy, both in general and specifically in, in this case, I'll talk mostly about dogs.
Where are our knowledge gaps? How can we, make rational decisions about which tumour types might benefit, which drugs we should use, how should we give them, etc. I think are, are great and again, especially as a, as a person who does a lot of research, it really does help us to identify those gaps, and those areas for future study that I think are very important as well.
So without further ado, let's go ahead and start talking about metronomic chemotherapy. So, I always put a conflict of interest disclosure, up, for all of my lectures and actually none of the, the conflicts of interest that appear on this slide are, are particularly relevant, for this hours of lectures. So, some of the topics that we'll cover in this hour include some of the targets of metronomic chemotherapy.
So in other words, what cells in the body are we actually trying to inhibit or impair or kill with metronomic chemotherapy and how might those be different from the, the goal of sort of more traditional chemotherapy. And then again, we'll discuss some of the current challenges. So again, which drug how should we give those drugs?
Which diseases might be most amenable to this kind of therapy? How are the best ways to sort of study these kinds of treatments? And what kinds of things might we be able to measure?
To be able to know if our treatment is actually working. So, let's talk a little bit about sort of conventional chemotherapy. And the other term that's, that's often used that you may hear me use in this lecture is, is what's called maximum tolerated dose or MTD chemotherapy.
And these are the types of chemotherapy that I think most of us would, would think about would come to mind when we think about chemotherapy, whether it's in dogs or whether it's in humans. So this is the administration of large doses of, these cancer drugs all at one time. With them a break following the treatment to allow recovery of any damage that may have occurred to some of the normal tissues in the body.
So, the goal behind this kind of MTD chemotherapy is really to give a dose of chemotherapy that's large enough that it's gonna kill some tumour cells and not do so much damage to the rapidly growing normal cells that the side effects are unacceptable. And then again, give a period of time. Where those normal cells have the potential to kind of fix themselves and regrow, and the hope is that the tumour cells, A may be more sensitive to these drugs, and B, may be less able to repair themselves.
And it's known that for many of the agents that we use like this, it is the rapidly dividing tumour cells, those cells that are rapidly proliferating, that tend to be the most sensitive to these agents. And again, the MTD, as I mentioned, is considered the highest dose that we can give of these medications in an infrequent basis that cause what would be considered an acceptable level of side effects. And of course what is considered acceptable is different in humans than it is in dogs, and it's even very different from dog owner to dog owner.
So that's always something that again is a, is requires some degree of personalization. So some of the other, cells in the body that we think of as potentially being these rapidly dividing cells, that have the potential to be injured as a result of chemotherapy include the bone marrow stem cells, which is why we can see things like neutropenia from chemotherapy, the gastrointestinal crip cells, which again is responsible for some of the gastrointestinal toxicities that we see, and the hair follicle cells. And again, as most of us probably are aware, alopecia.
It's not a major side effect that we see in most dogs. The exception being the non-shedding breeds, the breeds that have to go to the groomers, to get cut periodically. And the main reason for the difference is because of the fact that only about a third of, most dogs' hair is in tealagen phase at any time, and those are the ones that are rapidly dividing.
So not that many are sensitive. So again, the, the goal behind these conventional NTD therapies is to give a nice amount of time following treatment to allow the recovery of some of the normal tissues. One of the things that we've learned though, that's really quite interesting and, and I'll sort of jump ahead here and say that one of the big targets for metronomic chemotherapy is actually not the tumour cells themselves, but it's actually the blood vessels that are necessary obviously to provide nutrients and oxygen to the tumour and remove waste products.
So one of the things that's been determined in the laboratory setting is when in fact you administer large doses of chemotherapy, this MTD type chemotherapy, in addition to killing tumour cells, you actually do do quite a nice job of killing some of the blood vessel cells, specifically the endothelial cells that are lining the blood vessels. But the unfortunate thing is that the kinetics of repopulation of those endothelial cells is exactly the same. As the kinetics of repopulation of the other normal blood cells.
So it's the same as, resolution of neutropenia, repair of the gastrointestinal crip cells. Those blood vessel cells come back with the same kinetics. So as a result, those prolonged breaks have the potential to, again, initially cause a lot of, of endothelial cell death, but then allow those endothelial cells to regrow, which kind of defeats the purpose a bit.
So the idea behind metronomic chemotherapy, and another term that we use routinely for this is called low dose continuous chemotherapy. So where conventional chemotherapy may use a dose of chemotherapy. With a very long break in between, so 3 weeks, let's say between treatments.
There are some treatments in veterinary medicine, certainly the CHP protocol, the way we used to treat lymphoma, the administration of viblastin when we give it weekly for mast cell tumours, where those breaks are not as long and we're able to perform therapy on a weekly basis, for example. In human oncology, some might consider that form of therapy to be a form of metronomic therapy. But when we're talking about metronomic therapy, we're talking about giving considerably lower doses on a much more frequent basis, generally on a daily to every other day type basis.
So how do these treatments actually work? So one of the major targets, as I mentioned of, of these metronomically delivered drugs are the endothelial cells. So actually, these are the, the cells that are lining the blood vessels that are actually present within the tumours themselves.
Interestingly, the endothelium is one of the, lowest proliferating tissues in the body. So there's very, very little cellular turnover of the endothelial cells except when you're forming new blood vessels, which is what's required for tumour growth. So one of the potential sort of therapeutic indices or therapeutic advantages here is that drugs like these metronomically delivered drugs, target rapidly dividing endothelial cells and really the only place that you find those is actually in the tumours.
In addition to targeting these cells that are actually directly in the tumours, it also appears that some of these drugs are able to inhibit the recruitment and mobilisation of what are called circulating endothelial precursor cells. So these, believe it or not, are actually hematopoietic derives. So these are bone marrow derived cells that have the potential to go out into circulation and then differentiate into endothelial cells when they hit the end organ.
And studies that have been done in, in animals like mice and even some studies in dogs have suggested that up to 30% of the endothelial cells that actually constitute the vasculature of a tumour are actually derived from the bone marrow and not from ingrowth from sort of next door blood vessels. So the abilities to suppress the circulating endothelial precursors can actually really significantly, add to the potential to inhibit blood vessel growth. So how do they do this?
So there is again some direct cytotoxicity. So these drugs at actually quite low concentrations can actually kill the dividing endothelial cells themselves. They can actually potentially kill some of these circulating endothelial precursors, but there's a third indirect mechanism by which these drugs may work, and that is through basically tricking the tumour cells into making a substance that has antiangiogenic properties, and this is a substance that's called thrombuspondin 1.
So when a lot of different tumour cells are incubated with very small concentrations of these cancer drugs, concentrations that are much too low to actually kill them or inhibit their growth, these tumour cells respond by increasing the production of this, this, this, compound or hormone called thrombuspondin, which is a very potent endogenous inhibitor of angiogenesis. So there's really a two-pronged mechanism here, both direct cytoxicity and enhanced thrombuspondin production. By which these drugs have the potential to inhibit angiogenesis.
So which drugs, at least in a petri dish or in mice, which are kind of like little furry petri dishes, have shown the ability to, exert an antiangiogenic effect when given on a metronomic schedule. Well, as you can see here, the list is actually quite long and it incorporates most of, of the drugs that we use on a routine basis in our veterinary patients, including cyclophosphamide, the vinca alkaloids like vilastin. The platinum drugs like cisplatin and carboplatin and doxorubicin.
So this is quite a nice list, but, those of you who are somewhat familiar with chemotherapy will, will notice one unfortunate secret here, and that is that the majority of drugs on these lists are given as an injectable drug. And utilising these kinds of drugs on a metronomic schedule where they're given every day, for example, is really not practical for an injectable chemotherapeutic. So much more practically, we tend to, to focus on drugs that can be given on an oral basis.
And certainly one of the ones that's been the best studied is cyclophosphamide and we'll speak a bit more of that one in the future, in the next few minutes here. So there are other targets of metronomic chemotherapy besides the blood vessels that may be very important. The answer there is yes.
So there's the possibility that at least certain subsets of tumour cells could be sensitive to these drugs. All that is certainly a question of some debate. There's the potential for metronomic chemotherapy to exert some non-specific anti-inflammatory effects.
There are other kinds of immune cells that may be important, but one of the things that's actually attracted the most attention is, The ability of at least metronomic cyclophosphamide to actually interfere with the function and number of a very specific subset of lymphocytes floating around both in the blood and the lymph nodes and the tumour tissue called regulatory T cells. We'll talk a lot more about regulatory T cells, but again, this is one of the other potential targets of metronomic chemotherapy that could be very, very important. So again, in, in human patients, most of the metronomic chemotherapy protocols that we know about these oral therapies, and usually they're not given as single agents.
So rather than getting the chemotherapy drug by themselves, they're often combined with other oral medications. One, combination that's commonly used is to combine these drugs with a non-steroidal anti-inflammatory drug. In humans, it's often drugs in the coccib class, in, in veterinary medicine, we often will reach for paroxicam, although there's some debate about whether there's any difference between paroxicam and the others in terms of efficacy.
And they may also be, combined with other kinds of anti-angiogenic therapies. So, monoclonal antibodies like bevacizumab, which is a, an antibody that targets vascular endothelial growth factor. Small molecule kinase inhibitors, kind of like the human equivalent of palladia, which we'll mention briefly in the future as well.
And again, these are generally, almost never used as first line therapy on the human side, unless they're being utilised in very, very, old, very frail patients with lots of comorbidities where the goal is really palliation. And again with these kinds of therapies. Often, a reasonable goal is not necessarily to cause massive tumour shrinkage, the way you might with MTD chemotherapy, but, but a very reasonable goal for this form of therapy is actually prolonged stable disease.
So, just to give you an example of one of the studies that was done very early on in the investigation of metronomic chemotherapy in people is a study in breast cancer where they used a combination of oral cyclophosphamide and oral methotrexate given on a metronomic schedule. And actually, in this group of, of very heavily pre-treated breast cancer patients, most of whom had metastasis, they actually did see objective tumour shrinkage in about a third of the patients. And another half had stable disease for at least 6 months.
So again, when you're in a situation of, of relapse and your treatment options are limited, this is actually quite a substantially, a substantial outcome. Again, this is also something that's very well tolerated. So all the visits were outpatients.
There were no serious adverse events. And again, sort of the outcome was relatively the same as what's observed, with third line, conventional chemotherapy. So this actually appeared very, very encouraging.
And again, at the very bottom there, you can see that cost was actually quite, quite low compared to most of what's done, in oncology. So that was also very encouraging and from a human perspective, Certainly has the potential to be maybe maybe exceptionally useful in third world countries where obviously the finances associated with cancer care are really limiting. Some more recent studies have actually looked at metronomic chemotherapy for a wide variety of different human cancers, and there's a variety of additional indications and additional combinations that are sort of constantly being looked at.
So again, this is a review from 10 years ago, and the, the number of, of these studies that are ongoing is really only increased. So what do we know about metronomic chemotherapy in veterinary patients? So, the sad news is that the number of, of really well-formed clinical trials that have been performed in veterinary oncology is quite low.
And most of, the way that, doses and drugs have been selected is completely empiric. So it's totally random. Well, let's try this drug because it looks like it works in people and let's just pick a dose and see how that goes, or, or dosing may have been picked based on tolerability rather than whether it works or not.
Again, there certainly are some clinical endpoints that have been evaluated, and again, lots and lots of anecdotal use as I mentioned in the beginning. So there's an awful lot of oncologists who'll say, oh yeah, I saw a, a brain tumour respond to metronomic chemotherapy once and I saw a prostate tumour. And unfortunately, that's, that's really kind of the nature of veterinary medicine in general, is a lot of anecdote and a lot of very small kind of non-controlled clinical trials.
But, this is an example where, depending on the drug that you reach for, there actually has been some reasonably good science done, to, to establish a dose and schedule that seems to be effective. So, Just to run briefly through the literature that's out there. So the, really the first paper that was published that looked at metronomic chemotherapy for, for the treatment of any disease in, in veterinary oncology was actually a little study that came out of CSU here, that looked at dogs with, with splenic he sarcoma, that received a combination of metronomic cyclophosphamide, at quite a, quite a range of doses, 12.5 to 25 milligrammes per metre squared.
And that, that cyclophosphamide was actually alternated with another oral medication called a topezide. And then concurrent paroxicam was administered. And again, keep in mind here, look at the very beginning of that first bullet.
This was a study of 9 dogs, so a very, very small pilot study that really has not been repeated. I'll editorialise about a topezide for just a second. So this is a drug that had actually had a couple of problems when it was being administered.
One was that, the, there is a pill form of this drug that's available at least in the US, but it's really frighteningly expensive, too expensive to be practical to use. So in this study actually dogs were administered an oral, an oral dosing solution of etopicide. And again, one of the things that we would certainly caution everyone out there not to do is administer oral liquid chemotherapy.
So that is an exposure hazard for the administrator, and it's actually sometimes quite hard to know that the dosing is appropriate. Half of it ends up in the dog and half of it ends up on the dog. And as a testament to that, actually, they did try to measure blood levels of opposide in most of the dogs in the study, and they were undetectable.
So it did not appear that that was a major contributor to the efficacy of this protocol, either. So, as far as toxicity goes, there were some dogs that had very mild gastrointestinal adverse effects, but that very well may have been a result of the paroxicam rather than the cyclophosphamide. And 2 out of these 9 dogs did develop sterile hemorrhagic cystitis, which we'll talk about a bit more.
So this is an inflammation of the bladder that results from a metabolite of cyclophosphamide called acroline. And the, the, presenting complaints of this really quite closely mimic. an infectious cystitis.
So hematuria, polyuria, accidents in the house, etc. You culture them, they're negative, and actually, usually it will resolve, with drug withdrawal, although it can take some time to do that. So in this again incredibly small study, those 9 dogs that were treated with, with metronomic cyclophosphamide had an outcome that was roughly equivalent to dogs that were treated with conventional MTD doxorubicin.
And again, keep in mind this is not a randomised study or anything else. These were compared with historical control dogs. And again, the, the number is quite, quite small there.
So just to back up a second, so what do I, what do I think about this? What do I talk to owners about when we're talking about this or talk to vets about? We, there are, there are multiple, multiple, multiple studies that have been done with doxorubicin and hemangiosarcoma with much, much higher numbers than this one small study with metronomic cyclophosphamide.
So I don't think that based on this one paper, I'm comfortable saying, oh look, it, it giving metronomic cyclophosphamide and paroxicam is just as good as giving doxorubicin. I still really consider doxorubicin-based chemotherapy to be the standard of care, but If I have an owner for whom injectable chemotherapy is right out. It's completely out of the question.
And it's a choice between doing nothing and doing metronomic chemotherapy. I would certainly consider doing metronomic chemotherapy in that situation. Second study, that actually some of the same investigators, performed, was a retrospective study that looked at a relatively large number of dogs with incompletely resected grade 2 soft tissue sarcomas.
And again, this was not a randomised study. This was a retrospective study that looked at 30 dogs that received actually a relatively low dose of cyclophosphamide with paroxicam, and then 55 control dogs that received no additional therapy. And again, in both of these studies, these drugs are generally continued indefinitely as long as there aren't any dose limiting toxicities.
So 40% of the dogs in this study again have low grade toxicity and the most common thing were GI signs. Again, in my experience, very likely to be paroxicam related, more likely than cyclophosphamide related. And again, about 10% of dogs develop sterile hemorrhagic cystitis, which is relatively par for the course.
Again, in this study, there was an indication that dogs receiving postoperative cyclophosphamide and peroxicam may have had a superior outcome than dogs that received no additional therapy. Again, all the caveats that apply to this are again retrospective study, not randomised, not prospective, no placebolar. Considerable differences in the way that these dogs were followed up postoperatively as well.
All these really sort of inject a note of caution when interpreting these results. So how do I frame this study with regards to the care of dogs with incomplete resected soft tissue sarcomas? Very much like the story with hemangio that I just mentioned.
So we know of other treatments for incompletely resected soft tissue sarcomas that are effective. Second surgeries to change clean margin dirty margins into clean margins are incredibly effective. Radiation therapy is incredibly effective.
And those two really, again, I would consider to be much closer to a standard of care. But again, if we're in a situation where neither of those two are possible for an individual patient, and it's choice between doing nothing and considering metronomic chemotherapy, again, I wouldn't hesitate to talk to the owners about metronomic chemotherapy. With all the caveats that I mentioned.
And again, I will talk to you a little bit more about dosing of, of cyclophosphamide that I think is more appropriate for either of these two indications. So again, in both of these studies, really, these doses were picked empirically. So there were no measurements of any biological effects other than, again, outcome and, and adverse effects, obviously.
So another study that came out a few years ago was, was a study looking at metronomic dosing of lamustine. And, in this study, again, retrospective study in a relatively large numbers of dogs, the dose of lamustine was picked again just by taking, you know, the, the dose that's, that's routinely used when it's given every 21 days, 60 milligrammes per metre squared, dividing that dose by 21 and saying, why don't we try that? So really not very scientific rationale for why they picked the dose that they did.
And this was a protocol. It was actually associated with some fairly substantial toxicities. So, gastrointestinal toxicity was observed, liver toxicity, thrombocytopenia, azotemia, and again, more than 25% of the patients that received this therapy actually needed to discontinue treatment as a result of adverse effects.
And again, for a treatment, a treatment type that sort of prides itself on being well tolerated, I think most of us kind of feel that this is probably unacceptable. Combining that with the fact that actually the, the likelihood of seeing meaningful improvement was actually quite low. Really makes this not something that I think most of us feel comfortable reaching for.
Another drug that's been looked at a little bit more recently is chlorambuil. And chlorambuil is another oral alkyating agent in the same class as cyclophosphamide. It's been used for years and years and years for the treatment of chronic lymphocytic leukaemia in dogs and things like low-grade gastrointestinal lymphoma in cats.
So it's a drug we've used for a long time in veterinary medicine and, and again, this was looked at not for hematopoietic neoplasia, but for dogs with solid tumours. At a dose of 4 milligrammes per metre squared, once daily. About a third of these dogs were concurrently treated with non-steroidals.
And again, this was actually quite well tolerated. So there was very little in the way of adverse effects. A very small number of dogs had very mild GI toxicity, which may very well have been NSAID related.
Overall response rate was about 11%. Again, really no biological endpoint measurement here to know what the drug was doing or how it was doing it or how well it was working. But the fact that they did see a few responses was certainly encouraging and tolerability was outstanding.
a second study from the same group, this is the group at Purdue University, actually took a look specifically in dogs with transitional cell carcinoma of the urinary bladder using the same dose of oxicam, I'm sorry, same dose of of chlorambuil. Most of these dogs did receive the concurrent non-steroidal, and although the likelihood of seeing meaningful tumour shrinkage was actually quite small. A surprising number of dogs had their tumours stopped growing, for varying periods of time.
And if you took all the dogs and put them together, both, the dogs that appeared to experience benefit and those that didn't, the median progression free interval, was about 4 months. Again, so this is a, sort of a test to this idea that even without meaningful tumour shrinkage, you still could be doing some good for these patients if you can make their tumours stop growing. So this is something that we feel comfortable offering, generally as a second or third line, treatment for transitional cell carcinoma of the urinary bladder.
So it definitely is something that's in our armamentarium. A third study from the same group actually said, gee, I wonder if we could give higher doses of chlorambusil to these patients and maybe it would work better. I'm not gonna talk a lot about this, but the short version is that actually there was a much higher incidence of adverse effects in dogs that received 4 I'm sorry, 6 milligrammes or 8 milligrammes per metres per day of chlorambuil.
And there wasn't a really good hint that it appeared to be improving efficacy. So based on that information, and again, purely based on tolerability, it appears that sort of they've, they've identified a, you know, quote unquote optimal working dose of 4 milligrammes per metre square per day. So again, what's really lacking in the literature about chlorambucil is any kind of mechanistic understanding about how it's working or why or if in fact it's working, the way we think it is.
So, what kinds of things could we actually do to better investigate that? Again, so for most of these agents, with the exception of cyclophosphamide, which I'll talk about in a, in a second, really the dosages of these medications have been selected empirically, and, or, or dosed to toxicity. We do like to have, have drugs, if possible, that we can see work by themselves.
So when we're doing these combinatorial studies at the very beginning, it's really hard to know what's doing what and what's responsible either for the efficacy that we're seeing or for the adverse effects that we're seeing. So if we can actually look at these on a single agent basis initially, I think it gives us a much cleaner picture of what's going on. And then again, what are some of the potential mechanisms of action?
So we may be interfering with regulatory T cell function, we may be interfering with blood vessel growth. Can we actually dissect that out in a, in a veterinary population? So there's lots of different ways that we can do that, and most of them I mentioned already, so I'm gonna go ahead and skip this slide.
So let's talk just for a minute about regulatory T cells and why they're important. So regulatory T cells, are, are a group of lymphocytes, as I mentioned, that circulate in the blood, that are present in the lymph nodes and are also present in tumour tissue. And the job of regulatory T cells are actually to be immunosuppressive in nature.
So, during evolution, the idea behind these cells is to provide a little bit of a break, a little bit of a counter regulatory function. Against sort of an overexuberant, immune response to any sort of invading pathogen or anything else. You could imagine that if a T cell response gets too out of hand, you could see autoimmunity, you could see cytokine problems, etc.
So it's the job of these regulatory T cells to actually make sure that those things stay a bit under control and they don't get out of don't get out of control. And they do this through the secretion of cytokines that have immunosuppressive function, as well as potentially having some direct contact effects as well. So again, a very, very important population of cells that tumours have actually learned how to subvert, in order to avoid, avoid detection by the immune system.
So again, in humans, this is actually associated with a poor prognosis and a variety of different kinds of tumour. These are increased in peripheral circulation comparing tumour and normal patients, and as well as being increased in tumour cells, sorry, in tumours themselves and in tumour draining lymph nodes. And there's some evidence that this appears to be the case in in dogs as well in diseases like osteosarcoma and mammary cancer.
And again, their goal is to impair the anti-tumor immune responses. So, here's one of the earliest studies that was performed looking at regulatory T cells and their modulation that actually looked at oral cyclophosphamide as a metronomic agent and actually measured. in this case, regulatory T cells in the tumour tissue, before and after therapy, and you can see that cyclophosphamide actually substantially depleted the regulatory T cell number within the tumour tissue in this group of patients.
So, a few years ago, my colleague, Barbara Miller actually, undertook a study to try and again, determine a rational, dose of cyclophosphamide to use in dogs in this case with soft tissue sarcomas, that actually are is able to biologically modulate some of these important factors like angiogenesis and, and immune function. And this was funded by the Morse Animal Foundation. So, this was a dose finding study that started at a dose of 12.5 milligrammes per metre square.
And then the plan was to increase that in 2.5 milligrammes per metre square cohorts based on tolerability and, and the presence or absence of a biologic effect. And then, then the goals were to potentially look not only at regulatory T cells, but also indicators of angiogenesis.
So these are all dogs with grade 1 or 2 soft tissue sarcomas, who had tumours that were easy to biopsy, so we could actually look and see what was going on inside those. They all had to be feeling OK. And importantly, none of these dogs was co-treated with anything else.
This was cyclophosphamide only during the period of study. So, dogs had, regulatory T cells measured in the blood, in the lymph nodes, and in the tumour tissue. They actually had circulating endothelial cells measured in their blood, and they actually looked at the blood vessel density in the tumours, through serial biopsy using immunohistochemtry as well.
So 11 dogs were treated, and again, most of these dogs had grade 1 soft tissue sarcomas. There were a few grade 2s, and these are fairly large tumours, as you can see, the median diameter was about 9 centimetres. All of these dogs had disease stabilisation, but again, this was only a 28 day study.
So it's very challenging to make any assumptions about whether that was a biologic effect due to the therapy or just due to the natural history of the disease. So we can't really say much about anti-tumor effects based on that. No adverse events.
So actually, all the dogs tolerated this extremely well. So no difference in, in age, sex, weight, etc. Between the two groups of patients receiving the cyclophosphamide, these two different doses, 12.5 or 15.
And as has been seen for other kinds of tumours, actually, dogs with sausage sarcomas did have significantly higher numbers of circulating regulatory T cells compared to normal dogs. When, it was looked at to determine if we could deplete, or reduce the number of circulating regulatory T cells in dogs with these soft tissue sarcomas. There was a trend Toward improved or reduced numbers and percentages of regulatory T cells in dogs receiving the 12.5 milligrammes per metre square per day dose.
But actually, that became statistically significant at a dose of 15 milligrammes per metre squared per day. So, based on depletion of circulating Tregs, it appeared that this higher dose, 15 milligrammes per metre squared today was superior. We wanted to make sure that this wasn't something that was just simply, causing lympopenia, just indiscriminately, depleting lymphocytes and that the effect was selective for regulatory T cells.
So as you can see here, there is no significant change in the total number of lymphocytes. And even no significant change in the total number of CD4 lymphocytes, which used to be called T-helper cells, of which, the regulatory T cells are a subset. So it did appear that this was fairly selective specifically for the regulatory T cells.
Lodocyclophosphamide also decreased tumour micro vessel density. So again, at a dose of 12.5 milligrammes per metre squared, there was a trend toward a reduction in micro vessel density that was not statistically significant, but that was statistically significant at a dose of 15 milligrammes per metre squad per day.
So when circulating endothelial cells were measured, again, there were no statistical differences in the number or percentage of circulating endothelial cells, and this really constitutes a ridiculously small percentage of the cells in circulation, so they're quite hard to accurately measure. But what was observed is again a strong trend toward reductions in circulating endothelial cells in those dogs, especially those dogs receiving, again, the higher dose of cyclophosphamide. So, in conclusion, we feel pretty comfortable saying that what appears to be an optimal dose of cyclophosphamide, maybe 15 milligrammes per metre squad per day because of its ability to selectively decrease regulatory T cell numbers, and decrease tumour micro vessel density.
There are really no adverse events associated with this, and, and there are a couple of caveats to that. So, most of the studies that have been performed previous to this one did use concurrent non-steroidals, and that implies that many of the adverse effects that may have been observed were from the non-steroidals and not necessarily from the cyclophosphamide. I have to say this has been my experience as well.
We very rarely see gastrointestinal side effects from metronomic cyclophosphamide if it's not co-administered with an NSAID. And again, previous studies had, had suggested again, something like a 10 to 20% incidence of sterile hemorrhagic cystitis. But it's important to keep in mind that this is a 22 day or 28 day study, and the median time to development of hemorrhagic cystitis is more like 50 days.
So, it's possible that these dogs were not on therapy long enough for that side effect to become common. So, really based on that, let me just back up a second. I, I, I'm quite comfortable recommending a dose of 15 milligrammes per metre squad per day.
So now, how does one administer that practically? So, obviously the most scientific and, and the most, precise way to do that is to have specially compounded cyclophosphamide capsules prepared for your patient. So here in the US we have 25 and 50 milligramme cyclophosphamides, that are commercially available.
But any smaller than that and you need to have them specially compounded. So, however, I think that a reasonable alternative is actually to calculate the weekly dose of cyclophosphamide, that would be appropriate based on that dose of 15 milligrammes per metre squad per day. And then administer A number of 25 milligramme capsules, a certain number of days a week, spread out equally through the week, to approximate that same dose.
Let me give you an example in case that's not clear. So, for example, if I have a relatively large dog who's calculated, weekly cyclophosphamide dose at 15 milligrammes per metre square per day comes out to be about 100 milligrammes per metre squared. That'd be about 1 metre square dog roughly.
. Then an option might be to give a 25 milligramme cyclophosphamide capsule 4 days per week. So, for example, Monday, Wednesday, Friday, Saturday, etc. So equally spaced out through the week.
Similarly, if a dog had a dose that that calculated to be 50. Milligrammes per week. One could administer cyclophosphamide at a dose of 25 milligrammes twice a week.
So there have been no comparisons of using this slightly less continuous fashion of administration versus the true daily administration. And as a result, I do feel that if it's possible to have these capsules compounded, that's probably the optimal way forward. But again, as an alternative, one might be able to consider this.
Take the available capsule size, calculate your weekly dose, and administer a pill that many days a week to achieve that weekly dose. So a second study that was published very recently actually looked at a combination of temazolamide, which is another oral alkylating agent with cyclophosphamide. At a dose that some might consider suboptimum.
I won't spend a lot of time talking about this, but again, this is a study that's worth mentioning because these folks did actually spend a lot of time trying to measure indicators of drug activity. So they looked at changes in, an angiogenic growth factor called vascular endothelial growth factor. They looked at changes in the thrombospondin1 levels and they looked at depletion of regulatory T cells.
And actually, what they found was that temizolamide by itself had no effect on regulatory T cells, and it only appeared to have an effect when co-administered with cyclophosphamide. But cyclophosphamide itself also had that same effect on regulatory T cells. Neither drugs had effects on vascular endothelial growth factor or thrombospondin one that could be measured.
So based on this, at the dose that they picked, which again was somewhat arbitrary, Temizolamide added to cyclophosphamide does not appear to result in any additive benefit over cyclophosphamide alone. Two studies have come out very, very recently that have actually looked at ways to mitigate the adverse effect that we see most commonly from cyclophosphamide, which again is sterile hemorrhagic cystitis. So we've known for quite some time that when cyclophosphamide is administered in a bolus fashion, so rather than again, given, spread out over time, but given in an MTD fashion, the incidence of sterile hemorrhagic cystitis can be significantly reduced with co-administration of furosemide.
So the thought is again that that actually dilutes the urine so that the concentration of acryline that's in contact with the bladder mucosa is reduced, as well as encouraging the dogs to urinate more frequently through the day. I think a lot of us have been somewhat hesitant to consider chronic administration of furosemide to these patients. Although dogs with heart disease have been on furosemide for years without any severe complications.
So, two different groups actually both practising in Australia, actually took it upon themselves to look at, chronic co-administration with furosemide, and cyclophosphamide and look for the impact on the development of sterile hemorrhagic cystitis. So the median furosemide dose, given on a daily basis was 1.4 milligrammes per kilo, and that's within the range that we typically recommend for bolus administration, 1 to 2 milligrammes per kilo.
In the single arm study that was performed, the sterile hemorrhagic cystitis incidence was 3.6%. So again, there was no control arm in this study, but based on the historical literature reporting an incidence of 20%, that seems like a fairly substantial reduction.
There was a two-arm study that was also reported, and again, this was not randomised or placebo-controlled, but, at least they did have somewhat of a control arm. That also suggests that a fairly substantial reduction in the incidence of sterile hemorrhagic cystitis. So it went from 30% to something like about 10%.
So, here at Colorado State University, we have not yet really adopted the policy of routinely, treating everyone with furosemide. But one of the things that we do do very carefully is we look at a lot of urine in these patients. So we'll generally recommend, checking the urine.
For evidence of microscopic hematuria once every 6 weeks or so, indefinitely as long as these patients are on treatment. And again, this is the one situation where free catch is actually preferable to cyst cystocentesis because of the risk of traumatic induction of a little bit of hematuria. And if we start to see repeatable microscopic hematuria, at this point again, historically, we have discontinued cyclophosphamide at that point.
Given this information that's now available, this is a situation where I might consider starting furosemide at that point, to see if simply reducing the, concentration of, of aquiline that's accumulating in the bladder is enough to prevent, this, microscopic hematuria from progressing to, to full-blown clinical sterile hemorrhagic cystitis. A second co-administration study that was reported recently looked at a combination of cyclophosphamide with palladia, the kinase inhibitor that's made by Zoettas. So there's some information from, from human studies with similar agents that, drugs like palladia by themselves may be able to exert an effect, a suppressive effect on regulatory T cells, and so we wanted to take a look at that.
So this study design actually used 2 weeks' worth of palladia by itself, followed then with the addition of cyclophosphamide after the 1st 2 weeks. So what we discovered was quite interesting that palladia itself appeared to be able to reduce the total number and percentage of circulating regulatory T cells in dogs with cancer. And then when cyclophosphamide was added, again, you see that starting at day 28, there was a trend toward additive suppression of regulatory T cell numbers.
And again, this did reach statistical significance at the 56 day time point. So, I, I should mention that in addition to potentially this additive effect on regulatory T cell number and percentage, tolerability was outstanding, so there was really no additive adverse effects associated with combining tocininib and cyclophosphamide, and both of these agents could be administered at full doses. So one of the things that we looked at as a measure of quote unquote immune stimulation was circulating levels of a, of a hormone or a cytokine called interferon gamma.
And what we actually saw was significant increases in interferon gamma, with tarinib by itself, with laia by itself, and then again, an additive increase when the cyclophosphamide was added. Interestingly, at that day 56 time period, the, the change in regulatory T cell, correlated quite nicely with the change in interferon gamma that we saw, suggesting that it was a direct relationship. So, a couple more studies I want to just mention in the last minute or two.
So there are two studies that have, have been published recently looking at metronomic cyclophosphamide, as an adjuvant, in, in treating canine splenic hemangiosarcoma, and in osteosarcoma as well. So, this is a study that actually comes out of Italy, that looked at a small number of dogs. Treated, with either doxorubicin-based chemotherapy by itself, with or without the addition of metronomic chemotherapy.
And in this case, the metronomic chemotherapy generally consisted of an NSAID, cyclophosphamide, and many of these dogs received a third drug called thalidomide, which is a drug that we certainly don't use in the United States partly because it's impossible to get and partially because of its potential teratogenic effects. Again, this was also not randomised, not placebo-controlled. And again, there was some difference in the way that the dogs in the two arms were followed.
Based on the Kapameyer curve, you see here, there did appear to be a survival advantage in those dogs that received doxorubicin-based chemotherapy followed by metronomic chemotherapy, if they were rendered or if they remained disease-free after the doxorubicin was finished. So, I, I can talk to my personal experience of, with this, and I can certainly speak for a number of my US colleagues who've sort of investigated this approach in an ad hoc basis. And I have to say that we see results that look nothing like this curve.
So again, in this curve, it looks like there's an awful lot of these dogs that are long-term survivors out beyond a year, and that simply has not been our experience. So again, I do think that for an owner who wants to do everything possible, would it be reasonable to follow up doxorubicin-based chemotherapy with metronomic chemotherapy? Sure.
I, I don't see a lot of downside to it, but I certainly would not expect the kinds of results that were reported in this study. And whether that was due to the incredibly small number of patients that they were treated, whether that was due to the addition of the addition of this, this thalidomide, very hard to be able to say. So a second study recently actually was a randomised study in dogs with osteosarcoma that took a look at conventional therapy, so amputation and carboplatin, and then dogs that were disease-free following carboplatin were randomised to either receive metronomic chemotherapy with paroxicam and cyclophosphamide or metronomic chemotherapy with peroxicam, cyclophosphamide and palladium.
And in this study, Again, one of the take homes was that there was no difference in outcome between the dogs, that received the palladia and the dogs that did not receive the Palladium. But more importantly, if you looked at the median survival times in these two groups of patients, they're really not significantly different from patients that receive conventional chemotherapy by itself. So in addition to the palladia arm not doing any better than the non-Palladia arm, and this was a randomised placebo controlled trial.
The addition of metronomic cyclophosphamide and peroxicam really did not appear to be of any benefit. One thing that I, that I will say is that I think the, the study was designed, using a dose of cyclophosphamide that many of us would consider suboptimal. I think that they went for a dose of something like 10 milligrammes per metre squared per day.
So could these results have been different if a more quote unquote appropriate dose of cyclophosphamide were utilised? Possibly. But based on the information here, again, the use of metronome chemotherapy as quote unquote maintenance treatment following conventional therapy for osteosarcoma does not appear to result in a survival advantage.
So, that's really the, the sum total of the information that, that we have about metronomic chemotherapy and veterinary oncology to date. So most of us, I think, feel that, that cyclophosphamide is really the go to agent for this, and most of us will co-administer an NSAID plus or minus palladia. We do watch very, very carefully, primarily for sterile hemorrhagic cystitis, and I would consider if microscopic hematuria was observed without clinical signs.
Starting, concurrent furosemide and then again watching very carefully after that. we usually consider, chlorambuacil to be a second line agent that we would consider using if cyclophosphamide is not tolerated, primarily because of, again, it does not have the same risk of sterile hemorrhagic cystitis. The caveat is, again, biologically, it hasn't been as well characterised, and at least here in the states, it's quite a bit more expensive than the cyclophosphamide.
So in conclusion, a few people I'd like to thank before we get to the questions. Again, my colleague, Barbara Miller, who's really been the scientist who's done almost all of the work looking at regulatory T cell depletion and changes in angiogenesis from cyclophosphamide, as well as her laboratory and clinical colleagues, Jenna Burton and Leah Mitchell. I'd also like to, to thank the clinical trials team here at the Animal Cancer centre who are actually responsible for completing most of these studies.
And as well, the laboratory of Doctor Steven Dao, who has a lot of the equipment necessary for performing the measurements that were done. And at this time, I think I've left about 5 minutes, for just a couple of questions if any of them have come in, and again, thank you very much for your attention. Thanks very much, Doug.
That was great. We do have a quick question that's come through from Greg. What is the smallest strength of chlorambusil tablets that you can get in the UK in tablet form?
So I have no idea what's available in the UK, but in the US, the smallest size that's available is 2 milligrammes. And again, similar to what we see with with cyclophosphamide, we're often in a situation where we, we may have special sized, capsules compounded, but 2 milligramme is generally the size that's available in the US. I cannot say what, what sizes are available in the UK.
I think it is 2 milligramme because it's used occasionally for cus with cats as well. So I think it is a 2 milligramme tablet, Greg, but and again, Greg, similarly to, to what we see with cyclophosphamide, this is a situation where compounding may not be necessary. So if your target dose is 4 milligrammes per metre squad per day, that may be 2 tablets every day, it may be 1 tablet every day.
If your calculated dose comes out to be 3 milligrammes, then you could give 2 tablets on one day and 1 tablet the next. This, again, based on the, the limited information we have remains somewhat of an inexact science. I think it's You know, it's OK for sort of a smallish dog, isn't it?
A very small cat is I suppose a bit more of a, an issue with them, but I think you can still potentially, you know, use it in cats as well, can't do you use chlorambuillin in cats, Doug? We certainly do. We use it routinely for the treatment of low grade gastrointestinal lymphoma.
And again, I mean, I think the, the, the hematopoietic tumours like lymphoma and leukemias are a bit of a different animal. And we think that these drugs are actually killing the tumour cells when we use it for those particular diseases. So it's a little bit apples and oranges, but tolerability is certainly excellent, both in cats and in dogs.
That's great. Thanks, Doug. Are there any other questions?
While you're just having a think about that, I have just put in the chat box our surveyMonkey. We're doing our annual survey, which has been great getting all the, the responses, you know, the, the constructive as well as the, the positive. We're always trying to get better.
So do take some time to do that survey. I think there is a, a, a small prize, for somebody who gets picked out of the hat at the end of it all. So, really appreciate it if you haven't already done it, go to the Zoom chat, box and you'll be able to download that and, and do that after the webinar.
Also, there'll be Doug's survey as well. Does anybody have any more questions? Otherwise, we will let Doug go, into the, mid-afternoon sun in Colorado.
You were, you were saying pretty warm over there at the moment, Doug. Yeah, it's in the mid-thirties here, so, we're trying to stay indoors as much as possible. We, well, for golfing fans anyway, we have the the British Open just up the road in Southport at the moment.
Are you a golfing man? Oh, I, I watch a bit of it and actually there's a last time I checked there was a former Colorado State University student on the leaderboard. Oh, is that Jordan Spieth?
Yeah, yeah. Oh, OK, yeah, no, I think he finished the day in the lead, so good news. Well, I don't think there are any more questions.
I think it is one of those topics. It's obviously, you know, we're, we're moving in the right direction, starting to collect data. We're, as usual, a little bit behind the, The medics, but certainly an area that's well worth continuing to investigate, isn't it?
Absolutely, couldn't agree more. I think we've had a quick question at the last minute, just got in, Ian, so we'll, we'll read this out and see if you can help. Years ago, as in the last century, I read about piroxicam affecting human bladder tumours and gave it a go, a very small trial indeed, but it did seem to produce tangible effects, but it made dogs sick.
Tried Meticam and it didn't seem to either make dogs sick. Or better. So, so there's a couple of little helpful hints with paroxicam that are actually very useful.
So one of them is, this is a medication where we almost always will need to have it compounded because it has an extremely narrow therapeutic index. So rather than the COX 2 specific agents like Medicam and Daramax and Rimidil. Oxxicam is more of a balanced COX 1, COX-2 inhibitor.
And in theory, that could be the reason why it's a more effective anti-tumor drug, but it's also responsible for a bit of an increased incidence of in side effects. And if we even round up that 0.3 milligramme per kilo dose a little bit, The, incidence of side effects really goes up quite, quite dramatically.
So when it's, when it's dosed appropriately, always, if you need to round one way or the other, always round down. Don't give it more than once a day. Always administer it with food.
Actually, the large majority of patients will tolerate it quite well. And again, we do find that if it's necessary, co-administration of omeprazole, or another proton pump inhibitor can often really help to mitigate any, gastrointestinal adverse effects that are observed. So the, the data is a bit mixed out there.
So there is some evidence that some of the other NSAIDs may occasionally have some effect against diseases like transitional cell carcinoma, but we certainly consider, I consider at least paroxicam to be sort of the first line, because it's, it's been the best studied. Great, Doug. Ian said, thank you.
I love the webinar as well. So thank you so much. Really appreciate your time.
I know how busy you are. I know how busy the clinic is. Certainly when I went in to see it, it was very, very busy.
So we really do appreciate your time and hopefully it won't be too long before we get you on another webinar. Oh, outstanding. Thanks again for the invitation.
It was great to talk to you tonight. Thanks, Doug. Thanks everyone.
Do do the two surveys and hopefully we'll see you again soon on a webinar. Bye bye.