| REGULATION OF PARATHYROID FUNCTION BY CALCIMIMETIC COMPOUNDS |
| E. Nemeth, Toronto, Canada |
| Chair:
W.G. Goodman, Los Angeles, USA |
J. Cannata-Andía,
Oviedo, Spain |
| |
|
Dr
E. Nemeth |
Slide 1
Dr Nemeth: I'm going to cover the basic science that led up to the discovery and development of calcimimetic compounds which we believe are a new, totally new, therapy to manage to treat secondary hyperparathyroidism in the context of renal failure. And the good news about the basic science is that you really don't have to learn any new basic science to understand exactly how these compounds work, in fact you already know all the basic science and here it is, it's endocrinology that we've known about for the last 50 to 60 years.
Slide 2
All you really have to know is how the body regulates systemic calcium homeostasis and of course you know the main player here is parathyroid hormone. Parathyroid hormone acting on bone and the kidney tends to cause various changes that increase plasma levels of circulating calcium. Calcium in turn has a negative feedback role on to the parathyroid glands to suppress parathyroid hormone secretion, so there's this reciprocal relationship between circulating levels of PTH and calcium and this is the main driver, the main regulator of systemic calcium homeostasis in the body.
Slide 3
We've also known for many, many years that extra-cellular calcium acts directly on parathyroid cells to control parathyroid hormone secretion but it's only in the last 20 years that we've understood how this direct effect of extra-cellular calcium occurs and what we know now is that extra-cellular calcium acts on a calcium receptor on the surface of parathyroid cells and it is this calcium receptor that enables parathyroid cells to detect and respond to very small changes in the concentration of extra-cellular calcium. And calcium receptor, it's not quite as simple as this box here I've shown, it is a little bit more complex and I've depicted it here schematically.
Slide 4
It is, what is familiar to many of you, it is a G-protein coupled receptor, characterised by seven transmembrane domains, a very large extra-cellular domain, a cytoplasmic domain, it's linked through G-proteins to the regulation of various signal transduction mechanisms. It is homologous to a variety of other G-protein coupled receptors particularly those in the so-called family 3, GPCRs. Included in this family are the metabotropic glutamate receptors and the GABA-B receptors, so the calcium receptor turns out to be a very common receptor that pharmacologists are very well familiar with and in fact, these G-protein coupled receptors turn out to be ideal drug targets, fully 40-50% of the drugs currently on the market target receptors like this, G-protein coupled receptors.
Slide 5
Now, you don't even have to know all of this to understand calcimimetics and how they work, in fact calcimimetics were discovered and in development before the receptor was even cloned and you don't have to be a genius to figure out what calcimimetics might be good for and if you had a compound that activated the parathyroid cell calcium receptor, it should inhibit parathyroid hormone secretion and therefore be useful in the treatment of hyperparathyroidism.
Slide 6
So the reason we called them calcimimetics in the first place is because we were looking for compounds that mimicked or potentiated the effects of extra-cellular calcium at the calcium receptor, hence the term calcimimetic. So this is how they really work and this is all you really need to know to understand why these drugs might be useful in the treatment of secondary hyperparathyroidism. In normal circumstances you have a calcium receptor on the surface of parathyroid cells and in hypercalcemic conditions you have calcium receptor activation of the calcium receptor and that inhibits parathyroid hormone secretion. In situations of hypocalcemia, the receptor is unoccupied by its physiological ligant, extra-cellular calcium and that results in a stimulation of parathyroid hormone secretion. Now in the presence of a calcimimetic drug, what you do is you essentially trick the parathyroid cell in to thinking that it's a situation of hypercalcemia and that's depicted schematically here. So even though you could have a hypocalemic condition, in the presence of a calcimimetic drug, the parathyroid cell perceives that its calcium receptor is occupied and therefore it shuts off parathyroid hormone secretion.
Slide 7
Now these are the first generation calcimimetic compounds that were developed, the first was 568 that in turn led to this compound here cinacalcet, this compound has recently been approved for the treatment of secondary HPT in the United States and goes under the name of Sensipar. You can see that these compounds are relatively simple they have one chyrol carbon right there so they exist as 2 stereoisomers and it is mostly the RNA antimer which is depicted here, that contains most of the biological activity. Sensipar is the purified isomer, it's the R-isomer and it has most of the biological activity and you can also appreciate why it might be called Sensipar because it sensitises the parathyroid to extra-cellular calcium and by that action shuts off parathyroid hormone secretion. Now, these first generation calcimimetic compounds are not true receptor agonists, they don't really mimic the effects of extra-cellular calcium per se, what they do is they potentiate the effects of extra-cellular calcium and they are what pharmacologists call positive allosteric modulators of a receptor. What they do is they increase the sensitivity of the calcium receptor to activation by extra-cellular calcium and that's depicted here.
Slide 8
These are results obtained in vitro studying secretion of parathyroid hormone from ovine parathyroid cells, in the white you can see the conventional, the classic inhibition of parathyroid hormone secretion as you increase the concentration of extra-cellular calcium. Now in the presence of cinacalcet, either 10 or 100 nano-molar, you can see a shift to the left in the dose-response curve to extra-cellular calcium, so the cell is becoming more sensitive to the inhibitory effects of extra-cellular calcium and even though it's experiencing a hypocalcemic situation outside the cell, the presence of a calcimimetic agent makes the parathyroid think it's really hypercalcemia and so PTH secretion is suppressed. But the defining characteristics of these so-called allosteric activators is the fact that they have very little effect, they have no effect at sever or extreme levels of hypocalcemia or hypercalcemia, they only work within physiologically normal levels of extra-cellular calcium, so if you get rid of the normal ligant, these compounds have no effect and this we believe imparts and enhances safety features and specificity to these compounds. They don't act like your typical receptor agonists, they only act if you have a little bit of your physiological agonists there. So what they do is they sensitise the parathyroid cell to the physiological ligant, extra-cellular calcium, shift the dose-response curve to the left and by that action inhibit parathyroid hormone secretion. Now you can see that in vivo too.
Slide 9
These are some studies using an animal model of chronic renal failure. These are rats with secondary hyperparathyroidism induced by a 5/6 nephrectomy and you can see that they have secondary hyperparathyroidism just by their basal levels of PTH, which are elevated at about 44, normally they're about here. So these animals, with what we might call moderate secondary parathyroidism, were given a single oral dose of the calcimimetic cinacalcet at one of these doses and you can see a very rapid decrease in circulating levels of PTH which is dose-dependant and then it comes back to the basal levels, so the magnitude and the duration of the fall in plasma level of PTH is dependant on the dose.
Slide 10
Now there are calcium receptors on other cells in the body, mostly the kidney and also the parafollicular cell or C-cell of the thyroid and this calcium receptor on the C-cell regulates calcitonin secretion however, it's important to know that these particular calcimimetic, as typified by cinacalcet, show a unique selectivity for targeting the parathyroid cell calcium receptor and this we believe is an inherent property of allosteric activators. Here I'll show you some rat data to highlight that point. This is a simple dose-response curve to one of the first generation calcimimetic compounds, 568, you see the exact same kind of dose-response curves with cinacalcet, the ED50 is about 1 mg/kg for inhibition of PTH. You do see a stimulation of calcitonin but the ED50 now is about 40 mg/kg, so these particular calcimimetic compounds can differentiate the parathyroid calcium receptor from other calcium receptors in the body, selectively homing to the parathyroid and inhibit parathyroid hormone secretion without effecting calcitonin secretion.
Slide 11
One of the issues inherent or one of the issues that has been raised in the context of secondary hyperparathyroidism is that you might lose calcium receptors on the parathyroid cells during the course of secondary hyperparathyroidism and so it was of interest to determine if you would change the potency of calcimimetic compounds in this pathological setting and the fact of the matter is that no, you don't, it doesn't matter if the animals are normal or if they have secondary hyperparathyroidism, the potency in these compounds and their appliance remains unchanged. You can see it here, these are rats that were either sham-operated or received the 5/6 nephrectomy and simply doing a dose-response curve with a calcimimetic compound, you can see that the efficacy is essentially the same as determined by the maximal decrease in PTH and also the potency of the calcimimetic compound in lowering PTH was unaffected, so the disease state does not effect the efficacy or the potency of calcimimetic compounds. Another feature about calcimimetic compounds is they seem to be effective despite the severity of the disease. We'll see some clinical results showing this later on. Here are some rat results that almost predicted what you would see in the clinic. These are animals again, with a 5/6 nephrectomy developing chronic renal insufficiency and then when they're placed on different diets you can essentially regulate the severity of PTH. The severity of hyperparathyroidism as indexed by the magnitude, the basal level of PTH.
Slide 12
This is a large scale, so you're going from somewhere around 50-60 picograms/ml, all the way to over 1000 picograms/ml and these animals were then given a single oral dose of the calcimimetic compound and despite the different degrees or severity of HPT, of hyperparathyroidism, there was a rapid decrease in each instance and when you express this as a percentage of the control, you can see that they all come down to the same level. So these calcimimetic compounds inhibit the regulated secretion of parathyroid hormone, they do not inhibit the so-called non-suppressible constitutive secretion of parathyroid hormone and they bring it down to the same extent despite the severity of the secondary hyperparathyroidism. Now these are just some clinical results which essentially highlight the same thing.
Slide 13
These are some older studies done in Doctor Kurokawa lab where patients on dialysis were treated with a single oral dose of the calcimimetic compound, either at a 100 or 200 mg and in each case, each one of these points is a different person, in each case you can see a rapid decrease in circulating levels of PTH which then came back, despite the differences in the severity of the hyperparathyroidism, the compound effectively reduced all levels of PTH in these patients but note that it doesn't come all the way back and that hinted at maybe another mechanism, that calcimimetics acting through the calcium receptor might be effected and that might be synthesis of parathyroid hormone and indeed that turns out to be the case.
Slide 14
These are in vitro studies showing that when you increase extra-cellular calcium, you decrease the message for PTH, so you're inhibiting the synthesis of PTH, as indicated in going from 1.8 to 3 ml of calcium here.
Slide 15
In the presence of a calcimimetic compound, you also inhibit synthesis of PTH despite hypocalcemia, again the parathyroid gland is being tricked into thinking that it's hypercalcemia outside, even though it's hypocalcemia but in the presence of a calcimimetic compound it perceives it as hypercalcemia and now PTH synthesis is inhibited. So calcimimetics do two things, at least, they inhibit the immediate secretion of PTH. They also inhibit the synthesis of PTH and there's another very important action of calcimimetics that I'd like to talk about now here and that is hyperplasia and this of course is what really plagues the nephrologist, this is what characterises secondary hyperparathyroidism and you know that as the kidney fails your vitamin D, your phosphate, your calcium all become deranged, these all send signals to the parathyroid glands to enlarge, to undergo cellular hyperplasia and that hyperplasia is what's resulting in your chronically elevated levels of parathyroid hormone secretion. So we did a very simple experiment to ask whether or not we could block the proliferative effect of other 5/6 nephrectomy and indeed we could.
Slide 16
In these experiments here, what you see are section 3 parathyroid gland treated with bromodeoxyuridine and so cells that are proliferating stay brown, there are very few cells undergoing proliferation in sham-operated animals but within a week after a 5/6 nephrectomy, you can see a very large increase in the number of proliferating cells. If however you treat the animals, you put them on a calcimimetic at the same time you do a nephrectomy, you'll notice that the number of proliferating cells decreases and in fact you completely halt cellular hyperplasia under these conditions.
Slide 17
So calcimimetics look like they can also not only inhibit synthesis and secretion of PTH, they can inhibit cellular hyperplasia. This experiment is nice because it shows that if you start treating with a calcimimetic very early on, you'll block hyperplasia but unfortunately as nephrologists, that's not the clinical situation, by the time the patient shows up in the clinic, they've already got secondary hyperparathyroidism, their glands are already enlarged, so we did another experiment and asked, well if we let secondary hyperparathyroidism develop, if we let the glands undergo hyperplasia, can we prevent it from getting any worse and that's what this experiment was designed to show. These animals here received a 5/6 nephrectomy and we're measuring parathyroid gland volume and also the total number of parathyroid cells and you can see that after 12 weeks following a 5/6 nephrectomy there's hyperplasia as indicated both by an increase in cell number and by an increase in cell volume, now if we let the animals go out another 8 weeks, so we're now looking at 20 weeks, you can see that the gland volume continues to increase, the cell number continues to increase, so the hyperplasia, the secondary hyperparathyroidism is getting worse, however, if at this time we start treatment with a calcimimetic compound administered either orally or by subcutaneous infusion, we completely prevent the further development of hyperplasia. Compare this bar here with these and this bar with these. There is no further increase in the progression of secondary hyperparathyroidism as indicated by hyperplasia of the glands, so a calcimimetic will halt the progression of secondary hyperparathyroidism in its tracks.
Slide 18
Now I'd like to wrap up and just briefly touch on some of the effects of calcimimetics on bone. Renal osteodystrophy of course is an extremely complex heterogeneous pathology, to talk about the effects of calcimimetics and their effects on bones, we could have a whole symposium on that, let me just touch on some of the highlights, here. Again we're looking at a model of 5/6 nephrectomy treated or not with the calcimimetic compound cinacalcet and we then did some histomorphometry at the tibial metathesis and then also in some cortical bone and in the sham-operated animals, you can see within the marrow nice fatty marrow, a lot of adipocytes, after a 5/6 nephrectomy, development of classic signs of osteitis fibrosis cystica, of the fibrotic matter here, also accompanied by cortical tunnelling, an increase in cortical porosity. However if the animals are treated with cinacalcet, 15 mg/kg once a day for the same amount of time, there is complete prevention of the marrow fibrosis and the bones have shown no cortical porosity, they're essentially identical to the control sham-operated animals.
Slide 19
You can see that here again using pQCT analysis, same experiment, cortical bones by pQCT after nephrectomy, you can see the cortical porosity here, the loss of bone mass. However animals treated with 15 mg/kg of cinacalcet looked just like control animals and down here are a variety of biomechanical measures which I won't go through but it's suffice it to say that not only do these bones look better histomorphologically and by QCT analysis, they also get stronger. So they're biomechanically more competent, bone quality increases.
Slide 20
Now in the final topic that I want you to appreciate about how calcimimetic compounds work, you have to remember something about the major hormone they are effecting and that's parathyroid hormone and as you know, parathyroid hormone is really a double-edged sword It giveth and it taketh away from bone. What you're most familiar with is this effect of parathyroid hormone: a chronic increase in parathyroid hormone that you see in secondary hyperparathyroidism, and the net effect of the chronic increase is a decrease in bone mineral density. This is the classic catabolic effect of parathyroid hormones, but we also know that the effects of parathyroid hormone on bone are extremely dependant upon the temporal changes that occur in the circulation and so if you have intermittent increases in parathyroid hormone once a day, once every other day, then the net effect on bone is in fact an increase in bone mineral density and it is of course this intermittent decrease in PTH which underlies the usefulness of drugs like forteo for the treatment of osteoporosis.
Slide 21
Well remember the time course and the effects of the calcimimetic agent on circulating levels of PTH. That's what they do, they cause intermittent changes in circulating levels of PTH and that is something that you can do with a calcimimetic that you've never been able to do before. Vitamin D, because it acts genomically, if it effects parathyroid hormone levels, it takes a long time, it will bring them down and it will keep them down but with a calcimimetic, what you're really doing, is you're lowering the baseline PTH but then you're causing pulsatile changes around that lowered baseline and calcimimetics will be the first time you as a nephrologist have been able to alter PTH like that and so you might ask, would I have different effects on bone, if I lowered PTH and pulsed it versus just lowering it and clamping it in a new level.
Slide 22
We did one experiment to assess that and the way we could do that is by giving a calcimimetic once a day per os, orally, so that would cause a transient decrease and we could compare that with the subcutaneous infusion of the same calcimimetic at the same dose which would cause a lowered PTH level and keep it down and here's just one result from that study. Again in rats, where we looked at the bone mineral density in the distal femour and what you see now, is that here's the normal animal, sham-operated, here is the animal that received the nephrectomy and there is of course a lowered BMD which doesn't increase very much over a 57 day period these animals are normal, they keep growing, so that goes up a little and here's the effect of lowering PTH and keeping it down. A constant lowered PTH induced by the administration of a calcimimetic, given by subcutaneous infusion and you can see that there's really no difference between the animal treated with vehicle however, if you give the calcimimetic agent PO once a day, so now you're causing pulsatile decreases in PTH you can see an apparent anabolic effect, the bones are getting bigger, the BMD is returning towards the control levels.
Slide 23
The effects of the calcimimetic on the spectrum of renal osteodystrophy may be very different from what you are used to with treatment with vitamin D and it's important then to just summarise some of these differences between what a calcimimetic does and what vitamin D does. The calcimimetic acts on a cell surface receptor, vitamin D acts genomically, calcimimetics inhibit both synthesis and secretion. Vitamin D inhibits just synthesis. Calcimimetics have rapid on-set and recovery, those of vitamin D are slow. I didn't show you changes in calcium and phosphate, you'll see those in a minute.
Slide 24
Slide 25
So in summary, the calcium receptor and calcimimetic compounds inhibit not only PTH secretion but additionally inhibit cellular proliferation and also the synthesis of parathyroid hormones, so they have multiple actions all acting through the calcium receptor and that's just summarising how calcimimetic compounds may be used in the treatment of secondary hyperparathyroidism.
Slide 26
Thank you!
