I can tell you. There are so many news recently published about C-type natriuretic peptide (CNP) for achondroplasia and other skeletal conditions that it is hard for me to choose where to start from.
But, wait a minute...
I know, I know, all we want to talk is about CNP. But be patient as I believe that a bit of background information can make it easier to understand CNP, fibroblast growth factor receptor 3 (FGFR3) and achondroplasia and how to put all the new information in context. So, let's start with a brief review of how bones grow.
How bones grow
As the seventeen readers of this blog know, bone growth is a long, structured and very complex process that occurs during the development of a child through adulthood. Influenced by dozens of local and systemic agents, the bone growth process could be seen as a Mozart's symphony, where many different instruments play together in perfect harmony to create wonderful art. If you stop to think about it, what is on stake and how it is achieved, you would conclude that it is a piece of natural miracle. Every single player in the growth process works in fine tuning to achieve what is planned in our DNA.
Our long bones grow from thin cartilage layers located in their extremities called growth plates. Within the growth plates, chondrocytes, the master cells of bone growth, will "wake up" from a dormant state, start a proliferating frenzy, become very enlarged and, in the end of their life cycle, will give place to osteoblasts, the bone builder cells (Figure 1) (1). As said above, this process is regulated by many local and systemic agents. When they are not in balance, the normal process is compromised either leading to stunted or to excessive bone growth.
Figure 1. Growth plate.
What happens in achondroplasia
In achondroplasia, FGFR3, one of the local agents that regulate how chondrocytes wake up, proliferate and enlarge, is working too much due to a mutation in its structure (2,3). Since the normal action of FGFR3 is to reduce chondrocyte proliferation and hypertrophy (enlargement) pace, when it is working excessively chondrocytes just stop their normal functions and bone growth is severely compromised.
FGFR3 is what is called a receptor enzyme. It is placed across the chondrocyte cell membrane, just like an antenna on top of the house roof (Figure 2). While a TV antenna will capture TV signals to deliver them to our TVs inside home, FGFR3 transmits chemical messages delivered by FGFs from outside the cell to the cell nucleus.
Basically, when a FGF binds to the part of FGFR3 that is outside the cell, it activates (turns on) FGFR3 starting a series of chemical reactions consisting of an enzyme activating the next one and further like in a domino chain, until the last one enters the cell nucleus (Figure 3). Inside the cell nucleus, this last enzyme will turn on local agents that will trigger (or stop) the production of proteins from the DNA, each of them with distinct functions. In chondrocytes, the signals coming from FGFR3 "tell" the cell nucleus to stop cell multiplying activities. If chondrocytes stop multiplying, bone growth is compromised (2,3).
Figure 2. FGFR3 is like a roof antenna, picking up signals from outside the cell and delivering them to the cell nucleus.
Figure 3. FGFR3 signaling pathways.
|From Su N et al. Bone Res 2014; 2: 14003. Reproduced here for educational purposes only.|
As you see in Figure 3, FGFR3 activation turns on several enzymatic cascades. The MAPK (for Mitogen-Activated Protein Kinase) cascade is the important one for us in the context of CNP. The MAPK cascade consists of the enzymes RAS, RAF, MEK and ERK (at the right in Figure 3) (3). ERK is the one which goes to the nucleus.
Several studies established that under FGFR3 activation MAPK is specially responsible for regulating the enlargement of chondrocytes (hypertrophy, see Figure 1) (2,3). The hypertrophic zone seems to be the most important layer in the growth plate in regards to bone growth. Under FGFR3 overactivation in achondroplasia, there are fewer chondrocytes proliferating and enlarging and it is here where CNP has a role. Let's see how it works.
CNP and FGFR3
CNP is produced within the cartilage growth plate and works as a bone growth promoter, so it has the opposite effect of FGFR3. When released, this small peptide binds its receptor on the chondrocyte cell membrane (just like FGFR3) and activates (turns on) a chemical pathway inside the chondrocyte that inhibit MAPK at the level of RAF (Figure 4) (2). Do you get the point? CNP works naturally reducing the activity of the FGFR3 pathway.
Figure 4. FGFR3 and CNP crosstalk.
|From Klag KA and Horton WA. Hum Mol Gen 2016; 25:R2-R8. Reproduced here for educational purposes only.|
CNP and bone growth
Loss-of-function mutations in the CNP gene or in the CNP receptor (called NPRB) that impair their normal function cause a very rare genetic bone dysplasia called acromesomelic dysplasia Maroteaux type, which has some features resembling achondroplasia (4,5). On the contrary, mutations leading to gain-in-function lead to bone overgrowth (6,7). The role of CNP in bone growth was further described in achondroplasia and CNP-null mouse models (8).
Turning CNP into a viable therapy for achondroplasia
Life is not easy for peptides. The growth plate processes are tightly regulated, as we have already learned, but in fact this is true for all organic processes running in our body. Circulating active proteins and peptides like CNP can initiate, increase, decrease or stop many chemical reactions so the body has several systems to clear these agents off the circulation to avoid them to cause undesirable effects. One of these systems is comprised by blood enzymes that target peptides like CNP (they are called endopeptidases or endoproteases). This is so true that once released in the blood stream, CNP will last for about only two minutes (what is called half-life) (9).
Around ten years ago, scientists had proved that CNP has a positive role in bone growth and that it could counteract the inhibitory effect of FGFR3 in achondroplasia (8), but with that short half-life of just 2 min, how would they manage giving CNP to restore bone growth? They were able to show that continuous infusion of CNP had a positive effect on bone growth (8), but having an infusion pump connected to the body did not seem to be a reasonable option when thinking about long term therapies at that time.
Therefore, there might be other solutions out there. CNP is part of a family of three closely related molecules called natriuretic peptides. One of them, the brain natriuretic peptide (BNP) was shown to be naturally more resistant to endoproteases due to a prolonged "tail" that CNP does not have (Figure 5). With this knowledge in hand, researchers developed a form of CNP which bears a prolonged tail similar to BNP (10).
Figure 5. Natriuretic peptides.
The modified CNP (called analogue), which we know by the name of vosoritide (BMN-111), not only retains the biological functions of the original peptide but also is more resistant to neutralization by the clearance enzymes, with a prolonged half-life of 20 minutes. Being able to circulate longer in the blood stream gave enough time to vosoritide to reach the growth plates to exert its expected function (10).
In fact, both pre-clinical studies in mice and monkeys resulted in additional growth and the recently published results of the phase 2 study in children with achondroplasia (10-12) demonstrated that once-daily subcutaneous (SC) injection of vosoritide resulted in increased bone growth velocity and additional growth compared to what would be expected without the treatment. Vosoritide is now being tested in more than 100 children in a phase 3 study (NCT03197766), with results to be available by the end of this year and, if successful, the drug could be on the market next year.
Now, let's take a look on the results of the phase 2 study, just published in the New England Journal of Medicine (NEJM) last week (12). Actually, the main results reported in this paper had already been released during the R&D Day event held by Biomarin in June 2018 and repeated during the JP Morgan conference on last January. Unfortunately, the links to those presentations are no longer available at the Biomarin's site but you can see a snapshot of the R&D Day presentation from June 2018 in Figure 6.
Figure 6. Effect of vosoritide in average growth velocity after 42 months in the 3rd cohort of the phase 2 trial (15mcg/kg).
|Snapshot from Biomarin's R&D Day presentation (Jun 2018).|
The phase 2 study paper contents are protected by copyright but the NEJM released a picture in their Twitter account showing one of the main results of the trial (Figure 7).
Figure 7. Increase of bone growth velocity after six months of starting treatment with vosoritide.
|From Savararayan R et al. NEJM 2019; image (corresponding to Figure 1A in the original article) obtained from NEJM's Twitter open access account and reproduced here for educational purposes only.|
In summary, vosoritide (15mcg/kg) was able to restore bone growth velocity in exposed children with achondroplasia closer to the average bone growth velocity seen in non-affected children (Figure 6). The effect was sustained after 42 months of exposure. I cannot display it here but in one of the pictures (Figure 1B in the original text) one could interpret that there is what it seems to be a slight trend to a reduction on the effect on bone growth over the 42 months (12).
Nevertheless, the study showed progressive improvement on the z-score for height with 15mcg/kg/day which, in real world language, means that the difference of the exposed children growth pattern compared to the standard growth curves decreased overtime (12).
One question that was raised in the beginning of the clinical development of vosoritide years ago was whether the drug would cause worsening of the body disproportion. Results from this study show that this was not the case, but by the other side, there was no significant improvement on this aspect of the dysplasia (12). Bear in mind that most of the disproportion is set in the first two years of life, and that the children in this study were at least six years-old on enrollment, possibly too late to see relevant effects in this aspect of achondroplasia.
On the safety side, it seems that vosoritide has a fair safety profile, with the majority of adverse events linked to injection site reactions, which were mostly mild in intensity. Exposure to biologicals may trigger an immune response by the body, which may produce antibodies against that drug. This is common in the treatment with monoclonal antibodies against cancer and other inflammatory conditions, so it is not surprising that anti-drug antibodies (ADA) were found in this study (12). However, it seems that the presence of ADAs would not have had an impact in the drug efficacy (13).
A question waiting answer: given that results from the 42 months of exposure were already available one year ago, why were results from beyond that cutoff not included in this study, which has just been published?
Is there space for improvement?
Well, vosoritide has been consistently showing results that, if confirmed in the ongoing phase 3 study (NCT03197766), may pave the way to be approved for the treatment of achondroplasia in the next year or so. These are wonderful news, as this therapy may help improving the quality of life of many children in the future. However, it seems that there is space for even better bone growth effects with CNP.
Ascendis Pharma is developing another CNP analogue (they call CNP-38 meaning a CNP molecule with 38 aminoacids) but using proprietary technology to improve how long their CNP circulates to exert its effects in the bones. They have created a carrier system (we can call it a transport or "taxi") for delicate molecules like CNP (Figure 8). Protected by Ascendis taxi, TransCon, their CNP was shown to have a much longer half-life compared to vosoritide. In fact, they have just published the complete results of their pre-clinical studies made with TransCon CNP, which included tests comparing their CNP with vosoritide (14). As a matter of information, in their study they have reproduced the molecule corresponding to vosoritide, which is a CNP with 39 aminoacids (thus CNP-39) to compare with their CNP-38. Let's take a look on their study.
Figure 8. TransCon carrier system.
|From Ascendis Pharma. You can learn more here.|
In brief, due to the characteristics of their taxi and their CNP structure, they found that their TransCon system provided stable exposure of their CNP for a week, without the plasma peak observed with vosoritide, so with minimal if any effect on blood pressure. The effect in bone growth was at least as good as with CNP-39 (Figure 9) (14).
Figure 9. Effect of TransCon CNP (40 and 100 mcg/kg) vs. placebo and CNP-39 on body length and bones.
|From Breinholt VA et al. J Pharmacol Exp Ther|
Ascendis has already conducted a phase 1 study with TransCon CNP in healthy volunteers, and they confirmed the long half-life of their analogue, which will allow a weekly dose, in contrast with the daily dose of vosoritide. There were no cardiac safety concerns. Ascendis should be starting their phase 2 study in children soon.
Is this the end of the story?
No, not at all as Daiichi Sankio, a Japanese pharma industry, is developing ASB20123, a new analogue of CNP. In this case, the compound is a fusion molecule where the active part of CNP is combined with a fragment of the peptide hormone ghrelin (Figure 10). This engineering makes CNP resistant to endopeptidases giving more time for it to exert its functions (15,16). Basically, it is the same principle used for Biomarin's and Ascendis' analogues.
Figure 10. Structure of ASB20123, a new CNP analogue.
|From Morozumi N et al. PlosOne 2019, reproduced here for educational purposes only.|
Figure 11. ASB20123 effects in growth in a mouse model.
|Growth curves of female juvenile ICR mice treated with ASB20123 sc during the 8 weeks of the dosing period and the 4 weeks of the washout period. Body weight (A), body length (B) and tail length (C) data are shown in the upper panels, and the photographs in the lower panel represent the gross appearance of mice at Day 56 (D). Each value represents the mean ± SD of 10 (for the dosing period) or 5 mice (for the washout period). NS: not significant (p > 0.05), *: significant difference (p < 0.05) compared to the control group using Dunnett’s test. From Morozumi N et al. PlosOne 2019 (open access), reproduced here for educational purposes only.|
The researchers also tested ASB20123 given through a SC pump, again providing sustained release of their analogue, with improved growth results. The argument is that the use of a SC pump may allow stable but lower concentrations of their analogue to promote bone growth without cardiac adverse events (especially hypotension) (16). Newer SC pumps seem to be more comfortable than older models and would spare children to have daily or weekly shots. I don't feel exactly comfortable about this approach yet but I think it is too early to draw conclusions about it.
The achondroplasia therapy landscape is becoming crowded. Now, there are two CNP analogues in clinical development, one of them, vosoritide, closer to marketing approval, pending results from their phase 3 study in the end of this year. The other, TransCon CNP, heading to phase 2. Therachon is developing TA-46, a molecule based on FGFR3 and designed to compete against the mutated receptor for the FGFs (17). A Japanese group has been working with meclizine, an old antiemetic drug that showed positive effects on bone growth (18). QED Therapeutics, a small biotech, started working with infigratinib, a molecule designed to block FGFR activation (19). The Japanese group from Daiichi Sankio has just introduced their CNP analogue (16). Osteocrin, a natural peptide, has shown to improve bone growth by blocking NPRC (a trap receptor for natriuretic peptides) rendering more time to CNP to exert its effects in bone growth (20). Investigators have also found that a family of drugs used to lower cholesterol, the statins, may also be used to improve bone growth in achondroplasia (21). Furthermore, there is already initial research exploring gene editing to treat achondroplasia (to be reviewed in a future article).
However, this is still not the end
With the mounting knowledge about the chemical pathways altered in achondroplasia and the research to put them in balance again, researchers have also started to explore the use of therapies initially designed for achondroplasia in other skeletal dysplasias where the FGFR pathways may have a relevant role. The most natural example is hypochondroplasia, which is also caused by mutations in FGFR3. But there are other initiatives.
For instance, a study showed that BMN111 (vosoritide) had positive effects in a model of Crouzon Syndrome, a craniosynostosis linked to a FGFR2 mutation (22). The MAPK pathway, key in achondroplasia, is also fundamental in the family of genetic disorders called RASopathies, in which enzymes of the MAPK pathway or their regulators have mutations impairing their normal functions, which causes a plethora of clinical complications. The RASopathies include Neurofibromatosis and Noonan Syndrome among several other disorders. A recent work with statins in Noonan Syndrome showed that they were able to rescue growth in that RASopathy (23). The same group working with ASB20123 tested CNP in a mouse model of cardio-facio-cutaneous syndrome, another RASopathy, with positive results (24).
Things are getting better. A few months ago, Chinese investigators published a study where they found that FGFR3 has an important role in the mechanism of genetic disorders linked to mutations in the gene SLC26A2, which include diastrophic dysplasia. They showed that inhibiting FGFR3 with infigratinib (the FGFR blocker in development by QED) improved bone formation and the phenotypes of two lethal forms of SLC26A2 disorders: achondrogenesis type IB and atelosteogenesis type II (25). If they were able to ammeliorate the phenotypes in those devastating forms of genetic disorders linked to sulphate transport, what would be the results in milder forms such as diastrophic dysplasia?
The big question is not anymore if there is, or will be, any treatment available. The question now is: in which other skeletal dysplasias therapies for achondroplasia could also provide benefit?
The interested community has been divided lately with diverging opinions about what these new potential therapies' purposes "really" are. Some claim that they would be just cosmetic, a threat to human diversity. A flow of accusations and harsh judgement over parents taking decisions about their children without their "consent" is being published in the social media, as decisions are not what parents always take, every single day, from the most banal to the most fundamental issue (whatever they are).
Embracing the change
Not long ago, there was nothing to be done after a diagnosis of a genetic disorder, but to resign, as there were no perspectives ahead.
Now, the time for resignation is over. Therapies for genetic bone growth disorders are on their way and they have nothing to do with reducing human diversity as some have been declaring lately. They have to do with providing a better life for affected children, future adults.
By restoring bone growth, many clinical complications seen in skeletal dysplasias may finally be prevented or minimized. This will result in better functioning and better quality of life for our beloved children as they grow up to become adults. Can you imagine your kid not having to undergo major surgeries at young age, or having to deal with life-long orthopedic and neurological complications? (26, 27) Wouldn't that be good enough?
And finally, if restoring bone growth in achondroplasia and other bone disorders will make treated individuals taller, even better, for they will be able to better face the many existent challenges in the world outside. If you are still not there yet, you should start thinking in embracing the change. It is coming.
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