segunda-feira, 19 de março de 2012

The last stronghold: the growth plate cartilage

Medicine has been efficiently tackling conditions caused by altered proteins the body produces, either because of a disease state or due to a mutation. Actually, doctors do have in hands a significant number of drugs of biological (antibodies) or synthetic origin which can be used to block these proteins to treat several diseases like cancer and rheumatic conditions. So now, illnesses like breast cancer can be defeated more easily and an affected woman has more chance to survive after the diagnosis if the right treatment is applied early. You can see more about an antibody against a protein produced by breast cancer cells here.
Achondroplasia is caused by a single substitution in one of the hundreds amino acids of the chain that form the protein, or enzyme, called fibroblast growth factor receptor type 3 (FGFR3). The amino acid change makes this enzyme more active than normal and, as the natural role of FGFR3 is to negatively regulate the bone growth pace, the result is that children bearing the altered protein will grow less than they could do. The medical and personal consequences are very well known.

Enzymes are proteins capable to cause or accelerate chemical reactions inside the body. They have distinct electric charges and patterns that make them good targets in terms of the creation of compounds or drugs that can react with them and block their functions. FGFR3 is no exception. In fact there is already an expressive list of antibodies and synthetic molecules capable of stopping FGFR3 activity (see the table below with a partial list).

Table. A list of drugs and antibodies with action against FGFR3.

If we already have so many usable tools, what is preventing achondroplasia to be readily treated? Why don’t simply pick one of the good antibodies available and give it to the affected child? FGFR3 would be blocked, the car brake would no more stop the car and growth would be rescued. It sounds pretty good, isn’t it?

The road is not that easy

 I know, I will be repeating a bit what is written in the older articles in this blog, but we will not lose anything by revisiting this topic. On the contrary, the idea here is not to just give information, but to share knowledge. With knowledge comes insight.

There are several important biological and economic reasons to explain why it has been so difficult to find good treatments for achondroplasia. In this article about the growth plate we will be looking only at the biological ones.

Of course, the growth plate it is not the only issue. One important reason which makes difficult to treat achondroplasia is related to FGFR3 structure. FGFR3 is one of a family of four receptor enzymes and shares great homology (similarity) with its brothers. Furthermore, for one of the most important reactive parts of its structure, which is called ATP pocket (reviewed here: ATP pockets), the homology is also significant with those of other receptor enzymes families. These patterns imply that many of the current synthetic drugs capable of blocking FGFR3 have also the same action in other FGFRs and in other enzymes. We want to block only FGFR3, as it could be dangerous to interfere in other body chemical reactions. Therefore, it is a huge challenge to design a drug to beat only FGFR3.

Antibodies against FGFR3 are more specific. Several have been created and one is currently being tested for some kinds of cancer where FGFR3 plays an important role stimulating the progression of disease. So, why are they not being used for achondroplasia? This is a great question and the explanation resides within the body. Several layers of defense have been mounted by our body to defend us from invaders, but in the case of the bones, any drug or compound will have to deal not only with the immune system but also with a very well protected stronghold, the cartilage growth plate.

The cartilage growth plate

Having this said, let’s take a look in the growth plate. We will do it slowly to help us reflect about the challenge. The best way to reach a place is to learn the most we can about the way to it first, so to be prepared for the hurdles of the path. Be prepared, for a snow mountain, pick some chains for the wheels, for a remote hot tropical beach is good to have an appropriate car to cross unpaved roads. And of course, bring the right clothes. You will not want to feel cold while going down the mountain on skis or swim using a fleece coat.

The growth plate protects the mighty chondrocytes

The cartilage growth plate is the source of bone growth. It is through the scaffold the chondrocytes create around themselves within the growth plate that the new bone will be built. This is a good step from where to start, so how the growth is achieved?
Take a look in these figures showing illustrated structures of the growth plate:

And now, just look at this picture for an actual growth plate structure, from the work of a pair of modern pioneers in the field of growth plate cartilage, Drs. Naski and Ornitz.

You now may have a nice understanding of how the growth plate is organized. There are several layers of distinct types of chondrocytes:
  • The resting zone
  • The proliferative zone
  • The pre-hypertrophic or maturing zone
  • The hypertrophic zone
The resting zone is comprised by sleepy chondrocytes. They will be turned on by some chemical stimuli and start to proliferate (multiply), organizing themselves in piles of flat cells following a longitudinal sense. Then, obeying other kind of chemical instructions, they will start to enlarge and will become round, beginning to produce large amounts of cartilaginous matrix. At some point, one of the proteins they produce, called vascular endothelial growth factor (VEGF) will trigger the formation of blood vessels in the surrounding area. These vessels invade the cartilaginous matrix and open way to new cells, the osteoblasts, precursors of bone. At the same time, the hypertrophic chondrocytes will enter programmed cell death, a phenomenon called apoptosis. The spaces they lay will be replenished by the incoming osteoblasts, which will begin to produce the bone matrix (in the page by Drs Naski and Ornitz – link above - you will be able to find a more detailed description of this process).

There is one concept we need to bear in mind, which is the fact that the resting and the proliferative zones of the growth plate do not have direct blood supply. It is likely one of the many ways Evolution found to protect this so sensitive tissue.

To reach the chondrocytes, any nutrient, hormone or other body messenger will have to navigate through the cartilage matrix, the tissue produced by these cells. This matrix is composed by several very large proteins called collagens and complex sulfated sugar molecules, all of them organized in an intricate net. 

This net is responsible to sustain the cartilage format, but it is also responsible for blocking undesired molecules or invaders. Here again, this seems to be another Evolution’s touch to protect the chondrocytes in their crucial mission.

So, how important messengers of the body such as growth hormone (GH) or nutrients make their way to the chondrocytes?

This is the first rule we should be pay attention to. GH and several other body hormones are small molecules, as many common nutrients are as well. An elegant study performed by Farnum et al. in 2006 showed that molecules weighing less than 50 kDa will tend to diffuse more easily across the growth plate.

This diffusion pattern explain why specific, ready to use antibodies fail to reach chondrocytes in therapeutic doses: they are too large, weighing more than 150 kDa. This is not a problem for peptides (such as the CNP), oligonucleotides and small tyrosine kinase inhibitors (TKI).

Another thought about the cartilage matrix is that it is likely that any given therapeutic compound will take time to reach the target due to the diffusion characteristics, so drug developers should think in how to better handle data coming from pharmacokinetics studies. For instance, half-lives (a way to measure the time a drug will circulate within the body) might not reflect fairly the drug distribution.

What does come next?

We know that several drugs, like those listed above, can reach the growth plate. Did you see the paper describing the effects of PD106067 in the cartilage? TKIs cross the growth plate and reach chondrocytes, so if we can create one of these compounds with exclusive action on FGFR3, it looks like we would be capable of rescuing bone growth in ACH.

However, for other reasonable therapeutic strategies, such as oligonucleotides or aptamers, the problem is related not to the size of the molecule but to its nature. As they have particular chemical characteristics, they will not be able to reach the growth plate by their own, because the immune defense would stop them very fast. They will need to be transported to the growth plate by carrier systems. We will talk more about this in the next article.

In summary, we have briefly reviewed the growth plate properties and challenges it poses for drug development. Some of the therapeutic approaches such as TKIs have been proven to reach the chondrocyte and the issue here is about to find the perfect TKI, that one addressing only FGFR3, a very difficult task with the current technology. Other feasible approaches will need help to get into the growth plate and reach chondrocytes.

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