New Medication Against Heart Attack

[iansmith]

Town without heart attacks
Italian resort's genetic anomaly leads to a drug that could reverse coronary disease
_
Brad Evenson
National Post

December 9, 2003

The tiny Italian resort town of Limone sul Garda sits tucked along the western shore of Lake Garda, its scenic streets lined with oleander, olive and lemon trees. For centuries, it was best known for its mild Mediterranean climate and its delicious cold-pressed olive oil. But since 1980, the town's most famous export has been blood.

If studies confirm the astonishing potential of a protein called ApoA1 Milano found in the bloodstreams of 40 of Limone sul Garda's residents, millions of people could someday be cured of heart disease.

"It's very exciting," says Jean-Claude Tardif, director of clinical research at the Montreal Heart Institute. "It's really potentially a revolution in the treatment of coronary heart disease."

Last month, researchers from the Cleveland Clinic in Ohio reported infusions of the Italian protein reversed decades of cholesterol buildup in the arteries of heart patients in just a few weeks. Experts predict it will become a blockbuster drug worth billions of dollars.

http://www.canada.com/national/nationalpost/news/story.html?id=93235391-40d9-47f1-a09e-ec318750ab9f

 

[Monique]

Garda.. good place to be.. little touristic, but alla.

But the ApoA1 I don't understand, they just found out that it works? It would have been my first guess since it is a major part of the lipoprotein aggregates. Out the top of my head it probably allows HDL to take up more cholesterol and bring it to the liver to be excreted (remember, we are unable to metabolize cholesterol, the only way out is excretion).

 

[adrenaline]

The manufacturers of angioplasty balloons and stents are peeing in their pants!

 

[zoobyshoe]

I'm not getting how they are making use of this. Are they taking blood from these villagers, separating this stuff out, and injecting it into other people?

 

[Monique]

Originally posted by zoobyshoe
I'm not getting how they are making use of this. Are they taking blood from these villagers, separating this stuff out, and injecting it into other people?

That would be waaaay to expensive, they have probably made some bacterial system into which they transfected (or would it be transformed?) the gene encoding for the protein with a strong promotor in front of it so that the expression is high, after which they can purify it.

 

[zoobyshoe]

Originally posted by Monique
That would be waaaay to expensive, they have probably made some bacterial system into which they transfected (or would it be transformed?) the gene encoding for the protein with a strong promotor in front of it so that the expression is high, after which they can purify it.

This is the way proteins are usually replicated?

 

[iansmith]

Originally posted by zoobyshoe
This is the way proteins are usually replicated?

Yes. Insulin is produce by a genetically engineered E. coli. Some other protein are produce by yeast. the system are inexpensive and efficients.

 

[zoobyshoe]

Originally posted by iansmith
Yes. Insulin is produce by a genetically engineered E. coli. Some other protein are produce by yeast. the system are inexpensive and efficients.

So, there is nothing you can think of that might make this medication particularly expensive to produce?

I'm just speculating as to whether the drug companies will find a rationalization to jack up the price unfairly because it is sure to be such a widely desired treatment.

-zoob

 

[Monique]

The only problem with the system is that the bacteria (also yeast?) will eventually loose the foreign piece of DNA.

This due to the stress that is generated by having to over-produce this specific protein that has no function for the micro-organism.

A cell that has lost the piece of DNA will have a significant growth advantage and will eventually take over the culture.

Also, impurities left after purification would be able to generate an immune response, I imagine.

But overall it is a really clever way to produce this kind of medication. Not just bacteria are used, also rabbits and maybe cows. The protein is specifically expressed in the mamary glands and will be excreted in the milk after which it can be purified. I guess they use these higher eukaryotes, since some proteins need post-translational modifications which don't take place in bacteria.

 

[Monique]

Originally posted by zoobyshoe
So, there is nothing you can think of that might make this medication particularly expensive to produce?

I'm just speculating as to whether the drug companies will find a rationalization to jack up the price unfairly because it is sure to be such a widely desired treatment.

-zoob

I don't think maintaining high grades cultures is cheap, but it is cheaper than isolating it from blood and the quantities that are produced allow mass scale distribution.

HAH! Drug companies jacking up prices.. do you know how much profit they can make? Let me give you an example: Genzyme, a Massachusets based company, started out with like 15 people or so. They took over a project from academic researchers, the whole idea of making the medicine was already there, little research was required but setting up a system to produce and quality proof the production.

Now, the researchers who did all the work get nill % off the profit into which all those years of effort and work was put into. The company hitched a ride and has now grown to ~5000 people.

So, you might wonder, how much is their profit? Let me tell you: 95%. Yes, 5% of the cost is what they actually have to spend for the production. The 95% is what goes into their pocket. Surprised? :)

 

[iansmith]

Originally posted by zoobyshoe
So, there is nothing you can think of that might make this medication particularly expensive to produce?

I'm just speculating as to whether the drug companies will find a rationalization to jack up the price unfairly because it is sure to be such a widely desired treatment.

There is the R & D involve for the pricing and this is the expensive part of the product according to their rational. But it is less expensive because it can be mass produce more efficeintly than if you have to isolate the protein from its source.

Insuline is to be insolated from pig pancreas. It was expnsive and the product was not readily available.

Originally posted by Monique
But overall it is a really clever way to produce this kind of medication. Not just bacteria are used, also rabbits and maybe cows. The protein is specifically expressed in the mamary glands and will be excreted in the milk after which it can be purified. I guess they use these higher eukaryotes, since some proteins need post-translational modifications which don't take place in bacteria.

This compagny can isolate spider web from goat milk

Nexia Biotechnologies
http://nexiabiotech.com/en/01_tech/08.php

They also trying to use plants instead of animals. Cheaper, easier to produce, not as energy demanding and less ethic problem.

 

[Monique]

Btw, the patent has already expired for this medication, but generic versions have not be made since the compition would be too great with the already well established firm.

 

[Monique]

Researchers actually once did a trial in which they injected people with HDL (the good cholesterol).

The problem with using this molecule is that it is an aggregate of lipoproteins and cholesterol and other proteins such as the apolipoproteins (Apo) and thus very hard to produce. It never took off the ground because of this and other routes have been chosen.

 

[zoobyshoe]

Originally posted by Monique
So, you might wonder, how much is their profit? Let me tell you: 95%. Yes, 5% of the cost is what they actually have to spend for the production. The 95% is what goes into their pocket. Surprised? :)

This kind of profit margin is just about unbelievable to me. I suppose the phenomenon of medical insurance is what makes it possible. Not that I understand the workings of that.

 

[zoobyshoe]

Originally posted by iansmith
This compagny can isolate spider web from goat milk

Et, bien? Pourquoi?

What does anyone want with spider webs? Why would goats be a better source than spiders?

 

[Monique]

Originally posted by zoobyshoe
This kind of profit margin is just about unbelievable to me. I suppose the phenomenon of medical insurance is what makes it possible. Not that I understand the workings of that.

Normally it would not be that outragious, figuring the years (decade in some cases) of R&D that has gone into the research.

They have to earn that back somehow, that is why brand medicines are so expensive: they want to make optimal use of the years that their patent allows them to be the sole producer and thus have no competititors.

In this case I think it is outragious, since the company has not spend all the years doing R&D, the academia has. And also it is the sole producer, even though the patent has expired. The thinking of the company: with this medicine we are now earning the resources to research other venues of disease.

In my opinion the company should set up a system, in which patients of the poorer countries where the insurance system is not able to cough up $300,000 per patient per year (and this continues life-long), are benifited with donations of some sort.

Right now the group of academic researchers who first came up with the data have set up a European Working Group which helps such patients. Since the irony is: with this life-long treatment a patient can lead an almost normal life, otherwise it will get serious physical abnormalities (a spleen weighing 13 kg for instance).

Maybe the company has something like this in place, I am not sure, I think they should :)

 

[iansmith]

Originally posted by zoobyshoe
What does anyone want with spider webs? Why would goats be a better source than spiders?

Spider Web is one of the more resitant fiber that human knows. It could be use for fireproofing materials, bullet proofing or any other material that need reinforcement.

People do collect the wed from spider but it is a long and intensive manual labor. One indidual spider does not give as much web as one goat. For the goat all you have to do is to acquire the milk and isolate the protein. They are still working on the efficienty of the method.

 

[Monique]

Originally posted by zoobyshoe
Et, bien? Pourquoi?

What does anyone want with spider webs? Why would goats be a better source than spiders?

Spiderweb fiber is one of the strongest compounds known to exist in nature. What you can do with that? Just imagine :)

Why goats are a better source.. well.. how much web does a spider make in a day and how much milk does a goat make a day.. quantities ar the issue here.

 

[rockind78]

Originally posted by Monique
The only problem with the system is that the bacteria (also yeast?) will eventually loose the foreign piece of DNA.

This due to the stress that is generated by having to over-produce this specific protein that has no function for the micro-organism.

A cell that has lost the piece of DNA will have a significant growth advantage and will eventually take over the culture.

Also, impurities left after purification would be able to generate an immune response, I imagine.

But overall it is a really clever way to produce this kind of medication. Not just bacteria are used, also rabbits and maybe cows. The protein is specifically expressed in the mamary glands and will be excreted in the milk after which it can be purified. I guess they use these higher eukaryotes, since some proteins need post-translational modifications which don't take place in bacteria.

In a microbiology or molecular biology lab for instance, when the scientiest wants to maintain the presence of foreign DNA in a system, they use antibiotic resistance genes, growing the cells in the presence of the antibiotic. So in otherwords, if you wanted the continuted expression of your desired protein, and it it was sizably feasible to fit the gene encoding it into a plasmid that was readily transformed into a cell at a high efficiency, than what would stop someone from cloning the gene into a plasmid containing an antibiotic resistance gene and grwoing your cells in approporiate media with a sufficient concentration of the relative antibotic?

In utilizing this type of methodology, how would the gene of interest be "lost"?

 

[zoobyshoe]

Originally posted by iansmith
Spider Web is one of the more resitant fiber that human knows. It could be use for fireproofing materials, bullet proofing or any other material that need reinforcement.

How far have they gotten with this? Is there an actual bullet proof vest made from spider webs in existence? Or have they simply demonstrated the feasabilities on paper?

People do collect the wed from spider but it is a long and intensive manual labor. One indidual spider does not give as much web as one goat. For the goat all you have to do is to acquire the milk and isolate the protein. They are still working on the efficienty of the method.

Yes, I can see now that if you can spin it from a liquid under controlled conditions it would be easier than collecting by hand. It's not the same thing as with the silk moth.

 

[Monique]

Originally posted by rockind78
In utilizing this type of methodology, how would the gene of interest be "lost"?

Antibody resistance would be generated in the other cells. Also, you'd have to use a lot of antibiotics since it is continually used up, I guess it is expensive?

In my lab too, we grow all our cell lines (not transfected though) without any antibiotics.

 

[Monique]

Originally posted by zoobyshoe
How far have they gotten with this? Is there an actual bullet proof vest made from spider webs in existence? Or have they simply demonstrated the feasabilities on paper?

Correct me if I am wrong, but I am pretty sure that bullet proof vests made from spiderweb fiber are already in use today. Another advantage is that the material is very light AND strong.

 

[zoobyshoe]

Originally posted by Monique
Correct me if I am wrong, but I am pretty sure that bullet proof vests made from spiderweb fiber are already in use today. Another advantage is that the material is very light AND strong.

Amazing. I would imagine they could make full body garments out of it, including full face and head protection, given that it is light.

Do you know if it's fireproof or merely fire resistant? It melts, maybe, in a hot fire?

 

[rockind78]

Originally posted by Monique
Antibody resistance would be generated in the other cells. Also, you'd have to use a lot of antibiotics since it is continually used up, I guess it is expensive?

In my lab too, we grow all our cell lines (not transfected though) without any antibiotics.

I'm not sure I am following you here. Let me give you an example. the laboratory goal for this semester of my molcular biology class is to clone a fragment of the fur gene for chinese hamsters. Once the PCR product has been cleaned up, digested, and ligated into a specific plasmid, the gene can then be replicated. This is accomplished through the fact that the plasmid we are using codes for ampicillin resistance and the lac genes . Additionally, the MCS (multiple cloning site) for this plasmid is located in the middle of the lac Z gene. After transformations, the cells are plated out on ampicillin fortified LB agar that is treated with IPTG and XGal. These are both lactose analogs. One cannot be metabolized but is rather contiuously recycled as an inducer for the lac genes. The other turns blue when it is cleaved. Once the cells grow up, if they grow up, we know just from looking whether or not

1) the plasmid was taken up, as evidenced by growth on the medium
2) whether or not the gene was properly ligated in by way of a color change of the cells.

My point behind all this, is that although it would be expensive (that I will grant you), how would the antibiotic resistance be conferred to the the cells not carrying the plasmid, especially in genetically modified cloning strains?

The selective pressure of the antibiotics I would think keeps the plasmid, and thus the gene, in the cells as long as the antibiotic concentrations were up to par and the cultures were kept fresh.

 

[Monique]

Originally posted by rockind78
My point behind all this, is that although it would be expensive (that I will grant you), how would the antibiotic resistance be conferred to the the cells not carrying the plasmid, especially in genetically modified cloning strains?

The selective pressure of the antibiotics I would think keeps the plasmid, and thus the gene, in the cells as long as the antibiotic concentrations were up to par and the cultures were kept fresh.

Ian would be able to give a better explanation, but bacteria are able to readily take up DNA from their environment. This is what causes many pathogenic bacteria to become resistent to all kinds of antibiotics. I read an article in the paper today where, I believe, in France a highly resistant bacteria is on the rise..

Since the bacteria who initially weren't resistant also don't have the insert, they would have a significant growth advantage after aquiring the antibody resistance gene and take over the culture since they grow faster without the stress of being a protein factory.

 

[iansmith]

If the antibotics is used from the start of the batch culture and kept at the concentration requires bacteria not carrying the plasmid will die and won't have an advantge. What could happen is random resistance because the batch is use over and over and the selective pressure is great. Bacteria not carrying the plasmid migth and resistant to the antibitotics.

As far as for uptake free DNA, some strain of bacteria are not good at it. Most E. coli used are recombianse negative in order to decrease recombiantion and circular DNA is harder to uptake than linear DNA.

There is a system that could be used without the need for antibitotics. Some plasmid are retained not matter what happen. These plasmid carry a poison and an antidote. The antidote is unstable when free and the poison is stable. If the plasmid is not segregated then the antidote is degraded and the poison kills the bacteria.

 

[Monique]

Have you ever seen those incubators? They are HUGE. Six meters (18 foot) high if I remember the picture correctly. It mustn't be easy to keep those contamination free.

 

[iansmith]

Originally posted by Monique
It mustn't be easy to keep those contamination free.

These are more or less closed system. If you have a good sanitaziton plan it can be kept contamination free.

Also you can also use media that are specific for certain species. these are called selective media and contain salt concentrion or coumpound that inhibit the grow of certain groups and species.