3) 2. Screening methods of doping with erythropoietin
ee
ee
Or not artificially increase the number of erythrocytes is an important advantage in endurance sports, because it can carry more oxygen to muscles. For this reason, synthetic EPO is a doping substance banned by the World Anti-Doping Code since 1990. However, the test to demonstrate the presence of synthetic EPO in urine was introduced in 2001.
Both types of EPO molecules are proteins that have the same basic elements, amino acids. However, these proteins on their surface are also chains of carbohydrates (sugars) that differ between natural and synthetic EPO. This small difference is the basis of drug test that can reveal the presence or absence of synthetic EPO in addition to natural EPO normally excreted by the body. Indeed, sugars are capable of altering the pH of the molecule. It then uses the fact that synthetic EPO is more acidic than natural EPO to distinguish them visually on a gradient of acidity.
This method is based on a technique called gel electrophoresis, iso-electrofocusing. Its principle is to perform a separation of proteins according to their charge. The resolution can be expected in the first dimension separation is of the order of 0.01 pH units and can be improved by the use of an area closer to the isoelectric point. If one refers to a numerical example, by separating the proteins on a gel 18 cm long with an immobilized pH gradient between 3 and 10; 2.57 cm vest in one pH unit, but if the same separation is made on a narrower pH gradient covering three units, this time each unit will be resolved pH of 6 cm.
The search for EPO in urine (urine test) takes 2 ½ days, and after the manipulation, the presence of endogenous EPO and possibly recombinant is highlighted by antibodies recognizing the two types of molecules. EPO (synthetic) and
NESP
(Novel erythropoiesis stimulating protein) are located in an area most basic, respectively, more acidic, the gel than endogenous EPO (negative sample).
Posted by biophysicist at 16:53 - 3) 2. Screening methods to erythropoietin doping - Comments [0] - Trackbacks [0]
SHARE:
3) 1. Different types of synthetic erythropoietin and differences with natural erythropoietin
ee
ee
As we have seen erythropoietin (EPO) is a hormone produced protein type whose role is to stimulate the proliferation of erythrocytes [1], the cells that transport oxygen in the blood to all organs. Its production is regulated by the amount of oxygen available in the body, decreased tissue oxygenation will induce a higher EPO production and therefore an increase in the number of erythrocytes in the blood.
EPO can also be produced in vitro through biotechnology, then it makes a drug for treating patients who can not produce or EPO naturally. The two molecules of EPO, natural and synthetic, act exactly the same way in the body, they bind to a receptor located on the surface of erythrocyte precursor cells and their proliferation gives a signal. The number of red blood cells available increases.
Pharmaceutical specialties:
Name
DCI
Aranesp ®
Darbepoetin alfa
Dynepo ®
Epoetin delta
EPOMAX ®
Epoetin Omega
Eprex ®
Epoetin alfa
Eprex ® 4000
Epoetin alfa
Neorecormon ®
Epoetin beta
EPO mimetic peptides
Phase 3 test
Recormon ®
Epoetin beta
Mircera ®
CERA
ee
a) The NESP (novel erythropoiesis stimulating protein) or Aranesp ®
NESP (novel erythropoiesis stimulating protein) is considered the most famous rHuEPO, and is also known under the name of Darbepoetin alpha. It differs from epoetin by the substitution of five amino acids to assemble glycosilées five channels instead of three.
§ natural EPO
3 chains glycosylated, 165 amino acids, 30.4 kDa (kilo Dalton), 40% glycosylated; 14 sialic acids.
§ NESP
5 channels glycosilées; 165 amino acids but mutated 5; 37.1 kDa glycosylated 51%, 21 sialic acids
Over the molecule is glycosylated, there is less affinity between EPO and EPOR (the receiver). So the NESP at a rate of association with the EPOR lower than that of EPO:
Number of events of association between two molecules per unit time:
k (on) = 5.0 x 10 (8) M (-1) min (-1) for NESP
k (on) = 1.1 x 10 (8) M (-1) min (-1) for EPO
With k (on) = rate constant of association (M-1. min-1) where min means minute and M = 10 ^ 6
EPO is approximately 5 times faster than NESP to associate with the epo-r.
EPO as a faster association rate, there are more internalized EPO per unit time, and therefore the rate of degradation of EPO per unit time is higher than that of NESP. Its half-life is inversely proportional to the association rate, which depends on glycosylated chains and sialic acids.
The half-life is proportional to the number of sialic acids and the percentage of glycosylation of the molecule.
The rate of internalization is the same for both ligands. The ligand after internalization is re-secreted intact to 60% and 40% is degraded in both cases.
In practice, the use of NESP requires only one injection weekly for epoetin several cons.
ee
b) CERA (continuous erythropeitin receptor activator).
CERA (Continuous erythropoietin receptor activator) is an erythropoietin molecule which is inserted a long protein chain, nearly doubling its weight. Its half-life is very elongated, allowing a monthly injection. It was developed by Swiss pharmaceutical company Roche originally to treat anemia (MIRCERA ®).
CERA results from the integration of a polymer of 30 kDa (a string Methoxy-polyethylene glycol) secured to the molecule of Epo and N-terminal lysine residue on the 52 [17]. Its molecular weight is approximately twice that of epoetin and its half-life in humans is considerably increased, since it is approximately 130 hours after injection. This substantial increase in the half-life allows the efficient administration of CERA every 3 to 4 weeks [18, 19]. Reducing the frequency of administration is particularly valuable in patients with renal insufficiency not on dialysis and oncology patients [2] and Hematology, receiving chemotherapy treatments every 3 to 4 weeks.
This medicine is available in vials and prefilled syringe at different doses, for strictly medical use.
It is therefore a so-called 3rd generation EPO, or EPO, which allows Beta to maintain and extend its biological activity.
While a E.P.O. normal injection requires two or three times a week, CERA can be injected as in sick once a month or, for those who like risk, every 15 days. The fact that few tests are validated for detecting allowed athletes to believe they were untouchable.
The CERA molecule is large enough that it can not pass into the urine. However, there is a notable difference between the urine of an athlete at rest or during exercise. This would explain why, in general, an athlete out of competition can finally claim to be invincible and invisible.
With a E.P.O. classic, athletes must juggle the law, by injections every 3 or 4 days, transportation to the site of injections should be done systematically. The third generation of EPO of course, provides comfort since a monthly injection is easier to do in secret.
ee
ee
c) The peptide mimetics of EPO
ee
Develop small peptides capable of mimicking the actions of EPO by activating the receptor might seem impossible, yet it was possible to identify peptides capable of mimicking the action of this cytokine both in vitro and in vivo in mice. To achieve this, the method used was to build a library of random peptides attached to the surface of phages. This bank was then incubated with the EPO receptor and led to the demonstration of a peptide agonist capable of binding to the receptor extracellular domain.
The first peptide was identified (emp1) is a cyclic oligopeptide whose amino acid sequence is completely different from that of rHuEPO. However, this peptide competes with the labeled for EPO receptor binding, it induces the proliferation of a cell line corresponding to the EPO and induces the formation of colonies in vitro erythroblast from normal human marrow. It was also shown that it induces the phosphorylation cascade triggered by the binding of EPO to its receptor [20]. Other peptides EMP were then synthesized and selected to obtain biological properties increasingly significant, especially a higher affinity for the receptor. The final product was chosen, too, an amino acid sequence completely different from that of EPO. It is as effective as rHuEPO in receptor binding experiments and in vitro proliferation. The peptide was then pegylated gave birth to the hematide ™ (Affymax). This product has a long half-life in rats and monkeys. It stimulates erythropoiesis and increases hemoglobin in animals and in healthy volunteers. His long life could eventually allow a single IV injection monthly. He is now in Phase II clinical development in anemic patients with renal and oncology.
Interestingly, the anti-EPO antibodies do not cross with hematide ™ and do not neutralize its biological activity in vitro. It has been shown in animals with circulating anti-EPO hematide ™ that stimulates erythropoiesis and is capable of increasing the hemoglobin level. This suggests that this compound can be used as treatment in patients with anti-EPO antibodies and PRCA. A clinical trial is currently underway.
The future development of mimetics of EPO nonpeptide could potentially lead to ESAs active orally.
.gif "IP Gear")
LATSModerator / FOUNDING Member / NPC Judge
