Transcript Protein replacement therapies

Manifestation of Novel Social Challenges of the European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Manifestation of Novel Social Challenges of the European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
József Tőzsér
Molecular Therapies- Lectures 5-6
PROTEIN REPLACEMENT
THERAPIES
TÁMOP-4.1.2-08/1/A-2009-0011
Protein-based therapies
• Replacement of missing or abnormal proteins (e.g.
Insulin)
• Influencing signal transduction pathways (e.g.
interferons)
• Supplementation of growth hormones (e.g. PDGF)
• Influencing the haemostatic system (e.g. tPA)
• Degradation of molecules with enzymes (e.g.
Asparaginase
• Use of protein vaccines (e.g. recombinant HPV
vaccine)
TÁMOP-4.1.2-08/1/A-2009-0011
Examples of proteins replaced in therapy
•
•
•
•
•
•
•
Insulin (diabetes)
Albumin (hypoalbuminaemia)
Lactate (lactose intolerance)
Factor VIII. (haemophilia)
Factor IX. (haemophilia)
Protein C (protein C deficiency)
Beta-glucocerebrosidase (Gaucher’s disease)
TÁMOP-4.1.2-08/1/A-2009-0011
Examples of protein therapeutic products
•
•
•
•
•
•
•
Humulin (insulin)
Novolin (insulin)
Flexbumin 25% (albumin)
Lactaid (lactose)
ReFacto (F VIII)
BeneFix (F IX)
Ceprotin (aktivált protein C)
TÁMOP-4.1.2-08/1/A-2009-0011
Therapeutic proteins
• The first therapeutic protein use was the utilization of
cow pox „protein vaccine" against smallpox by Jenner
(1796)
• The first therapeutic protein used was insulin by
Banting and Best (1922)
• More than 200 peptide or protein has been approved
in USA for use in therapy
• Therapeutic proteins may come from different sources
TÁMOP-4.1.2-08/1/A-2009-0011
Therapeutic proteins – sources
Protein
•
•
•
•
•
•
•
Albumin
Insulin
Factor VIII
Factor IX
Kalcitonin
Anti-venom
β-glucorerebrosidase
Original source
Human blood
Pig, bovine pancreas
Human blood
Human blood
Salmon
Horse, dunkey blood
Human placenta
TÁMOP-4.1.2-08/1/A-2009-0011
Therapeutic proteins – sources
• Natural sourse is usually sparse and expensive
• It is difficult to satisfy demands
• Hard to isolate the product
• May lead to immune intolerance (e.g. in case of
animan proteins)
• Potential viral and pathogen contaminations
• Nowadays most of the protein drugs are made
by recombinant techniques
• Cheaper, safer, unlimited source
TÁMOP-4.1.2-08/1/A-2009-0011
Insulin, the first protein replacement drug
• In January 1922, Banting and Best used first insulin to
treat a 14-years-old patient named Leonard Thomson
• He became more ill as the consequence of the
injection, but his blood glucose level decreased,
therefore the improvement of the preparation
technique was decided
• 6 weeks later a better extract was able to decrease
the blood glucose level from 520 mg/dL to 120 mg/dL
within 24 hours.
• Leonard lived for additional 13 years, he died of
pneumonisa at the age 27
TÁMOP-4.1.2-08/1/A-2009-0011
Structure of insulin
Two polypeptid chains,
A.chain:
21 aminoacid residues
B-chain:
30 aminoacid residues
Chains are held together by
a disulfide bridge
Insulin gene is located at
chromosome 11
TÁMOP-4.1.2-08/1/A-2009-0011
Protein therapeutics – from blood
• The human body contains approx. 6 liters of blood
• 60-70% of blood is plasma, 8-9%- proteins. Therefore
it is an important protein source
• Human plasma contains about 10,000 different
proteins
• About 20 proteins make up the 99% of the total protein
content of plasma
• Annually several million liters of outdated
transfusion plasma is genereated, therefore it is an
excellent and relatively abundant protein source
TÁMOP-4.1.2-08/1/A-2009-0011
Examples of blood protein products
•
•
•
•
•
•
Factor VIII (correction of haemophilia)
Factor IX (correction of haemophilia)
Albumin (correction of hypoalbuminaemia)
Intravenous IgG (in infections)
Antithrombin III (correction of coagulation disorder)
Alpha I-PI (correction of emphysema)
TÁMOP-4.1.2-08/1/A-2009-0011
Cohn fractionation
• Cohn fractionation was originally developed in 1946.
• In Cohn fractionation plasma proteins are selectively
precipitated by using ethanol, salt, temperature
change.
• Separation of the fractions is achieved by
centrifugation.
TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
Recombinant technologies
• They were developed in the 1970s and 1980s
• Paul Berg (1973): discovery of restriction enzymes
• Herbert Boyer (1978): cloning of the human insulin
gene into E. coli - Genentech
• Fundamentally two major approaches
– Expressin in isolated cells
– Expression in transgenic plants or animals
TÁMOP-4.1.2-08/1/A-2009-0011
Hemostatic cascade
XIa
Ca2+
Extrinsic pathway
VII
+
TF, Ca2+
VIIa
TF,
Ca2+
Common pathway
XI
IX
IXa,
VIII
PL
Ca2+
X
XII
XIIa
Intrinsic
pathway
Xa
V
PL
Ca2+
prothrombin
XIII
thrombin
fibrinogen
All components of the intrinsic pathway are in the bloodstream.
Extrinsic pathway is initiated by the TF that is normally excloded from the
Bloodstream.
Cascade (avalange) model.
fibrin
crosslinked
XIIIa +
Ca2+
TÁMOP-4.1.2-08/1/A-2009-0011
Haemophilias A and B
• Hemophilias A and B are caused by deficiencies in
factors VIII or IX, respectively
• Affect ~1 in 5,000 males in Hemophilia A,
~1 in 30,000 males in Hemophilia B
• Inherited as a recessive X-linked trait in both cases
(Mother would be an unaffected carrier)
• Treated by administration of factor VIII or factor IX
concentrates
• Recombinant factor VIII or IX
• Gene therapy trials
TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
Haemophilia A – gene therapy
• Only FVIII level is  1 % of normal causes severe
symptoms (spontaneous bleeding into joints, vital
organs), therefore even low levels of proteins are
beneficial
• Tight control of FVIII production is not required
• Broad therapeutic index minimizes the risk of
overdose
• Delivery to bloodstream does not require expression
in the liver
• Domain B is not required for function, in its absence
the expression levels are higher
TÁMOP-4.1.2-08/1/A-2009-0011