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CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL FARMACO
Adriana Maggi
BASI MOLECOLARI DELL’AZIONE DEL FARMACO
BIOTECNOLOGIE FARMACOLOGICHE
LEZIONE 11
Ingegneria animale II
Produzione di biofarmaci
Utilizzo di vettori tessuto specifici per
esprimere proteine terapeutiche nel latte
o nel siero di animali transgenici
The cost of making one transgenic animal
ranges from $20,000 to $300,000
It has been estimated that one transgenic
animal can produce, in its lifetime, $200 to
$300 million worth of pharmaceuticals.
AviGenics comparison of production inputs and costs for monoclonal
antibodies* using traditional cell culture
versus using transgenic poultry or goats
Traditional Cell
Culture
Poultry Eggs
Goat Milk
170,000
250
21,000
Capital Equipment Cost,
or Cost per Animal
(dollars)
100 Million
1,000
10,000 to 50,000
Equipment Maintenance
Costs, or Keeping Cost
per Animal (dollars)
100,000
10
2,500
100
0.10 to 0.25
2 to 20
Raw Material Volume
(kg)
Unit Cost per Protein
(dollars per gram)
*100 kg of raw material per year
Source: AviGenics www.avigenics.com
Nutritional food.
Partial list of bioactive components in milk with human health implication
Milk protein components
Whey proteins
Casein
Lactoferrin
-Lactalbumin
Peptides
Whey proteins
Casein
Milk fat components
Other
———Cancer———
Conjugated linoleic acid
Vaccenic acid
Sphingolipids
Butyric acid
13-methyltetradecanoic acid
Calcium
Lactose
Vitamins A and D
Oligosaccharides
Nucleosides
Ether lipids
———Cardiovascular Health———
Conjugated linoleic acid
Stearic acid
Omega-3 fatty acids
———Hypertension———
Whey proteins
Whey proteins
Milk-fat globule membrane proteins
Peptides
———Immune Response———
Conjugated linoleic acid
———Bone Health———
Conjugated linoleic acid
Probiotics
Calcium
Vitamin D
Calcium
Potassium
Probiotics
Calcium
Phosphorus
Vitamin K
Pharming products currently
in development.
Animal
Drug/protein
Use
sheep
sheep
sheep
sheep
sheep
pig,
pig
goat
goat
goat
goat
cow
cow
cow
alpha1 anti trypsin
CFTR
tissue plasminogen activator
factor VIII, IX
fibrinogen
tissue plasminogen activator
factor VIII, IX
human protein C
antithrombin 3
glutamic acid decarboxylase
Pro542
alpha-lactalbumin
factor VIII
fibrinogen
cow
collagen I, collagen II
cow
lactoferrin
cow
human serum albumin
chicken, cow, goat
monoclonal antibodies
deficiency leads to emphysema
treatment of cystic fibrosis
treatment of thrombosis
treatment of hemophilia
treatment of wound healing
treatment of thrombosis
treatment of hemophilia
treatment of thrombosis
treatment of thrombosis
treatment of type 1 diabetes
treatment of HIV
anti-infection
treatment of hemophilia
wound healing
tissue repair, treatment of rheumatoid
arthritis
treatment of GI tract infection,
treatment of infectious arthritis
maintains blood volume
other vaccine production
Giugno 2006
APPROVAZIONE DA PARTE DELL’EMEA DEL PRIMO FARMACO
IN UN BIOREATTORE ANIMALE:
ANTITROMBINA UMANA PRODOTTA IN CAPRA
(per la terapia della deficienza ereditaria di antitrombina, una
patologia che affligge 3000-5000 soggetti)
Nature Biotechnology, 24,8:877
Alcuni farmaci in fase avanzata di studio
prodotti in bioreattori animali
Ditta
GTC Biotherapeutics
(Framigham Ma USA)
prodotto
indicazione
antitrombin
hereditary antitrombin
deficiency
fase sviluppo
Approved EU
phase III USA
Pharming
C1 inhibitor (rabbit) Hereditary angiodema
(Leiden Netherlands) h Lactoferrin (rabbit) infection and inflamm
hfibrinogen (rabbit) tissue sealant
phase III
preclinical
preclinical
Bioprotein Technologies Rothavirus particles roth infection
(Paris)
(rabbit)
preclinical
PharmAthene
protexia (goat)
neural tissue
preclinical
Risposta agli xenobiotici ‘umanizzata’
• SXR e’ un recettore intracellulare che lega
ed induce la trascrizione di CYP3A un citocromo
essenziale nel metabolismo dei farmaci
• PXR e’ l’omologo murino del recettore umano
SXR
• Diverse specie animali rispondono ad un
determinato xenobiotico (inducendo CYP3a) in
modo specifico; questa caratteristica e’ insita
nelle differenze tra i diversi recettori
(Es SXR e PXR)
Strategia per il Knock out di PXR
Strategia per l’inserzione di SXR
Risposta agli xenobiotici ‘umanizzata’
• L’ablazione di PXR e la sua sostituzione con
SXR ha permesso di creare un modello di topo
‘umanizzato’ per il metabolismo dei farmaci.
• il modello ‘umanizzato’ permette di prevedere
il metabolismo di un determinato farmaco e di
studiare l’interazione tra farmaci diversi
Modelli per lo screening di farmaci
Animali reporter
Adriana Maggi
Metodologie innnovative in drug
discovery: animali reporter
Nature Review Drug Discovery
March 2005
REPORTER SYSTEMS
TO STUDY GENE EXPRESSION
IN VIVO
ER
ER
POL II, TF,
co-regulators
ER ER
ERE
transcription
Reporter
GFP
Reporter: easily
detectable protein
REPORTER SYSTEMS: FROM CELLS TO MICE
THE REPORTER/
DETECTION SYSTEM
THE PROMOTER
TRANSGENESIS/Kin
Strategia per la generazione del topo reporter ER-luc
RAPID TURNOVER!
THE ACTIVITY OF ESTROGEN RECEPTORS OBSERVED
IN ALIVE MICE: LUCIFERINE BIODISTRIBUTION
5 min.
10 min.
15 min.
20 min.
25 min.
30 min.
“Optical Imaging” after
estradiol injection (50mg/Kg s.c.)
CTS/S
30
6 HOURS
24 HOURS
100
ABDOMEN
50
15
50
3 HOURS
CTS/S X (10-3)
TIME 0
HEAD
2
CHEST
TAIL
25
1
TIME 0
3 HOURS
6 HOURS
24 HOURS
IS THE ERE-LUC MOUSE A FAITHFULL REPORTER OF
THE ACTIVITY OF ESTROGEN RECEPTORS?
ANALYSIS OF RESPONSIVE TISSUES
CO-LOCALIZATION OF ERs AND LUCIFERASE BY
IMMUNOCYTOCHEMISTRY STUDIES
TIME-COURSE, DOSE-RESPONSE AFTER ADMINISTRATION
OF ESTRADIOL
PARALLEL MEASUREMENT OF ENDOGENOUS ESTROGENRESPONSIVE GENES (PROGESTERONE RECEPTOR)
ANALYSIS OF LUCIFERASE ACTIVITY AFTER TREATMENT
WITH ICI 182,780 TO MEASURE THE CONTRIBUTION OF
ERRs
Ciana et al. Mol. Endocrinol. 2001, Nature Med. 2003
ERE-Luc mouse a model to study ER transcriptional activity
Embryo’s development
12.5
13.5
immature
day1
15.5
14.5
16.5
17.5
18.5
19.5
adult
day10
proestrus
estrus
diestrus 1
diestrus 2
REPORTER MICE
A NEW APPROACH
FOR THE DEVELOPMENT OF THERAPEUTICS
DRUG DEVELOPMENT – PRECLINICL PHASES
Preclinical studies
Pharmacology:
pharmacodynamics and organ-specificity of action
drug disposition ADME
Toxicology:
single, repeated-dose toxicity testing
special toxicity tests
day 0
day 6
day 12
day 1
day 7
day 13
day 2
day 8
day 14
day 3
day 9
day 15
day 4
day 10
day 16
day 5
day 11
day 17
Bioluminescence analysis
of ERE-Luc cycling female
mice
day 18
day 19
day 20
day 21
250
cts/s
200
Control
E2
ER activity in LIVER
of adult cycling ERE-Luc mice
treated with:
150
100
50
0
cts/s
200
150
E2
T low
T high
E2 5.5 ug/kg/day
Tamoxifen 5.5 ug/kg/day
Tamoxifene 666 ug/kg/day
Raloxifene 5.5 ug/kg/day
Raloxifene 2111 ugkgday
100
50
0
cts/s
200
150
E2
R low
R high
100
50
days
0
(each data point represents the
photon emission mean of a
minimum of 5 mice)
DRUG DEVELOPMENT – PRECLINICL PHASES
Preclinical studies
Pharmacology:
pharmacodynamics and organ-specificity of action
drug disposition ADME
Toxicology:
single, repeated-dose toxicity testing
special toxicity tests
ERE-LUC mouse in preclinical studies
Adsorption, Distribution, MEtabolism
With reporter mice the study of drug levels in plasma becomes meaningless:
drug dosage can be directly based on the pharmacological response even
in case of prolonged treatments
Drug metabolism may maintain some interest to evaluate the enzymes
responsible for the process
DRUG DEVELOPMENT – PRECLINICL PHASES
Preclinical studies
Pharmacology:
pharmacodynamics and organ-specificity of action
drug disposition ADME
Toxicology:
single, repeated-dose toxicity testing
special toxicity tests
ERE-LUC mouse in preclinical studies:
toxicology
the actual knowledge of the entire spectrum of drug targets facilitate
the prediction of side effect
gender pharmacology
effects in embryos
effects during lactation
differential effects in all of the target organs during development,
maturity and aging
14
12
3
*
2 *
1
*
0
ADULT
8
*
BRAIN
10
*
*
2
0
SUCKLING
CONTROL
5
*
0
5
*
*
*
2,5
2,5
*
10
* 7,5
7,5
6
4
L.U./mg prot. (x100,000)
LIVER
L.U./mg prot. (x10,000)
L.U/mg prot. (x100,000)
MICE LACTATING FROM
MOTHERS EXPOSED TO DDT
0
ADULT SUCKLING
p,p’-DDT
o,p’-DDT
Di Lorenzo et. al. Endocrinology 2002
IMAGING OF ERE-LUC PREGNANT MICE
16,5 DPC
18,5 DPC