BASI MOLECOLARI DELL’AZIONE DEL FARMACO
BIOTECNOLOGIE FARMACOLOGICHE
LEZIONE 11Ingegneria animale II
CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL FARMACO
Adriana Maggi
Produzione di biofarmaci
Utilizzo di vettori tessuto specifici per esprimere proteine terapeutiche nel
latteo 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.
Traditional Cell Culture Poultry Eggs Goat Milk
Raw Material Volume (kg) 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
Unit Cost per Protein (dollars per gram) 100 0.10 to 0.25 2 to 20
AviGenics comparison of production inputs and costs for monoclonal antibodies* using traditional cell culture versus using transgenic poultry or goats
*100 kg of raw material per yearSource: AviGenics www.avigenics.com
Nutritional food.
Milk protein components Milk fat components Other
———Cancer———Whey proteins Conjugated linoleic acid CalciumCasein Vaccenic acid LactoseLactoferrin Sphingolipids Vitamins A and D-Lactalbumin Butyric acid OligosaccharidesPeptides 13-methyltetradecanoic acid Nucleosides
Ether lipids Probiotics———Cardiovascular Health———
Whey proteins Conjugated linoleic acid CalciumCasein Stearic acid Vitamin D
Omega-3 fatty acids———Hypertension———
Whey proteins Calcium
Potassium———Immune Response———
Whey proteins Conjugated linoleic acid ProbioticsMilk-fat globule membrane proteins
———Bone Health———Peptides Conjugated linoleic acid Calcium
Phosphorus
Vitamin K
Partial list of bioactive components in milk with human health implications
Pharming products currently in development.
Animal Drug/protein Usesheep alpha1 anti trypsin deficiency leads to emphysemasheep CFTR treatment of cystic fibrosissheep tissue plasminogen activator treatment of thrombosissheep factor VIII, IX treatment of hemophiliasheep fibrinogen treatment of wound healingpig, tissue plasminogen activator treatment of thrombosispig factor VIII, IX treatment of hemophiliagoat human protein C treatment of thrombosisgoat antithrombin 3 treatment of thrombosisgoat glutamic acid decarboxylase treatment of type 1 diabetesgoat Pro542 treatment of HIVcow alpha-lactalbumin anti-infectioncow factor VIII treatment of hemophiliacow fibrinogen wound healing
cow collagen I, collagen II tissue repair, treatment of rheumatoid arthritis
cow lactoferrin treatment of GI tract infection, treatment of infectious arthritis
cow human serum albumin maintains blood volume
chicken, cow, goat monoclonal antibodies 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 prodotto indicazione fase sviluppo
GTC Biotherapeutics antitrombin hereditary antitrombin Approved EU (Framigham Ma USA) deficiency phase III USA
Pharming C1 inhibitor (rabbit) Hereditary angiodema phase III(Leiden Netherlands) h Lactoferrin (rabbit) infection and inflamm preclinical
hfibrinogen (rabbit) tissue sealant preclinical
Bioprotein Technologies Rothavirus particles roth infection preclinical(Paris) (rabbit)
PharmAthene protexia (goat) neural tissue preclinical
Risposta agli xenobiotici ‘umanizzata’• SXR e’ un recettore intracellulare che legaed induce la trascrizione di CYP3A un citocromoessenziale nel metabolismo dei farmaci
• PXR e’ l’omologo murino del recettore umanoSXR
• 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 conSXR ha permesso di creare un modello di topo‘umanizzato’ per il metabolismo dei farmaci.
• il modello ‘umanizzato’ permette di prevedereil metabolismo di un determinato farmaco e di studiare l’interazione tra farmaci diversi
Modelli per lo screening di farmaci
Animali reporter
Adriana MaggiAdriana Maggi
Metodologie innnovative in drug Metodologie innnovative in drug discovery: animali reporterdiscovery: animali reporter
Nature Review Drug DiscoveryNature Review Drug DiscoveryMarch 2005March 2005
GFP
POL II, TF, co-regulators
REPORTER SYSTEMSTO STUDY GENE EXPRESSION
IN VIVO
ERER
ERE
transcription
Reporter: easily detectable protein
ER
ER ER
Reporter
REPORTER SYSTEMS: FROM CELLS TO MICE
THE PROMOTER
TRANSGENESIS/Kin
THE REPORTER/
DETECTION SYSTEM
Strategia per la generazione del topo reporter ER-luc
RAPID TURNOVER!
5 min. 10 min. 15 min.
20 min. 25 min. 30 min.
THE ACTIVITY OF ESTROGEN RECEPTORS OBSERVED IN ALIVE MICE: LUCIFERINE BIODISTRIBUTION
24 HOURS
TIME 0 3 HOURS
6 HOURS
HEAD30
15
24 HOURS
TIME 0
3 HOURS
6 HOURS
ABDOMEN
50
100
50
CTS
/S
CHEST2
1
TAIL
25
CTS
/S X
(10-3
)
“Optical Imaging” after estradiol injection (50g/Kg s.c.)
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 ESTROGEN-RESPONSIVE GENES (PROGESTERONE RECEPTOR)
ANALYSIS OF LUCIFERASE ACTIVITY AFTER TREATMENT WITH ICI 182,780 TO MEASURE THE CONTRIBUTION OF
ERRs
IS THE ERE-LUC MOUSE A FAITHFULL REPORTER OF THE ACTIVITY OF ESTROGEN RECEPTORS?
IS THE ERE-LUC MOUSE A FAITHFULL REPORTER OF THE ACTIVITY OF ESTROGEN RECEPTORS?
Ciana et al. Mol. Endocrinol. 2001, Nature Med. 2003
ERE-Luc mouse a model to study ER transcriptional activity
Embryo’s development
immature adult
day1 day10 proestrus diestrus 1estrus diestrus 2
12.5 13.5 14.5 15.5 16.5 17.5 18.5 19.5
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 1 day 2 day 3 day 4 day 5
day 6 day 7 day 8 day 9 day 10 day 11
day 12 day 13 day 14 day 15 day 16 day 17
day 18 day 19 day 20 day 21
Bioluminescence analysisof ERE-Luc cycling female
mice
ER activity in LIVERof adult cycling ERE-Luc mice
treated with:
E2 5.5 ug/kg/dayTamoxifen 5.5 ug/kg/day
Tamoxifene 666 ug/kg/dayRaloxifene 5.5 ug/kg/dayRaloxifene 2111 ugkgday
(each data point represents thephoton emission mean of a
minimum of 5 mice)
0
50
100
150
200
250
2 4 6 8 10 12 14 16 18 20 22
cts/
s
ControlE2
0
50
100
150
200
2 4 6 8 10 12 14 16 18 20
cts/
s
E2T lowT high
0
50
100
150
200
0 2 4 6 8 10 12 14 16 18 20
cts/
s
E2R lowR high
days
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 facilitatethe 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
L.U
/mg
prot
. (x1
00,0
00) LIVER
0
1
2
3*
*1214
L.U
./mg
prot
. (x1
0,00
0)
0
2
4
6
*
* 8
L.U
./mg
prot
. (x1
00,0
00)
*
*
0
2,5
5
7,5
10
*
*
*
0
2,5
5
7,5
10
* *
*
p,p’-DDT o,p’-DDTCONTROL
ADULT ADULTSUCKLING SUCKLING
BRAIN
MICE LACTATING FROM MOTHERS EXPOSED TO DDT
Di Lorenzo et. al. Endocrinology 2002
IMAGING OF ERE-LUC PREGNANT MICE
16,5 DPC 18,5 DPC