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