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METABOLIZAM LEKOVA

Metabolizam je mehanizam za eliminaciju egzogenih (nepoželjnih) jedinjenja iz organizma i kontrolu nivoa endogenih
(poželjnih) jedinjenja u organizmu.

U toku metabolizma lekova u ćeliji se ne stvara energija, niti gradivni elementi, već je osnovni cilj lakša i brža eliminacija (ćelija teži da se što pre oslobodi leka)!

OSOBINE METABOLITA
POLARNIJI I HIDROSOLUBILNIJI OD
POLAZNOG JEDINJENJA
VIŠE JONIZOVANI PRI FIZIOLOŠKOM pH
MANJE VEZANI ZA PROTEINE PLAZME I
TKIVA
MANJE DEPONOVANI U MASTIMA
TEŽE PROLAZE KROZ ĆELIJSKE MEMBRANE

LAKŠA ELIMINACIJA LEKA IZ ORGANIZMA

1

REAKCIJE METABOLIZMA
REAKCIJE I FAZE - REAKCIJE
FUNKCIONALIZACIJE (oksidacija, redukcija, hidroliza)
REAKCIJE II FAZE - REAKCIJE SINTEZE ili REAKCIJE KONJUGACIJE (enzimi transferaze) REAKCIJE III FAZE

VEĆINA ENZIMA I FAZE JE SMEŠTENA U
MIKROZOMIMA, A VEĆINA ENZIMA II
FAZE U CITOZOLU HEPATOCITA
(izuzev glukuronil transferaza koje se t e. nalaze u mikrozomima)

AKTIVNOST METABOLITA
NEAKTIVNI

METABOLITI LEKA

AKTIVNI
TOKSIČNI
TOKSI NI

2

Primer formiranja aktivnih metabolita benzodiazepini
Na osnovu formiranja aktivnih metabolita i njihovog t1/2, benzodiazepini se dele na:
Kratko - delujuće (imaju 3-OH grupu koja se direktno konjuguje dajući neaktivne metabolite)
Dugo - delujući (obično imaju alkil grupu na N1 i podležu reakciji oksidativnog N-dealkilovanja- OND dajući aktivne metabolite sa dugim t1/2 ).

1.
2.

CH 3
N

H

O

OND
Cl

N

DIAZEPAM

t1/2 =30h

O

N
Cl

N

NORDIAZEPAM

t1/2 =50-100h (1-6 dana)

Nordiazepam je glavni aktivni metabolit većine dugo-delujućih BDZ.

Metabolički enzimi -CITOHROM P450

3

CYP450 ima centralnu ulogu u oksidativnom metabolizmu lekova (odgovoran je za 70-80% ukupnog metabolizma I faze), različitih zaga ivača iz spoljašnje sredine i drugih ksenobiotika, a učestvuje i u metabolizmu i sintezi mnogih endogenih jedinjenja
(steroidni hormoni, holesterol, liposolubilni vitamini, masne kiseline – uključujući i njihove derivate prostaglandine i leukotrijene).
CYP450 je zajednički termin za veliku familiju hemtiolatnih proteina (više od 50), koji su primarno smešteni u glatkom ER hepatocita (ali su prisutni u odre enoj količini u svim ćelijama organizma, izuzev zrelih eritrocita i skeletnih mišićnih ćelija). Pojedini enzimi su smešteni u unutrašnjoj membrani mitohondrija.

NOMENKLATURA
Hemoproteini – hem
(prostetična
grupa)
Hem = protoporfirin
IX - Fe2+/Fe3+

4

oznaka familije enzima (gena)

CYP 2D6 → individualni broj

podfamilija

Kod čoveka je do sada otkriveno ukupno 17 familija. Familije 1, 2 i 3 su uključene u metabolizam ksenobiotika.

Relativan udeo izoenzima u metabolizmu lekova

ISPITIVANJE METABOLIZMA
LEKOVA

5

ZNAČAJ
MNOGI LEKOVI SVOJE EFEKTE,
NEŽELJENE EFEKTE i/ili TOKSIČNOST
ISPOLJAVAJU PREKO METABOLITA!
METABOLIČKI STATUS
METABOLIČKI PROFIL

PROIZVODI METABOLIZMA
1º METABOLITI:
LEK ENZIM 1º METABOLIT
2º METABOLITI:
1º METABOLIT ENZIM

2º METABOLIT

METABONATI: NASTAJU IN VITRO
HEMIJSKIM, NEENZIMSKIM PROMENAMA
METABOLITA

PRAĆENJE METABOLITA
Biološki materijali: plazma, urin (ostali)
Priprema biološkog materijala: T-T, SPE
Kvalitativna i kvantitativna analiza: HPLC,
GC, MS, HPLC-MS, GC-MS, MS-MS,
NMR, MS-NMR

6

PREDKLINIČKA

IN VITRO
Primenom in vitro eksperimentalnih modela (različiti preparati jetre i drugih organa) IN VIVO
Na EKSPERIMENTALNIM ŽIVOTINJAMA
(miš, pacov, zamorac, kunić, pas,majmun, svinja) 2. Kod LJUDI
U in vivo ispitivanjima, metaboliti se uobičajeno prate i odre uju u različitim biološkim tečnostima, npr. plazmi ili urinu.
1.

KLINIČKA

Ispitivanje metabolizma in vitro

Primer: metabolizam diazepama - reakcije I faze

7

REKOMBINANTNI ENZIMI
- cDNK CYP450-ekspresioni sistemi -

NEDOSTATAK
Ne može se proceniti kvantitativan doprinos pojedinačnih izoenzima metabolizmu leka u jetri, jer se ne uzima u obzir njihov različit sadržaj, aktivnost u prisustvu drugih enzima, kao i interindividualne varijabilnosti u aktivnosti.

MIKROZOMI
- uz primenu inhibitornih monoklonskih At MIKROZOMI IZ HUMANE JETRE (CYP450)
+
Supstrat
+
Monoklonska At
Supstrat
Proizvod
% inhibicije kvantifikuje ulogu ispitivanog izoenzima u metabolizmu leka

8

Legenda:

IN VITRO - IN VIVO KORELACIJA
Za definisanje in vitro – in vivo korelacije potrebni su kvantitativni modeli zasnivani na Michaelis –
Menten -ovoj kinetici
Iz Michaelis – Menten -ove jednačine se izračunava CLint
In vivo parametri koji se dovode u vezu sa parametrima in vitro sistema su CL, t1/2, Css

Model za kvantitativno predvi anje interakcija
Iz Michaelis – Menten -ove jednačine izračunava se CLint i CLint(i)

Ii =

CLint
CLint( i )

9

Ispitivanje metabolizma in vivo

FAKTORI KOJI UTIČU NA
METABOLIZAM LEKOVA IN VIVO
FIZIOLOŠKI FAKTORI:
STAROST/UZRAST
POL
TM
TRUDNOĆA

SPOLJAŠNJI FAKTORI:
ISHRANA
PUŠENJE
ALKOHOL
SUPSTANCE IZ OKRUŽENJA
KOMBINOVANA TERAPIJA

PATOLOŠKI FAKTORI
GENETIČKI FAKTORI

FIZIOLOŠKI FAKTORI
STAROST/UZRAST
PEDIJATRIJSKA POPULACIJA
Po ro enju smanjena aktivnost enzima i I i II faze.
Aktivnost enzima I faze brzo raste i u toku 2-3 nedelje dostiže vrednosti višestruko veće nego kod odraslih.
Aktivnost enzima II faze raste znatno sporije (sve do 23 godine kada dostiže vrednosti kod odraslih) – izuzetak je konjugacija sa sulfatnom kiselinom i glicinom.

10

GERIJATRIJSKA POPULACIJA
Smanjena brzina eliminacije lekova zbog smanjene renalne ekskrecije i smanjene brzine metabolizma (fiziološke promene – smanjen protok krvi kroz jetru i masa jetre, smanjen minutni volumen srca, relativna hipoksija,...).
Mnogo je više redukovana aktivnost enzima I faze
(reakcije oksidacije), nego enzima II faze.

POL
Za pojedine lekove uočene razlike u farmakokinetici izme u muškaraca i žena. Razlike u resorpciji, BR i distribuciji su posledica razlika u fiziologiji i sastavu organizma (npr. sekrecija HCl i brzina pražnjenja želuca je manja kod žena; sadržaj masnog tkiva je veći kod žena, dok je mišićna TM veća kod muškaraca). Tako e su uočene razlike i u metabolizmu pojedinih lekova, ali se ekspresija metaboličkih enzima ne razlikuje izme u polova, kao što je to slučaj kod pojedinih životinjskih vrsta. Lekovi koji pokazuju razlike u farmakokinetici izme u polova kod ljudi
LEK

UOČENE RAZLIKE

Paracetamol

Veće conc. nepromenjenog leka kod žena zbog smanjene konjugacije sa glukuronskom kiselinom

Aspirin

Viša aktivnost esteraza kod muškaraca – niži nivoi leka u plazmi Hloramfenikol
Fenitoin
Tetraciklin

Viši nivoi u plazmi kod žena

Hlordiazepoksid
Diazepam

Niži CL kod žena u pore enju sa muškarcima

Eritromicin

Viši CL kod žena

Lidokain

Veće t1/2 i Vd kod žena

Nortriptilin

Intenzivniji metabolizam kod muškaraca; kod žena prisutni viši nivoi nepromenjenog leka u plazmi

Propranolol

Niži CL kod žena zbog smanjene konjugacije sa glukuronskom kiselinom Rifampicin

Viši nivoi u plazmi kod žena; jača urinarna ekskrecija nepromenjenog leka

11

HORMONI
Nedostatak testosterona smanjuje aktivnost
CYP450, dok je pokazano da estrogeni smanjuju oksidaciju pojedinih lekova. T e. pokazano je da kinetika pojedinih lekova zavisi od faze menstrualnog ciklusa žene (nivoa progesterona).

TRUDNOĆA
U trudnoći može biti povećana aktivnost pojedinih izoenzima CYP450.

DO NOT take this drug if there is any possibility that you are, or may become, pregnant. Just one dose can cause severe birth defects. Thalidomide was first marketed in Europe in the late 1950's. It was used as a sleeping pill and to treat morning sickness during pregnancy. At that time no one knew thalidomide caused birth defects!!! From Tragedy to Promise
Thalidomide
is synthesized

1954

Thalidomide withdrawn 1956

Introduced in
Germany as sedative 1961

Shown to inhibit
TNFα expression

1965

Report showing effectiveness in patients with leprosy 1991

FDA approves for ENL

1994

1998

Anti-angiogenic properties shown

Fast-track approval of IMiDs

2000

Today

Reports of effectiveness in
Multiple myeloma

12

PATOLOŠKI FAKTORI
OBOLJENJA JETRE
Redukovan metabolizam lekova.

OBOLJENJA BUBREGA
Poremećene su i neke metaboličke reakcije.

INFLAMACIJA
Medijatori akutne faze suprimiraju aktivnost CYP450.

HIPOKSIJA, SRČANA INSUFICIJENCIJA i dr. bolesti koji smanjuju protok krvi kroz jetru, smanjuju aktivnost enzima I faze (manji uticaj na enzime II faze).

SPOLJAŠNJI FAKTORI
PUŠENJE
POLICIKLIČNI AROMATIČNI CH IZ DIMA CIGARETE
INDUKUJU CYP1A2 – ubrzan metabolizam teofilina kod pušača.

ALKOHOL
HRONIČNO UNOŠENJE ALKOHOLA INDUKUJE
CYP2E1. Paracetamol se posredstvom ovog enzima prevodi u hepatotoksične metabolite.

13

ISHRANA
GLADOVANJE povećava hepatotoksičnost paracetamola.
DIMLJENA HRANA (policiklični aromatični CH) indukuje CYP1A2.
SOK OD GREJPFRUTA (naringin, furakumarin) inhibira CYP3A4 u tankom crevu – povećava BR mnogih per os primenjenih lekova.

14

Efekat soka od Grejpfruta na koncentraciju Felodipina u plazmi

tbl 5mg felodipina sa sokom

bez soka

Review DG. Bailey, et al. Br J Clin Pharmacol 1998,46:101-10.

KOMBINOVANA TERAPIJA
– INDUKCIJE / INHIBICIJE ENZIMA
IZOENZIM

INHIBITORI

INDUKTORI

CIMETIDIN, AMIODARON,
FLUOROHINOLONI, FLUVOKSAMIN,
FURAFILIN

POLICIKLIČNI AROMATIČNI
CH (DIM CIGARETE),
DIMLJENA HRANA

KETOKONAZOL, AMIODARON,
FLUKONAZOL, FLUVOKSAMIN, IZONIAZID

BARBITURATI, RIFAMPICIN

CYP2C9

CIMETIDIN, FLUOKSETIN, FLUVOKSAMIN,
KETOKONAZOL, OMEPRAZOL

KARBAMAZEPIN

CYP2C19
CYP2D6

HINIDIN, FLUOKSETIN, CIMETIDIN,
AMIODARON, PAROKSETIN, RITONAVIR

NIJEDAN POZNAT

CYP2E1

DISULFIRAM, DIETILDITIOKARBAMAT

IZONIAZID, ETANOL

CYP3A4

MAKROLIDNI ANTIBIOTICI, ANTIMIKOTICI
AZOLI, SOK OD GREJPFRUTA,
FLUOKSETIN, FLUVOKSAMIN, CIMETIDIN

KARBAMAZEPIN, FENITOIN,
RIFAMPICIN,
FENOBARBITON

CYP1A2

15

Efekat rifampicina i eritromicina na dispoziciju ciklosporina

KLINIČKI ZNAČAJNE INTERAKCIJE
JAKI INHIBITORI CYP3A4 (makrolidni AB, antimikotici derivati imidazola)
+ ASTEMIZOL, TERFENADIN ILI CISAPRID = srčane aritmije (torsades de pointes)
TERFENADIN POVUČEN SA TRŽIŠTA U EU i USA – zamenjen svojim metabolitom FEKSOFENADINOM
RIFAMPICIN + ORALNI KONTRACEPTIVI = smanjena efikasnost kontraceptiva diltiazem, azolni antimikotici, makrolidni AB, inhibitori proteaze + STATINI
(simvastatin) = rabdomijaliza

GENETIČKI FAKTORI – GENETIČKI
POLIMORFIZAM
Poly, morphe = mnogoobličnost, postojanje različitih oblika
Genetički uslovljene razlike u ekspresiji odre enog metaboličkog enzima unutar populacije – prisustvo enzima u različitim strukturnim varijantama i/ili količinama, kao posledica promena na nivou gena.
Nasledno uslovljene razlike u aktivnosti enzima dele bolesnike u dve ili više subpopulacija, koje se me u sobom jasno razlikuju po kapacitetu za metabolisanje odre enih lekova supstrata genetički polimorfnog enzima.
Bi- ili polimodalna distribucija odgovarajuće mere enzimske aktivnosti in vivo

16

Fenotipovi:
PM (poor metabolizers) – oskudni (spori, slabi metabolizeri)
EM (extensive metabolizers) – ekstenzivni (normalni, brzi, obimni, katalitički kompetentni) (IM - intermediate metabolisers)
UM - ultrarapid metabolisers

Polimorfizam CYP2D6

Polimodalna distribucija metaboličkog odnosa debrizokvin/4-OH debrizokvin u populaciji Šve ana

17

ODRE IVANJE FENOTIPA I GENOTIPA
ODRE IVANJE FENOTIPA
Procena aktivnosti enzima in vivo primenom probnih supstanci
– praćenje nepromenjenog leka i metabolita u urinu, plazmi i salivi.
METABOLIČKI ODNOS (metabolic ratio): nepromenjen lek / metabolit breath test - 13C i 14C obeležene probne supstance
14CO u izdahnutom vazduhu
2

13CO

2i

Koktel pristup (coctail approach)

Probne supstance za ode ivanje aktivnosti izoenzima CYP 450
IZOENZIM

SUPSTRAT

METABOLIT

CYP 1A2

kofein

Demetil derivati

CYP 2A6

kumarin

7-OH-kumarin

CYP 2C9

tolbutamid
S-varfarin

hidroksitolbutamid
6-OH- i 7-OH-S-varfarin

CYP 2C19

S-mefenitoin omeprazol 4'- OH-S-mefenitoin
5-OH-omeprazol

CYP 2D6

debrizokvin dekstrometorfan sparteine

4-OH-debrizokvin dekstrorfan 2,3- I 5,6-dehidrospartein

CYP 2E1
CYP 3A4

hlorzoksazon

6-OH-hlorzoksazon

14C-N-metilerithromicin

N-demetileritromicin+14CO2
1'-OH- i 4-OH-midazolam

midazolam

ODRE IVANJE GENOTIPA
Analiza gena odre enog metaboličkog enzima primenom različitih PCR (Polymerase Chain Reaction) tehnika i drugih metoda.

18

Učestalost fenotipova PM i UM za pojedine CYP450 izoenzime me u različitim rasama i etničkim grupama

Belci

PM

UM

Azijati

PM

UM

Crnci

PM

UM

Etiopljani/Arabljani

PM

UM

učestalost (%)
CYP 2D6
CYP 2C9
CYP 2C19

5-10
0.2-1
2-4

1-10
n.i.
n.o.

1
2-3
10-25

0-2
n.o.
n.o.

0-20
n.o.
1-5

2
n.o.
n.o.

1.8-2*
n.o.
2*

10-29
n.o.
n.o.

n.i. – nisu identifikovani;
n.o. – nisu odre eni;
* - odre ivanje izvršeno samo me u Arabljanima

POSLEDICE GENETIČKOG
POLIMORFIZMA
A) Enzim katalizuje deaktivaciju leka:
PM su osetljiviji na toksične efekte nepromenjenog leka (prilagoditi doziranje)
B) Enzim katalizuje aktivaciju leka (u slučaju prekurzora): UM osetljiviji na toksične efekte metabolita (prilagoditi doziranje) Primeri klinički značajnih polimorfizama: primena standardnih doza lekova kod PM
IZOENZI
M

LEK

Fenotip

REGISTROVANE KLINIČKE
POSLEDICE

varfarin
CYP2C9

CYP2C19

Fatalna krvarenja

glipizide, tolbutamide Produžena hipoglikemija

omeprazol

Veći stepen izlečenja peptičkog ulkusa i infekcija izazvanih H. Pylory

progvanil*

PM

Neefektivna profilaksa malarije

TCA antipsihotici Izraženi ekstrapiramidni neželjeni efekti

kodein**

CYP2D6

Pojačani neželjeni efekti, izražena kardiotoksičnost Odsustvo analgezije

*Prekurzor aktivnog metabolita ciklogvanila
**Analgetičku aktivnost duguje aktivnom metabolitu morfinu

19

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