Insert preclinical drug research

Preclinical Drug Research – INSERT
Norvir®
Molecular weight = 720.95
 lipinsky’s rule of five  MW: 200-500 = too big = bad absorption
— a very large compound will not pass the membranes = less absorption
 Freely soluble in methanol and ethanol, soluble in isopropanol and practically insoluble in
water
— no solubility in water
— but we don’t have information about the absolute bioavailability  why?
o it is insoluble in water  you cannot administer it IV
o IV with methanol is also not possible = massive irritation
 you cannot determine the absolute bioavailability

Route of administration
 gelatin capsules
 oral solution
 2 formulations: for the pedriatic population
 baby cannot get infected during the pregnancy (when the mother is infected)
 during labor there is a risk that the baby can get infected  get in contact with the blood of
the mother
— solution: caesarean section
 a lot of of pedriatic patients
— dose is too high + pill itself its too large
— oral solution with a flavour: compound is too bitter + babies/kids will like this more
 they will not spill it out
 comparison of bioavailability in both solution
— relative bioavailibility
 bio-equivalence:
— comparison with a reference drug (with the same mode of action)  To know if your
new drug is better than the one already on the market
— mechanism of action: inhibitor of both the HIV-1 and HIV-2 proteases

Clinical pharmacology
 Potency  low affinity for the target is not good
 exogenous targets  no off-target effects
 very high therapeutic index  concentration where we expect an efficacy compared to the
concentration where you see adverse effects (cytotoxicity)  difference of 1000-fold
— The IC50 of viral replication ranged from 3.8 to 153 nM
— cytotoxicity studies on several cell lines showed that >20 M was required to inhibit
cellular growth by 50% resulting in an in vivo therapeutic index of at least 1000.

Cross-resistance to other anti-retroviral
 among protease inhibitors, variable cross-resistance has been recognized
 cross-resistance between ritonavir and reverse transcriptase inhibitors is unlikely

Safety pharmacology
 no data

,  exogenous compound + 1000 fold difference (therapeutic index) + no drugs for an effective
HIV treatment  you can deviate from the guidelines
 Examen: what should you normally perform  CORE BATTERY
— name of tests + explanation

Pharmacokinetics
 ritonavir has been studied in healthy volunteers and HIV-infected patients
 The absolute bioavailability of ritonavir has not been determined
 peak concentrations of ritonavir were achieved approximately 2 hours and 4 hours after
dosing under fasting and non-fasting conditions, respectively.
— non-fasting: will start interfering with the absorption (plasma concentration will be
lower)
 after 4 hours you achieve plasma peak concentrations  effect will appear later
 effect of food on oral absorption
— no difference between the oral solution and the capsule
 metabolism
— isopropylthiazole oxidation metabolite is the major metabolite and has antiviral
activity similar to that of the parent drug  !! concentrations of this metabolite in
plasma are low
— liver microsomes have demonstrated that CYP3A is the major isoform + CYP2D6
contributes
— lot of metabolisms  drug will be cleared  less compound + less effect
o but one of the metabolites is active
 contribution will be low to the efficacy, because the concentration of the
active metabolite is low
— what other in vitro tests can be performed?
o S9 fractions (entire cytosolic fraction present) or primary hepatocytes (the
cells itself) or liver slices
 liver microsomes are very valuable for inhibition and affinity studies
 S9 fraction is good for phase 2 enzymes
 you need the complete cells (hepatocytes) for induction studies
— how can you asses that ritonavir affinity CYP3A and CYP2D6
o recombinant enzymes
o liver microsomes  how could you know this?  specific inhibitors for
CYP3A
 first you add the inhibitors, then the drug  if the concentration
doesn’t change, it will be metabolized by this enzyme
 inhibitor is already bounded to the CYPs and that’s why the
drug cannot bind anymore
 indirect way for assessing this
 not always specific  can bind with a different affinity to other
enzymes
 CYP2D6 = polymorphic  you need to do sequencing + check what
the polymorphic gene is  maybe change the dose
 slow  reduce dose to avoid toxicity
 high  increase dose to see an effect
 elimination
 only a small proportion is cleared by the kidneys  faeces = route of elimination
— what type of study was used?  mass balance study