Studies On The Inhibitory Effect Of Pharmaceutical Excipients On Cytochrome P450 3A

The cytochrome P450 is a superfamilly consisting of a number of hemo-enzymes.Among them,there are three major families(CYP1,CYP2 CYP3) contributing to many drug metabolism.In human liver and small intestine,cytochrome P450 3A(CYP3A) is the most important subfamily among the cytochrome P450 superfamily,and accounts for 30%of the total P450 content in the adult human liver and for 70%in the human intestinal.The major congener of the CYP3A family is CYP3A4,which is involved in the metabolism of more 50 %of the currently marketed drugs and in the oxidation of a variety of endogenous substrates.Recently,certain concerns have been raised due to the findings that some substances can affect drug metabolism through interaction with some components of the microsomal system thus leading to inhibition of cytochrome P450 3A.For example,Grapefruit Juice could inhibit CYP3A4 activity and increased some drug bioavailability including dihydropyridines, terfenadine,saquinavir,cyclosporine,midazolam,triazolam and verapamil.In addition,some flavoids in herb medicine had also the same effects on CYP3A4.Pharmaceutical excipients are substances other than the pharmacologically active drugs that are included in the final pharmaceutical products.These substances are used as co-solvents,binders,diluents, lubricants,coloring,flavoring or coating agents for the drugs.Often these substances are regarded as inert from a therapeutic sense.However,pharmaceutical excipients may influence the absorption,distribution,metabolism,or excretion of the active drugs,which lead to either decrease or increase the bioavailability of drug and then cause significant pharmacological effects clinically.Although there are a handful of papers dealing with these potential effects,the general effects of most of the excipients on drug metabolism are yet unknown.To evaluate the effects of pharmaceutical excipients on CYP3A activity and acquire the potential inhibitory pharmaceutical excipients,we designed several experiments.Firstly,we examined the effects of 22 common excipients with fixed concentration on recombinant cytochrome P450 3A4(rCYP3A4),which was expressed in baculovirus infected Sf9 cells and supplemented with recombinant P450 reductase and cytochrome b5,using midazolam as the probe.Based on the primary study,we continued to evaluate the inhibitory potency and mechanism of some pharmaceutical excipients on rCYP3A4.Secondly,to further understand the effects of the excipients on CYP3A in in vivo system,we still used midazolam as CYP3A probe and study their effects on the pharmacokinetics of midazolam and its major metabolite 1'-hydroxymidazolam with different administration modes.Moreover,we further evaluated the impacts of PEG400 with different dose and administration mode on CYP3A in vivo. Thirdly,according to the experimental results in vitro and in vivo,we examined the inhibitory potency and mechanisms of pharmaceutical excipients on CYP3A,using rat liver and intestinal microsomes as in vitro model systems,and confirmed the ralationship between the inhobitory potency and the phyiscal-chemical characteristics of excipients.Lastly,based on the former study results,we selected two pharmaceutical excipients,which not only inhibited CYP3A activity but also increased midazolam bioavailability,to examine their impacts on other CYP3A substrate bioavailability.Using bamidipine as model drug,we investigated the effects of PEG400 and Tween 20 on its pharmacokinetics,and confirmed whether they could increase bamidipine relative bioavailability.In the in vitro study,using rCYP3A4 as in vitro cell model to select the potential inhibitory pharmaceutical excipients on CYP3A activity,the results showed that 15 of 22(68.2%) tested excipients could inhibit the activity of CYP3A4 more than 50%in six category pharmaceutical excipients,particularly the surfactants and polymers.Surfactants could completely inhibit CYP3A4 activity,for example,the inhibition rates were 99.40%,99.50% and 99.80%for RH40,SLS,and Triton X-100,respectively.Olive oil was the only excipient having no effects on the CYP3A4 activity.Each from six catogory excipients was selected for examining its inhibitory[potency through 1'-hydroxymidazolam formation.The IC50 values in vitro of selected excipients were 10.77,0.11,0.25,0.29,219.93,6.61,4.10 mg·mL-1 for PEG400,F68,RH40,SLS,PEG2000,lecithin,Vit.C respectively.The IC50 values of surfacts were relative lower.In the rCYP3A4 cell coincubation system,F68 inhibited midazolam biotransformation via the 1'-hydroxylation pathway by apparently mixed type competitive-noncompetitive mechanism,with mean K_i values of 0.16 mg·ml-1, and mean K'_i values of 0.08 mg·ml-1,respectively.Based on the results in the rCYP3A4 cell model study,10 pharmaceutical excipients were selected to study the effects on CYP3A in rats in vivo through the pharmacokinetics of midazolam and its primary metabolite 1'-hydroxymidazolam.We continued to use midazolam as in vivo CYP3A probe,ketoconazole(KTZ) as positive control,and three ratios including the ratio of AUC_i/AUC and CLz/F of MDZ or the ratios of AUC_0-∞ (1'-hydroxymidazolam/midazolam) as indexes of CYP3A activity,respectively.In the in vivo studies,most selected excipients significantly inhibited the activity of CYP3A by decreasing the ratios of AUC_0-∞(1'-hydroxymidazolam/midazolam).For examples,single dose administration of PEG400,F68,Tween 20,EL35,RH40,SLS,Lecithin and Vit.C could decrease the ratio of AUC_0-∞(1'-hydroxymidazolam/midazolam) from 1.14 to 0.34,0.57, 0.32,0.20,0.45,0.44,0.20 and 0.33,respectively(p＜0.05).On the other hand,after multiple dose excipients were administered,PEG400,F68,Tween 20,S40,RH40,SLS,Lecithin could also decrease the ratio of AUC_0-∞(1'-hydroxymidazolam/midazolam) from 1.14 to 0.39, 0.52,0.33,0.82,0.70,0.65 and 0.66,respectively(p＜0.05).However,single or multiple dose administration of excipients had complicated effects on the ratios of AUC_i/A UC and CLz/F of midazolam.Further study on different dose and administration mode of PEG400 showed that PEG400 could also decrease the ratio of AUC_0-∞(1'-hydroxymidazolam/midazolam) and had the same effects on the ratios of AUCi/AUC and CL/F of midazolam as the former results.According to the results in vitro and in vivo,we investigated the inhibitory potency and mechanisms of 9 pharmaceutical excipients on CYP3A,using midazolam as CYP3A probe and rat liver microsomes as CYP3A expressing system.The data displayed that the IC₅₀ values in vitro of selected excipients were 17.51,7.63,2.06,3.14,2.81,0.01,0.06,10.21, 7.13 mg·mL-1 for PEG400,F68,Tween 20,S40,EL35,Triton X-100,SLS,Lecithin,Vit.C respectively.In addition,all excipients inhibited midazolam biotransformation via the 1'-OH pathway by apparently mixed type mechanism,with mean K_i values of 27.22,4.91,2.72, 1.36,1.27,0.0075,0.11,3.75 and 1.19 mg·mL^-1,and mean K'_i values of 16.78,7.40,6.37, 1.11,4.47,0.02,0.037,20.00 and 6.12 mg·mL^-1,for PEG400,F68,Tween 20,S40,EL35, Triton X-100,SLS,Lecithin,Vit.C respectively.Samiliar to the rat liver microsomal study,we further investigated the inhibitory potency and mechanisms of 9 pharmaceutical excipients on CYP3A,using midazolam as CYP3A probe and rat intestinal microsomes as CYP3A expressing system.The data displayed that the IC₅₀ values in vitro of selected excipients were 10.28,0.63,0.39,2.23,0.64,0.01,0.16, 12.58,1.51 mg·mL^-1 for PEG400,F68,Tween 20,S40,EL35,Triton X-100,SLS,Lecithin, Vit.C respectively.In addition,all excipients inhibited midazolam biotransformation via the 1'-OH pathway by apparently mixed type mechanism,with mean K_i values of 2.58,0.53, 0.60,1.30,0.58,0.0078,0.071,7.35,1.60 mg·mL^-1,and mean K'_i values of 22.02,2.43, 1.02,3.06,1.79,0.027,0.32,38.50,1.40 mg·mL^-1,for PEG400,F68,Tween 20,S40,EL35, Triton X-100,SLS,Lecithin,Vit.C,respectively.On the basis of studies using midazolam as probe to assess the inhibitoin of pharmaceutical excipients on CYP3A,we continued to investigate the impacts of PEG400 and Tween 20 on other CYP3A substrate bioavailability,bamidipine as model drug and ketoconazole as postive control.The results showed PEG400 and Tween 20 elevated barnidipine AUC_0-∞ 1.15 and 1.11 times,compared to the negative control,and confirmed that they could increase bamidipine bioavailability minorly.In conclusion,22 pharmaceutical excipients were choosed to investigate the inhibitory effect on CYP3A in recombinant CYP3A4 cell model,10 adjuvants were selected for animal research and then 9 excipients were selected for mechanism study using rat liver and intestinal microsomes.The results indicated that most pharmaceutical exicipients could decrease the ratios of AUC_0-∞(1'-hydroxymidazolam/midazolam) and inhibited CYP3A activity with apparently mixed type mechanism in rat liver and intestinal microsomes, however,different administration modes and doses had different influences on midazolam bioavailability.Furthermore,the inhibitory potency and constants of excipients changed with their physical-chemical characteristics.Therefore,pharmaceutical excipients are generally regarded as inactive ingredients, however some excipients are not "inert" adjuvants and could change drug metabolism through effects on cytochrome P450 activity,such as CYP3A.This paper raises awareness of the ability of excipients to inhibit drug metabolism.This information may be important when selecting substances for drug manufacturing.Moreover,as patients often receive many medications simultaneously,the excipients from one medication could impact the activity of another active drug in a different medication.