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Dear all,
i am working in drug metabolism and pharmacokinetics lab and i conduct
CYP Liability studies of NCEs. With one of the molecule i am facing a
problem of Solubility. The molecule is showing very poor solubility in
all the buffers, that are generally used for conducting CYP inhibition
studies. This molecules is showing very good pharmacokinetics profile
in in-vivo situation. At the same time molecule is showing some
inhibition towards particular human CYPs. Because of solubility problem
i could not able to increase the concentration of my test compound in
my in-vitro CYP inhibition studies. After certain concentration (Around
1 uM) i am getting saturation in inhibition indicating that the
compound is not going beyond that concentration. I tried different
combinations of buffers to solubilise the drug and to conduct the
study. But some how the compound is not soluble. Few buffers i tried
where solubility is improved, but i have to compromise on CYP
activities.
Can any body help me how to go ahead with such molecules. please
suggest me ways and means to conduct such important studies for
molecules like this.
Syed.Mujeeb
Senior Research Associate,
DMPK,Discovery research,
DRL, Hyderabad, India
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Dear Muzeeb,
You could use acetonitrile up to 2% in the assay. Also, please go
through article published in DMD regarding the use of different
solvents for the assay.
I hope this helps,
Good luck,
Samiulla
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Dear Muzeeb
First and foremost reason to conduct the CYP liability studies in drug
discovery is to predict the in vivo drug interaction from in vitro and
scaling up to the clinics.
Solubility is such a problem, you can not circumvent it in discovery.
Instead of increasing the solubility of the NCE (Which you have tried)
rather find a way to solve the objective.
From your question, it looks like that you want to predict the IC-50 (by
keeping the substrate concentration constant and varying the inhibitor
concentration) for the molecule and scale up to in vivo situation - Here
you are finding the solubility problem for the molecule.
What I would like to suggest is that instead of finding the IC-50 of the
molecule, try to find the nature of inhibitor (competitive or non
competitive or un competitive) and calculate the Ki value at its highest
solubility concentration using the Dixon plots or secondary derived
plots and then make use of the Ki for predicting the in vivo drug
interaction by the formula I / Ki
Where I is the free (UN bound) concentration of the inhibitor (NCE in
question) with in the premises of the enzyme and Ki is the in vitro
dissociation constant of the inhibitor for the particular enzyme.
It is found that a ratio of I / Ki greater than 2 suggests that there is
a 80% likely hood of drug interaction in vivo.
To validate it, do a drug interaction study using a preclinical species
using the NCE and co administered drug (probe substrates for the
particular enzyme) and see the elevation of AUC for the probe substrate.
For more information, you can down load a article from the net -
AAPS PharmaSci 2002;
Which Concentration of the Inhibitor Should Be Used to Predict In Vivo
Drug Interactions From In Vitro Data?
Kiyomi Ito, Koji Chiba, Masato Horikawa, Michi Ishigami, Naomi Mizuno,
Jun Aoki, Yasumasa Gotoh, Takafumi Iwatsubo, Shin-ichi Kanamitsu,
Motohiro Kato, Iichiro Kawahara, Kayoko Niinuma, Akiko Nishino, Norihito
Sato, Yuko Tsukamoto, Kaoru Ueda, Tomoo Itoh, Yuichi Sugiyama
Volume 4(4) article #25
[http://www.aapsj.org/view.asp?art=ps040425 - db]
Any further suggestion on this issue is most welcome.
Ansar Ali Khan, M.Sc.,
Research Scientist,
Drug Metabolism and Pharmacokinetics,
Glenmark Research Centre,
Plot No.-A-607, TTC, Industrial Area,
Mahape, Navi Mumbai-400 709,
India.
Phone No: 91-22-5590 2491/92 Extn.308
Fax No.: 91-22-5590 2318
Dear Syed.Mujeeb,Back to the Top
You could dissolve your compounds in Methanol, Acetonitrile or DMSO and add to incubation mixture providing the % organic solvent is <1%.
There are several articles regarding the effects of organic solvents
on the activities of CYP P450 Isoforms. i.e:
J Easterbrook, C. Lu, Y Sakai and A.P. Li, (2001) Drug metabolism and
disposition, Vol. 29, No. 2, 141-144.
Regards
Mahmud Kajbaf , PhD
Medicines Research Centre
Via A. Fleming, 4
37135 Verona
Italy
Tel: +39 045 9219104
Fax: +39 045 9218153
E-Mail: mahmud.2.kajbaf.aaa.gsk.com
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What about decreasing the concentration of your microsomes or S9s or
what
have you by diluting them into the buffer that you are using. Wouldn't
that
show effectively the same thing?
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