The lack of pharmaceutical property information results in improper evaluation of the biological activity, bioavailability and pharmaco- and toxicokinetic profile of drug-like molecules in drug discovery. Consequently, potentially useful drug candidates are discarded prematurely, or too much time is spent on candidates that do not succeed. In the drug delivery group, we wish to understand the mechanisms that govern important pharmaceutical properties such as drug solubility, dissolution, absorption, distribution, metabolism, elimination and toxicity (ADMET) at the molecular and cellular levels through a multidisciplinary approach (see Fig. 1).
Fig.1. By utilizing our extensive panel of in vitro models (including for example single uptake or efflux models as well as integrated models of uptake, efflux and metabolizing enzymes), and combining these with our high throughput assays considerable amounts of data have been and are continuously generated (A). The data are analyzed using high capacity mass spectroscopy (B) and are used in the development of mechanistic cell kinetic models (C). The results give detailed information about the uptake, accumulation, metabolism and elimination of drugs in target tissues, such as intestine, liver and kidney (D) and are analyzed in PB/PK models to predict drug fate and drug interactions in humans (E).
Our research is fully integrated but can for clarity be divided into four areas:
- Drug solubility and dissolution
- Intestinal drug transport and prediction of drug absorption
- The role of drug transporting proteins
- Drug optimization
The research of each area is described below.
Understanding drug solubility and dissolution
Our research in the field of poorly soluble compounds was recently extended to studies of the impact of the solid state on solubility in biorelevant dissolution media (BDM) of drug like molecules. We have shown that the solid state can be manipulated through chemical approaches which result in improved dissolution and solubility. In a recent project, we demonstrated that by adding bulky side chains to a series of ciprofloxacin analogues, the crystal lattice was disrupted (as measured by decrease in melting point) and the dissolution rate and apparent solubility in BDM were increased (as measured by the solubilization ratio). Another strategy to manipulate the solid state of poorly soluble compounds showing solid-state limited solubility is to produce its corresponding glassy material. In a collaborative project between the Drug Delivery and Pharmaceutics research groups, the molecular properties of importance for glass-forming ability were identified. With the aim to better understand glass forming ability and stability, a Molecular Dynamics simulation project was initiated in collaboration with the Department of Cell and Molecular Biology, Uppsala University.
Fagerberg, JH, Tsinman, O, Sun, N, Tsinman, K, Avdeef, A and Bergström, CAS. Dissolution Rate and Apparent Solubility of Poorly Soluble Compounds in Biorelevant Dissolution Media, Mol Pharm, 7 (5), 1419-1430, 2010.
Zaki, NM., Artursson, P. and Bergstrom CAS. A modified physiological BCS for prediction of intestinal absorption in drug discovery. Mol Pharm 7 (5), 1478–1487, 2010.
Understanding intestinal drug transport and predicting drug absorption
The drug delivery group has a high international standing for its pioneering development of cell culture and computational models and techniques for studies of intestinal drug transport and predicting oral drug absorption, including the Caco-2 model. All these techniques and models are available in our laboratory, where they are applied in various collaborative projects with drug discovery scientists. Recent research in the drug absorption field include basic studies into the role of the paracellular transport pathway and the development of a computation server that predicts drug-likeness, solubility, permeability and maximal absorbable dose for compound libraries, usually selections of pharmacological screening hits obtained from collaborators. The implemented models are based on our own extensive database of high quality experimental measurements and have been used to prioritize compounds from various hit selections for further optimization. In other investigations we are establishing structure-property relationships for diverse compound series in collaboration with the Department of Medicinal Chemistry, Uppsala University, whereby we identify structural motifs that promote high permeability or limit P-glycoprotein-mediated efflux of e.g. peptide mimetics and the influence of series of bioisosteres on protein binding and drug metabolism.
Linnankoski, J., Mäkelä, J., Palmgren, J., Mauriala, T., Vedin, C., Ungell, A-.E., Ranta, V-.P., Lazorova, L., Artursson, P., Urtti, A. and Yliperttula, M. Paracellular porosity and pore size of the paracellular space of human intestinal epithelium and selected cell models. J Pharm Sci, 99(4), 2166-2175, 2010.
Lazorova, L, Hubatsch, I , Ekegren, JK, Gising, J, Nakai, D, Zaki, NM, Bergström, CAS, Norinder, U, Larhed, M, Artursson, P. Structural features determining the intestinal epithelial permeability and efflux of novel HIV-1 protease inhibitors, J. Pharm. Sci. 2011, [Epub ahead of print].
Understanding the role of drug transporting proteins
Current successful research strategies in this research field include comprehensive mapping and proteomics-informed mechanistic modeling of drug interactions with uptake and efflux transporters in pharmaco-kinetically important human organs such as the intestine, liver and kidney. In this project, we have discovered dozens of new drug interactions with drug uptake and efflux transporters in hepatocytes, see Fig.2. The potential clinical importance of these drug interactions is now investigated, especially with regard to the effects of genetic variation and the risk of drug-induced liver toxicity.
Proteomics-informed modeling of hepatic drug kinetics and interactions
Fig.2. In the liver, active transport of drugs into and out of the hepatocytes affects the intracellular concentration of drug and thus also the amount of drug available for metabolic and/or biliary elimination. The protein expression levels of various transporters in the liver affect the capacity for drug transport and thus the transport kinetics of the drug. In 2010, we determined the global protein expression of membrane proteins in the human liver. By relating transporter expression in the human liver to the expression levels and function in our in vitro expression systems, we are able to estimate the maximal function of the transport protein in the human liver.
By cloning and stable expression of the ten most important drug transporting proteins in the human hepatocyte, maps of the interplay between various drug uptake and efflux proteins in the liver cells will be obtained. This already allowed the identification of new specific and general inhibitors of three common ABC-transporters involved in drug resistance e.g. against antiviral and anticancer drugs, see Fig.3. A similar effort with regard to drug uptake transporters in the human liver is under completion. In addition, common genetic variants of transport proteins with altered (reduced) function were recently expressed and studied.
We also recently developed new cell culture models which co-express drug transport proteins and common drug metabolizing enzymes, such as CYP3A4, in collaboration with the Department of Physiology and Pharmacology, Karolinska Institutet. These models will be used to quantify the interplay between drug transport and metabolism in activating and eliminating drugs and toxins in the human liver. Our experimental data on protein expression (in collaboration with the Department of Proteomics and Signal Transduction MaxPlanck Institute, Martinsried) and drug transport are used for mechanistic modeling of hepatocellular drug kinetics and the results based on in vitro experiments are in excellent agreement with published in vivo data. A major advantage of the mechanistic modeling approach is that the models provide parameters that are inaccessible in clinical settings, such as free intracellular drug concentrations in the liver. Similar models which are currently under development aim at quantifying the dose-dependent contribution of active and passive transport in organs such as the intestine and liver.
Drug inhibition patterns: Affinity overlap of ABC transporters
Fig.3.The three major ABC-transporters Pgp, MRP2 and BCRP have broad substrate
specificities and mediate drug resistance against e.g. antiviral and anticancer drugs. In this study, we explored the chemical space of registered oral drugs to obtain the inhibition patterns of these three ABC transporters. In total, 66 (54%) of the investigated compounds inhibited one or more of the three ABC-transporters. The numbers of specific inhibitors were 11, 8 and 4 for P-gp, BCRP and MRP2, respectively. A large overlap was observed between P-gp and BCRP, with 40 inhibitors in common. Computational models that predicted specific and general inhibitors were constructed.
Matsson, P., Pedersen, J., Norinder, U., Bergström, C.A.S and Artursson, P. Identification of novel specific and general inhibitors of the three major human ATP-binding cassette transporters P-gp, BCRP and MRP2 among registered drugs. Pharm Res 26, 1861-1831, 2009.
Sugano, K. , Kansy, M., Artursson, P., Avdeef, A., Bendels, S., Di, L., Ecker, G.F., Faller, B., Fischer, H., Gerebtzoff, G., Lennernaes, H., Senner, F. Coexistence of passive and active carrier-mediated uptake processes in drug transport. Nat Rev Drug Discov. 8, 597-614, 2010.
Ahlin, G., Chen, Y., Ianculescu, AG, Giacomini, KM, Artursson, P. Genotype dependent effects of inhibitorsof the organic cation transporter, OCT1: Predictions of metformin interactions. Pharmacogenomics J (Epub ahead of print, 2010)
Karlgren, M., Ahlin, G., Bergström, CAS., Karlsson, J., and Artursson, P. A combined in silico and in vitro strategy for identifying and predicting drug interactions at the human liver specific organic anion transporter OATP1B1 (SLCO1B1). Pharm Res (submitted, 2011).
Understanding drug optimization
In 2009, the Drug Delivery Group founded Uppsala Drug Optimization and Pharmaceutical Profiling Platform (UDOPP) with support from Uppsala University Innovation. The mission of UDOPP is to provide top class collaborative research in the fields of drug discovery and chemical biology and a prerequisite for this is an appropriate characterization and understanding of the physicochemical and ADMET properties of identified probes. In 2010, UDOPP became a part of a new research infrastructure in Sweden, the Chemical Biology Consortium Sweden (CBCS). Since its inauguration, UDOPP has harbored eight academic collaborations as well as numerous collaborations with small and big pharma. Through UDOPP, the Drug Delivery group has been able to widen its competence field with experts in bioanalytical chemistry, drug metabolism and assay development as well as acquired additional equipment, e.g. sensitive high throughput analytics such as a second HPLC-MS-MS equipment. Importantly, through UDOPP, the group now has access to all standard ADMET assays, such as metabolic stability, CYP induction, plasma protein binding etc. Further, through collaboration with clinicians at the Department of Surgery (Akademiska sjukhuset), we isolate human hepatocytes on a weekly basis.
There are as yet no submitted manuscripts from our collaborations within UDOPP.