Browsing by discipline "Biopharmacy"

Intracellular drug sequestration is useful to understand when designing new drugs with intracellular targets. The knowledge of the intracellular distribution can also help to understand the side effects and pharmacokinetics of a drug, as well as the lack of response in e.g. some multidrug resistant cancer cells. Intracellular concentrations are also important to know when predicting the role of active transport in the overall transport process when binding site of the transporter is intracellular. The literature review describes the mechanisms causing intracellular drug sequestration along with the consequences of intracellular drug sequestration and methods that are used to study it. Alterations of intracellular distribution of anticancer drugs in multidrug resistant cancer cells are also described as an example of the many factors affecting the distribution pattern of the drugs inside cells. Understanding these mechanisms is valuable when designing strategies to overcome the multidrug resistance. The most commonly applied methods for studying intracellular concentrations of drugs are based on fluorescence microscopy. In experimental work, subcellular fractionation protocol is introduced and applied to determine the concentration of CDCF, clotrimazole and celiprolol in vitro in the plasma membrane and cytoplasm of MDCKII cells. CDFC and celiprolol are substrates of the MDR1 transporter and clotrimazole is an inhibitor. Concentrations in the fractions were measured in wild type cells and in MDR1-transfected cells with and without MDR1 inhibitor verapamil to see if the transporter had an effect on the concentrations. Also the effect of lipophilicity of the drug on partition between plasma membrane and cytoplasm was reviewed. Celiprolol showed a typical behaviour of the MDR1 substrate whereas CDCF and clotrimazole did not. Clotrimazole as a lipophilic compound was accumulated more to the plasma membrane than less lipophilic CDCF and celiprolol. Lipophilicity affected also to the ratio of Km (or Ki)(determined from the concentration in extracellular fluid) and Km (or Ki)(membrane) (determined from the plasma membrane concentration) values, with clotrimazole Ki(membrane) value being larger than respective Ki value, and CDCF and celiprolol Km(membrane) values being smaller than their respective Km values.

Investigating the role of cell membrane proteins has increased over the last decade, as drugdrug interactions and genetic polymorphisms have been found to cause changes in drug pharmacokinetics and dynamics. In this study the characteristics of the OATP1B1 transporter were reviewed and new in vitro research method to study protein functions was developed. Human Embryonic Kidney cells (HEK) is a human derived mammalian cell-line that is widely used in the study of OATP1B1 transporter. The Sf9 cell line is isolated from Spodoptera frugiperda insect and is one of the standard in vitro tools in a genetic engineering study. In the experimental part of this thesis the goal was to express OATP1B1 transporter in Sf9 and HEK293 cell lines. The wild-type SLCO1B1-gene encoding the OATP1B1 was virulent with baculovirus into the cells by the Bac-to-Bac® Baculovirus Expression System. For expression in the Sf9 cells, the aim of the study was to clone the SLCO1B1-gene into the pFastBac vector. The cloning was not successful in this study although attempts were made for several approaches. The expression of OATP1B1 transporter in HEK293 cells was successful. HEK293 cells expressing OATP1B1 transporter are well suited for the study of the SLCO1B1-gene. The in vitro method developed in this study remains in the research team as a tool to investigate the polymorphisms of the SLCO1B1-gene, the inhibition of the transporter and possible drug interactions.

Methods for the assessment of the bioequivalence (BE) of drug products are generally well-documented and the approaches for such studies are described in guidances issued by regulatory authorities throughout the world. While in general, the BE requirements of most regulatory bodies have much in common, in various instances specific issues and approaches may differ. In the literature part of the master's thesis these differences in the selected regulatory BE guidelines (Europe, United States and World Health Organization) was examined and also the scientific reasons behind these differences were considered. It was found that the prime differences were in the BE related issues in which the scientific community are not in agreement (multiple dosing, highly variable drugs, moieties to be measured (parent/metabolite), food effect studies etc.). The differences were also related to drug products that have biopharmaceutical, bioavailability (BA), pharmacokinetic, and pharmacodynamic properties that preclude the use of standard approaches that are outlined in regulatory guidelines. In the future the push for international harmonization of regulatory standards is hopefully leading to worldwide discussions and changes regarding BE and other components of the drug approval process (both new and generic drugs). Expensive in vivo BE studies are usually needed for generic drug products or if a formulation is significantly altered during clinical trials. In this master's thesis a pharmacokinetic model (based on a compartmental absorption and transit model, CAT) was constructed and tested to predict relative BA, to assess the risk of bioinequivalency and to probe properties of drugs suitable for the use of the model. Also the errors and uncertainties related to the model were discussed. GI tract physiology, formulation type and drug solubility, dissolution, absorption and elimination rates were taken into account in this pharmacokinetic simulation model. In the model formulation differences were described by dissolution rate constant (Kd) (calculated from experimental dissolution data) and gastric emptying rate (GE) (varies for different formulations). Hence, when integrated with a pharmacokinetic compartment model it was possible to get predictions of concentration-time profiles of different formulations. Generalised rules in BE assessment were used to estimate the risk of bioinequivalency. The resolution power of the model and the errors related to the model was evaluated by theoretical pharmacokinetic simulations. Generally, the simulations suggested that the model predicts the risk in the BE study most accurately when the drug belongs to the class I/III in the biopharmaceutical classification system (BCS) or to the class II when saturation solubility is not the limiting step in the absorption. Used Kd value is valid if dissolution data is accurate (method discriminative). Also, there has to be enough information about the formulation (type, disintegration, excipients). Otherwise it has to be considered if these factors effect on the resolution power. The weaknesses of the simulation models are assumptions. Hence, when exploring the results it has to be estimated case by case, if they affect on model's ability to separate formulations (reliability of the risk assessment and the ability to predict relative BA). This model is useful tool in formulation development and regulatory applications.

The major problem in cancer treatment is toxic side effects of the chemotherapy. Typically less than 1 % of the administered free drug reaches target cells while the rest damages non-diseased cells. Toxic side effects often limit dose escalation of anticancer drugs which leads to incomplete tumor response, early disease relapse and possible the development of drug resistance. Liposomes can be targeted in cancer tissue with passive or active targeting. In passive targeting the liposomes accumulate in abnormally formed cancer tissue through the process of extravasation and enhance the concentration of liposomal drug in solid tumor. To further improve the anticancer efficiency of passive targeted liposomes is to couple a targeting ligand to the surface of the drug carrier (i.e. active targeting). The ligand specifically binds to a surface epitope on the target cell leading to the accumulation of the liposomal drug inside the tumor cells. The aim of this study was to investigate the cytotoxicity of targeted immunoliposomes. In experimental part the liposomes were constructed using cetuximab (C225, Erbitux®) antibody and evaluated for specific cellular uptake and cytotoxicity in vitro. Cetuximab antibody is specific and selective inhibitor of HER-1 -protein (ErbB-1, EGFR, epidermal growth factor receptor). HER1 -protein is frequently expressed in high levels in human carcinomas (for example in lung and colorectal cancers, head, neck and breast cancers and in pancreatic, ovarian, prostate and bladder carcinomas). Specific immunoliposome uptake and cytotoxicity were studied in SKOV-3cells (ovarian adenocarsinoma cell line) which overexpress the EGF -receptor. Monkey kidney epithelial cells (CV-1) were used as a control cell line which represents non-diseased cells. Active targeting and cellular uptake of liposomes were investigated in cell uptake studies. Non-targeted pegylated liposomes were used as control liposomes. Specific binding of the cetuximab antibody to EGF -receptor was noticed in competition studies. The in vitro cytotoxicity of doxorubicin containing immunoliposomes was studied with Alamar Blue® cell viability assay. Liposome size was determined at intervals of about two weeks during the experimental part. In conclusions, antibody targeted immunoliposomes showed greater cellular uptake and cytotoxicity in EGFRoverexpressing target cells (SKOV-3) than the corresponding non-targeted liposomal drug. Immunoliposomes showed greater cytotoxicity after five days incubation, which can be a consequence of liposome formulation and slow rate of release of doxorubicin. In contrast, antibody targeted liposomes did not show specific cellular uptake or cytotoxicity in CV-1 control cell line. In clinical cancer therapy actively targeted liposomes could improve the therapeutic effectiveness of the liposomal preparations. Many studies have shown that ligand-bearing liposomes will selectively bind to target cells in vitro, but only few studies have shown the possibility in vivo.

Raman spectroscopy is based on vibrations that occur between the atoms of a compound. The overall structural energy is derived from the electronical energy as well as vibrational, rotational and translational energy. In Raman spectroscopy the vibrational and rotational energies are essential. Usually the excitation energy used in Raman spectroscopy can be either in the region of visible light or NIR. The sample absorbs the energy and energy is also scattered back to all possible directions. Elastic scattering is called the Rayleigh scattering. In that case the back-scattered photons have an equal energy as the original excitation energy. However, some of the scattering happens inelastically and it forms the basis of Raman-phenomena. If the detected photons have smaller energy than the original, it is called the Stokes scattering. If the energy is bigger, it is anti-Stokes scattering. Raman is typically very rare and weak phenomenon. The spectral features in Raman spectra consist of the intensities and energies of the back scattered photons. Raman spectroscopy provides very accurate and detailed structural information on the molecule. It is basically a label-free technique with minimal need for sample preparation and the measurements can also be carried out e.g. through container walls. Further, Raman is quite insensitive to hydrous samples and it is suitable to solutions and biological assessments. However, there are some drawbacks that are formed by the luminescence phenomena i.e. fluorescence. Strong fluorescent backgrounds can mask the relevant Raman features in spectra because Raman and fluorescence are competetive processes. For instance many drug molecules have such structures that they cause strong fluorescence. It is also one of the reasons that pharmaceutical applications and measurements have been partly limited due to this problem. There are applications to improve and enhance a Raman signal. For example resonance phenomena and SERS are favored. To solve the fluorescence-related problems there are also means; one can change the laser wavelenght, photobleach the sample or apply different kinds of data manipulation techniques to the spectral data achieved. There are drawbacks with these methods. They can be slow, complex, damage the samples and still insufficient fluorescence suppression is a problem. In this study a novel time-gated CMOS-SPAD detection technique is applied to non-fluorescent and fluorescent drug measurements. The new detection system has a programmable on-chip delay time and it is synchronized with a picosecond pulsed laser. The scattered photons can be measured in the time scale when they are simultaneously measured in traditional energy and intensity wise. Raman scattering occurs in the timescale of sub-picoseconds while the fluorescence phenomena happen typically in the order of nanoseconds. This time difference can be exploited effectively to suppress the fluorescence. In the literature review of this study the basis of vibrational spectroscopy is introduced - especially Raman spectroscopy. The techniques related, as well as the novel time-resolved technique are covered. Further, different kinds of applications in the field of Raman spectroscopy are reviewed, mainly pharmaceutics-related and biologically relevant applications. In the experimental work the focus was to compare a continuous-wave 785 nm laser setup coupled with the CCD-detector to the pulsed picosecond 523 nm laser coupled with the CMOS-SPAD-detector. The measurements were performed on different kinds of drugs, both non-fluorescent and fluorescent. The aim was to obtain information on the effectiveness of CMOS-SPAD-technique on fluorescence suppression for solid drugs and solutions. Secondary goals were to collect knowledge on the similarities and differences between the Raman setups used for solution measurements, to optimize and discuss the key elements of setups for solids and solutions and to show preliminarily the applicability of the CMOS-SPAD-system on fluorescent drug's solutions as well as find out the requirements related to quantitative assessments using Raman spectroscopy. In drug research there is also constant need for reliable in vitro cell assays. The assessments made in this study may prove useful to the future applications e.g. measurements with living cells. An effective fluorescence suppression was achieved to strong fluorescent backgrounds using the novel time-resolved CMOS-SPAD-detection system coupled with the pulsed picosecond 532 nm laser. The setup is potentially a convenient tool to overcome many fluorescence-related limitations of Raman spectroscopy for laboratory and process analytical technology (PAT) use in the pharmaceutical setting. The results achieved encourage to consider that with careful calibration and method validation there is potential for quantitative analysis, biopharmaceutical and biological applications e.g. in vitro cell studies where most Raman techniques suffer from strong fluorescence backgrounds. Other potential fields for future applications can be also considered.

Biopharmaceutical Classification System (BCS) is a scientific framework for classifying drug substances based on their aqueous solubility and intestinal permeability. When combined with dissolution of the drug product, the BCS takes into account three major factors that govern the rate and extent of drug absorption. For a BCS biowaiver, the in vitro dissolution study may be used as a surrogate for in vivo bioequivalence studies. Currently, BCS I drugs are accepted as biowaiver candidates by EMEA, FDA and WHO. EMEA and WHO also accept class III drugs in some conditions. The main difficulty in classifying drugs according to BCS is the determination of permeability. Biopharmaceutics Drug Distribution Classification System (BDDCS) was introduced to provide a surrogate for permeability. If the major route of elimination is metabolism, then the drug exhibites high permeability. There are two parts in this master thesis. BCS and BDDCS are discussed and evaluated in the literature part. The focus is in the BCS III drugs. The purpose of the experimental part is to evaluate BCS III drug, hydrochlorothiazide as a biowaiver candidate. Solubility of the drug substance and dissolution of the drug product was determined. Aim of the permeability studies with Caco-2 cells were to study if hydrochlorothiazide permeates by passive diffusion across the monolayer. Importance of paracellular diffusion was evaluated by opening tight junctions with EDTA. Influence of dissolution rate was evaluated by theoretical simulation. According to the results of this study, hydrochlorothiazide has good solubility in aqueous buffer. It has been reported to diffuse passively across the epithelial cells but in this study permeability increased when concentration decreased. This may be due to active transport. Hydrochlorothiazide diffuses partially through the tight junctions. Dissolution of the hydrochlrothiazide tablet was very rapid. Drug eliminates almost entirely by metabolism, it is also BDDCS class III drug. EMEA and WHO accept BCS III drugs as biowaiver candidate if dissolution rate is very rapid. According to this, hydrochlorothiazide could be suggested as a biowaiver candidate. There are also other issues to be considered, for example excipients used in tablets. Since hydrochlorothiazide has been discovered to be absorbed in the upper part of the small intestine, the influence of excipients is especially important. This possible influence should be evaluated before the final decision of biowaiver.

The liver is the major site of drug metabolism and excretion. Within the liver endogenous and exogenous compounds are eliminated through many metabolizing enzymes. Drug removal is not only dependent on metabolic enzymes, but also on transporters. Before cellular metabolism can occur, a drug must first enter the hepatocyte. Very lipophilic drugs enter the cell membrane through passive diffusion, but polar or ionized organic compounds can enter the cell membrane only by transporters. Transporters in the basolateral membrane of the hepatocyte facilitate drug entry and access to drug metabolizing enzymes. Transporters in the canalicular domain (apical) of the hepatocyte faclitate removal of drugs or metabolites from the cell interior. Recent studies have shown that transporters can mediate drug-drug interactions, and transporter genes are subject to genetic polymorphism which may affect pharmacokinetic parameters of a drug, such as absorption, distribution, and excretion. This Master's thesis consists of two parts, a literature review and an experimental section. In the literature review two transporters, OATP1B1 and MRP2, are discussed in detail. OATP1B1 is expressed on the basolateral and MRP2 on the apical membrane of the hepatocyte. These transporters are responsible for the vectorial transcellular hepatobiliary transport of various organic anions in humans. The experimental section aims at modelling vectorial hepatobiliary transport of three compounds in a double-transfected (OATP1B1/MRP2) MDCKII cell line. All three compounds studied, rosuvastatin, estrone sulphate, and estradiol glucuronide, are substrates of both transporters. Wild type (WT) MDCKII cells were used as a control. Tight junctions form a barrier between cells. This barrier regulates the paracellular passage of, for example, water, ions, large molecules, and drugs. In the experimental section the tight junctions were reversibely opened to distinguish between trans- and paracelluar routs of transport of the three compounds studied. Permeation of rosuvastatin and estradiol glucuronide in the basolateral to apical direction was faster in the double-transfected cell line compared to the MDCKII-WT cell line. Permeation of estrone sulphate, however, behaved unexpectedly in the double-transfected cell line. The permeation of this compound was almost equal in the apical to basolateral and basolateral to apical direction. The reason for this unexpected finding remains unclear. By opening the tight junctions the permeation of all compounds in both cell lines was increased, indicating that the compounds studied preferred the paracellular route and the importance of transporters were reduced. The double-transfected MDCKII cell line is a useful in vitro model of hepatic vectorial transport of organic anions in humans.

Both primary pharmacokinetic (PK) parameters, volume of distribution (Vd) and clearance (CL), undergo considerable developmental changes in infancy and childhood, necessitating compensatory changes in dosing regimens if therapeutic effect without toxicity is to be reached and maintained. Neonates exhibit higher body water content and decreased plasma binding capacity compared to adults, producing increased Vd values for many drugs. Due to immaturity of glomerular function and low metabolic enzyme expression, CL tends to be significantly decreased in neonates. Both Vd and CL undergo simultaneous but independent maturation during development. Performing pediatric clinical trials is challenging due to ethical and practical constraints. Modeling and simulation approaches, such as population pharmacokinetic (POP-PK) and physiologically based pharmacokinetic (PBPK) modeling, are beneficial aids in planning and performing clinical studies in children. The aim of the literary review is to assess the developmental phenomena that cause pediatric pharmacokinetics to differ from adults, the clinical consequences arising from these differences, and present ways to apply POP-PK and PBPK models in pediatric drug research. In the experimental work, two modeling approaches for the prediction of pediatric pharmacokinetics are explored. First, the performance of the commercial PBPK software Simcyp in simulating a drug-drug interaction between cyclosporine A (CsA) and ketoconazole (KTZ) is assessed. Second, a method for in vitro-in vivo extrapolation (IVIVE) of CL in children is developed and evaluated. The aim is to assess the suitability of both modeling methods in pediatric drug research. Simcyp predicted the general age-related trends in the CsA CL and CsA-KTZ interaction well for the most part. However, the values of the simulated CL terms and magnitude of the simulated interaction were significantly under- and overpredicted, respectively. Due to limited clinical data, though, Simcyp performance could not be fully validated. The method developed here for IVIVE of pediatric CL values yielded successful predictions in most cases, with in vitro data from hepatocytes performing slightly better when compared to microsomal data. Success in extrapolations performed for adults correlated well with success in the pediatric extrapolations. Therefore, in drug development, the method developed in this work would be most useful after adult PK data is available, before the first pediatric clinical studies.

Multidrug resistance protein 1 (MDR1, p-glycoprotein) belongs to the ATP-binding cassette transporter family and it's encoded by ABCB1/MDR1 gene. It is a protein which transports many different kinds of compounds out of cells, for example from endocytes to the lumen with the use of energy from ATP. MDR1 is there for a restrictive factor for several orally administered drugs. It`s important to have knowledge about MDR1-inhibitors, in order to avoid harmful drug-drug and food-drug interactions that might affect medical treatment. The purpose of this master's thesis was to optimize an in vitro MDR1-vesicle uptake method and use it to screen inhibitors from compound libraries. To optimize the method, the effect of cholesterol loading on ATP-dependent transport of test substrate N-methylquinidine (NMQ) was evaluated, transport kinetics of the vesicles and kinetics of known inhibitors were also tested. With the optimized method, screening was done with a library of 25 food additives and a library of 42 synthetic compounds. The chemical structures of the synthetic compounds were analyzed manually in order to find factors that could explain their ability to inhibit MDR1. Only one inhibitor was found among food additives: curcumin. Other additives didn't increase or decrease the ATP-dependent transport of NMQ. Several inhibitors were found from the library of synthetic compounds, also a couple of compounds were found to increase the active transport of NMQ. Results indicate, that the additives used in this study have low risk to cause MDR1 mediated interactions, if curcumin is excluded. The inhibitory effect of curcumin should be investigated in in vivo-situation, because vesicle-based in vitro-results have tendency to overestimate results. Screening results of the synthetic compounds gives more confirmation to the usefulness of the screening method. The MDR1-inhibition screening method described in this Master`s thesis is valid, and it can be used to screen different compound libraries for MDR1-inhibitors. In the future it could be used to screen different kinds of compounds, which might end up inside humans and cause interactions with drugs.

Nanofibrillar cellulose (NFC) can form hydrogels with high water content (> 98 %). It has been studied for drug release, and it has been used as a cell culture matrix, due to its similar structure to extracellular matrix (ECM). In addition it has been found that they has no cytotoxicity. Iontophoresis is the application of an electric current over a defined area for the purpose of enhancing permeation across a membrane for ionized drug species. The aim in the experimental work in this Master's thesis is twofold. First, to find out the suitable drug loading concentrations into NFC hydrogels, which can provide a good release profile, a release study with two model drugs, propranolol and ketoprofen, loaded into three types of NFC hydrogels at three different concentrations, was carried out for this purpose. Second, to see if NFC hydrogels are applicable as a drug reservoir in iontophoretic transdermal drug delivery applications, an iontophoresis study was carried out using porcine ear skin model in vitro for human skin with propranolol loaded into NFC hydrogel of type A. In addition, Stella models were used as an aid to find suitable ways to predict the release and permeation behaviour of models drugs in the abovementioned context. The UPLC results from the release study show for both model drugs, the wt. % released had linear correlation with squareroot of time. At 6 hours, more than 70 wt. % propranolol was released from hydrogel reservoir. For ketoprofen, the release varied between 30 - 87 wt. %, where higher initial loading concentrations produced a decrease in the wt. % released from hydrogel. The iontophoresis study did not show a significant difference between the tested current densities (0.50 mA/cm2; 0.25 mA/cm2) produced on the wt. % of drug released. Simulation models could be run with the mathematical equations for diffusion controlled drug release. In conclusion, the NFC hydrogels show potential as drug reservoir for drug release. Additional experimental data using other types of drug reservoirs should be obtained for a better understanding of the suitability of NFC hydrogels as a drug reservoir in iontophoretic transdermal drug delivery.