Browsing by discipline "Biofarmaci"

Liposomes are nanosized drug delivery vesicles composed of phospholipid membranes. They present an attractive drug delivery system due to their bioavailability and flexibility. Liposomes can be prepared by different techniques. They can carry both hydrophobic and hydrophilic molecules and their surface can be modified with targeting molecules. Coating the liposome surface with the PEG derivative makes their pharmacokinetics easier to predict. There are several liposome-based medicinal products already on the market. Triggering of drug delivery systems by different external or internal stimuli allows precise control of drug release. Light-triggered drug release is an attractive alternative due to the easy control and regulation of the stimulus. The problem with light-triggered therapy has previously been the need to use high-energy ultraviolet light that penetrates badly to the tissues and is not safe. In TTA-UC process the low-energy red or green light is converted to high-energy blue light. In this process photosensitive molecules are excited by visible light and after that the energy is transferred from sensitizer to annihilator molecules. Collision of two annihilators leads to the excitation of the other molecule while the other returns back to its general energy state. The excitation breaks up with fluorescence. In this process the highly permeable and safe red light is converted to blue light which has enough energy to induce drug release. The aim of this work was to optimize liposomal preparation method and prepare a pegylated and stabile liposome formulation for TTA-UC process. Hydrophobic light sensitive molecules were loaded into the phospholipid membrane as much as possible. One of the problems in this work was to find proper methods to measure the concentrations of these molecules. The lipid composition for formulation was chosen after thermostability studies. As a quality control, the size, capability to load calcein and phase transition temperature of liposomes were measured. The quality control of light sensitive molecules was operated too. In this work, the formulation for TTA-UC was prepared. In further studies TTA-UC process happened with sufficient efficacy. The formulation was pegylated and stable in physiological conditions and the concentrations of the molecules were high enough. This was the very first time to get TTA-UC to happen in this kind of liposome formulation that may be useful as a drug carrier. Long-term stability studies and further optimization of TTA-UC method are needed in the future. Some drug release studies are important to arrange in the future, too.

Elucidation of transporter- and/or metabolic enzyme-mediated drug interactions is important part of early drug development. However the knowledge about clinical consequences of transporter-mediated drug-drug interactions is still limited and more investigation is needed to improve our understanding. MDR1 transporter, widely distributed on the pharmacokinetic barriers in the body (e.g. intestine) and has been shown no limit the bioavailability of drugs. Substrates of MDR1 are exposed to limited intestinal drug absorption and intestinal drug-drug interactions due to inhibition of the transporter. In predicting the clinical significance of an interaction, the principal obstacle has been the limited ability to appropriately scale the preclinical data into in vivo situation. In vitro-in vivo correlations on the extent of MDR1's influence on absorption and standardized predicting methods for drug-drug interactions using the inhibitory constants (IC50 and Ki) would greatly increase the value of in vitro studies. Current in vitro and in silico methods for prediction of the influence of MDR1 on intestinal absorption and related drug-drug interactions are discussed in the literature review. In addition, the latest regulatory draft guidances (FDA, EMA) are reviewed. Aliskiren has been shown to be a sensitive MDR1 substrate in vivo and high affinity substrate for the transporter in vitro. The objective of the experimental work was to study the MDR1-mediated transport of aliskiren and the related drug-drug interactions in vitro and in silico. Vesicular transport assay was used to obtain kinetic parameters for aliskiren (Km and Vmax) and inhibitor potencies (IC50) for ketoconazole, verapamil, itraconazole and its metabolite hydroxyitraconazole. Ki was further calculated for itraconazole and hydroxyitraconazole. Aliskiren showed high affinity to MDR1 transporter with a Km value 5 µM, consistent to what was reported previously in different assay systems. The interactions between aliskiren and the inhibitors in vitro correlated to the observed interactions in vivo in humans. In addition, hydroxyitraconazole was shown to be a potent inhibitor of MDR1-mediated transport of aliskiren in vitro. This suggests that hydroxyitraconazole may contribute to the pronounced interaction observed between aliskiren and itraconazole in a clinical interaction study. A compartmental absorption and transit (CAT) model with added enterocyte compartments and MDR1 efflux was used to describe the influence of MDR1 on intestinal absorption of aliskiren in humans. The integration of kinetic parameters (Km) from in vitro studies requires further optimization on how to describe the intracellular drug concentrations in the model. Aliskiren is however suitable MDR1 probe substrate to be used in in vitro and in vivo trials in humans and therefore gives a good basis for developing vitro-in vivo predictive models.

Background: Antibiotics have been an important factor in the dramatic decrease of infectious disease mortality in the 20th century. Bacteria are, however, very quick to respond to the changes in their environment because of their short life cycle. Thus, the development of bacterial antibiotic resistance is a natural consequence of the enormous worldwide antibiotic use. The current situation is that the antibiotic resistance develops faster than novel antibiotics are found and developed. The three main resistance strategies of Gram-negative bacteria are: modification of the antibiotic target, enzymatic inactivation of the antibiotic and reduce of the intracellular antibiotic concentration by changing the function of the outer membrane. To decrease the intracellular antibiotic concentration bacteria use efflux pumps. RND efflux pumps are the most important family of efflux pumps regarding antibiotic resistance. They typically function as a part of a tripartite structure which allows the efflux of antibiotics to the extracellular space. Multiple inhibitors have been developed against RND efflux pumps but none has reached the clinical stage of drug development. Objectives: Development and testing of a 384-well plate method for screening efflux pump inhibitors for E. coli (BAA1161) efflux pumps. Methods: Verifying that the absorbance measurement is a sensitive enough method for measuring the bacterial (BAA1161) growth in 384-well plate format. The antibiotic chosen to be used in the screening method was piperacillin and the positive control efflux pump inhibitor was mefloquine. Determining the minimum growth inhibiting concentrations (MICs) of piperacillin and mefloquine in 96- and 384-well plate formats. Verification of the synergistic growth inhibitory effect of piperacillin and mefloquine with the checkerboard method in 96- and 384-well plate formats. Determining the positional effect in the 384-well plate. Determining the highest DMSO concentration without effect on the growth of BAA1161. Screening of 126 natural compounds in 384-well plates to test the developed method. Screening was done in quadruplicates based on the growth inhibitory effect of the natural compounds when combined with piperacillin. Dose-response assay was conducted in combination with and without piperacillin with the compounds that showed growth inhibiting effect during screening. Results and discussion: Absorbance measurement was sensitive enough method for measuring the BAA1161 growth in the 384-well plate. MIC value of mefloquine was 32 μg/ml in both plate formats. Piperacillin’s MIC was 1024 μg/ml in the 96-well plate, but on the 384-well plate there was variation in the MIC. Piperacillin and mefloquine showed synergistic effect on BAA1161 growth inhibition in the checkerboard assays. Positional effect could not be determined, because of the variation in the BAA1161 growth inhibition effect of piperacillin. This randomly occurring phenomenon were piperacillin inhibited BAA1161 growth completely or almost completely with sub-MIC concentration was encountered in all the subsequent experiments in the 384-well plate format. One possible reason for this phenomenon, occuring in the 384-well plate format, could be piperacillin heteroresistance of BAA1161 strain. In the test screen, four compounds, which all included gallic acid ester, showed promising activity. These compounds were: epigallocatechin gallate, hamamelitannin, isopropyl gallate and octyl gallate. In the dose-response assay, hamamelitannin’s and octyl gallate’s effect was synergistic with piperacillin. Conclusions: The developed method can be used to screen novel efflux pump inhibitors. However, to increase the reliability of the method, further optimization is required to eliminate the variability in the effect of piperacillin. When plate format of a method is changed, factors which could affect the functionality of the method in the new format should be carefully assessed. Based on the test screed, gallic acid esters are interesting compounds which combined effects with antibiotics should be studied in the future experiments.

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.

Nanolääkkeiden pinnalle elimistössä muodostuva biomolekyylikerros eli proteiinikorona vaikuttaa muun muassa jakautumiseen, toksisuuteen ja soluvuorovaikutuksiin. Koronan ominaisuuksien tuntemus jakautumisen eri vaiheissa on siten edellytys tehokkaampien ja turvallisempien nanolääkkeiden kehittämiselle, mutta kehitystyötä on hidastanut soveltuvien menetelmien puute. Turvallisuuden ja tehon ennakoinnin osalta on korostettu leimavapaiden in vitro -menetelmien tarvetta. Tutkielmassa kehitettiin multiparametriseen pintaplasmoniresonanssilaitteistoon ja laskennalliseen mallinnukseen perustuva menetelmä liposomien koronan tiheyden ja paksuuden määrittämiseen. Toisin kuin koronan tutkimiseen yleisesti käytetyt menetelmät, valoon perustuva kajoamaton ja leimavapaa menetelmä ei vaikuta koronan rakenteeseen. Näin voidaan tutkia myös löyhemmin sitoutuneista proteiineista muodostunutta pintakerrosta, mikä vastaa keskeisimpään kirjallisuuskatsauksessa todettuun menetelmäpuutteeseen. Menetelmää sovellettiin neljän biosensorille immobilisoidun liposomiformulaation pinnalle ihmisen seerumissa muodostuvan koronan tutkimiseen. Sen avulla oli mahdollista määrittää ensimmäistä kertaa tiiviin ja löyhän koronan tiheys ja paksuus laimentamattomassa seerumissa. Tulokset tukevat käsitystä ns. erotteluhypoteesin kuvaamasta erillisestä löyhästä proteiinikerroksesta ja avaavat uusia mahdollisuuksia sen biologisen merkityksen arviointiin. Lisäksi voitiin määrittää ensi kerran opsoniinimolekyylien sitoutumiskinetiikka liposomien pinnalle, minkä avulla voidaan arvioida nanolääkkeiden taipumusta poistua verenkierrosta ja aktivoida sisäsyntyinen immuunipuolustus. Menetelmä soveltuu siten liposomien koostumuksen ja pinta-arkkitehtuurin optimointiin prekliinisessä lääkekehitysvaiheessa.

Gene therapy is the therapeutic delivery of nucleic acid sequences into cells, where they can replace a gene that is missing, mutated or poorly expressed. It is a potential treatment to cure e.g. genetic diseases, viral infections and various cancers. The nucleic acid needs to be delivered across the cell membrane and into the nucleus to affect the gene expression. Anionic nucleic acids need a cationic carrier, such as a cationic liposome, to enable their delivery into the cells. The liposomes used in gene delivery usually contain both a cationic lipid to associate with the nucleic acid and a neutral helper lipid to stabilize the structure. The liposome-nucleic acid complex is called a lipoplex. The cationic carrier must include or function as a cell-penetrating enhancer (CPE) to be able to translocate across the cell membrane into the cytosol and to the nucleus. The experimental part of this work was aimed at developing and characterizing an innovative poly-cationic liposomal platform for gene delivery, using a novel synthetic CPE. The CPE used in this study is an oligo-guanidyl derivative (OGD) that had either 4 (OGD4) or 6 (OGD6) cationic charges. Liposomes were surface-engineered with OGD, obtaining a cationic formulation that was then exploited for DNA loading. The study has two main characterization steps: Step 1 was to decorate liposomes with OGD by post insertion using increasing amounts of OGD, and determine the vesicle size and zeta potential by dynamic light scattering (DLS). Step 2 involved DNA loading by post insertion into the cationic liposomes with increasing amounts of DNA. The lipoplex size and zeta potential was determined by DLS, the complexation by electrophoresis, and the thermodynamics of the cationic liposome/DNA association by isothermal titration calorimetry (ITC). The measurements were performed in isotonic buffers (HEPES pH 7.4 and citrate pH 5) and in lower ionic strength TRIS buffer (pH 7.4). The aim of the characterization studies was first to find a liposome composition that includes just enough OGD to obtain a sufficiently high zeta potential and a uniform, sufficiently small size. The optimal formulation contained either 10 % of OGD4 or 5 % of OGD6 of the total lipid amount. The second step was to find the highest stable DNA loading for the lipoplexes. All the characterization studies were performed on OGD4 lipoplexes in TRIS buffer. The optimal OGD4/DNA N/P (nitrogenous/phosphorous) ratio was found to be around 5. Further investigation is needed to determine the best lipoplex composition and manufacturing method using an isotonic buffer. A DNA release study remains to be performed prior to further in vitro and in vivo studies.

Cancer immunotherapies aim to target the immune defence mechanisms of the body specifically and efficiently against the tumour tissue. Cancer vaccines and oncolytic viruses are forms of active immunotherapies, which require patients having a properly functioning immune system. The vaccines are based on the administration of tumour antigens into the body to which the immune system reacts. However, often the response is not robust enough. The oncolytic viruses in turn kill the cancer cells which causes the release of antigens from the tumour tissue. Viruses usually elicit a strong immune response but sometimes it is targeted too much against the virus instead of the tumour. Oncolytic vaccine is a composition of an oncolytic virus and a cancer vaccine. Tumour antigens can be coded to the genome of the virus therefore, when the virus invades tumour cells they start to produce the antigens. Eventually the cancer cells are also destroyed due to viral replication. The antigens can be tumour-associated that is, they are expressed in healthy tissues too. Their usage is not always efficient which is why an interest towards utilizing tumour-specific antigens has been increased. Considering the expression of antigens, tumour tissue is very heterogenous and distinctive between patients. Hence, utilizing mutated patient unique neoantigens would enable the development of personalized tumour-specific oncolytic vaccines. Genetic modification of viruses is complicated thus, an easier way to insert the neoantigens to the virus has been invented. The developed oncolytic vaccine platform is called PeptiENV, and it is designed to use with enveloped viruses. The idea is to fuse tumour-specific antigens onto the envelope of the virus and eliminate the need of gene insertion. The aim of this study is to investigate in vivo the efficacy of PeptiENV in preventing tumour growth and eliciting a tumour-specific immune response. An object is also to observe survival times of the treated animals. Furthermore, the preservation of infectivity is studied in vitro. The research was executed with two potential oncolytic viruses, vaccinia virus (VACV) and herpes simplex virus type 1 (HSV-1). The PeptiENV complex was formed by using an artificial tumour antigen, ovalbumin epitope SIINFEKL, which was attached to the viral envelope with cell penetrating peptide (CPP) or cholesterol anchor. The preservation of infectivity was examined by measuring cell viability of PeptiENV infected cells. Animal experiments instead were performed with a mouse melanoma model created with B16-OVA cells, which express ovalbumin and therefore the antigen epitope SIINFEKL. PeptiENV was compared to control treatments which were virus, SIINFEKL peptide and complexation medium only. Treatments were administered as intratumoural injections. Tumour growth was followed by measuring the size of implanted tumours every other day. With flow cytometry, tumour-specific immune response was assessed by acquiring the relative amount of SIINFEKL-specific CD8+ T cells in the tumour tissue. Euthanizing dates were registered in order to observe the survival of the mice. According to the in vitro results, conjugation of peptides to the virus does not affect infectivity. In addition, the in vivo studies show that PeptiENV VACV CPP prevents tumour growth the most. Difference in tumour growth between PeptiENV VACV CPP and control treatments is significant. Mice injected with the same treatment also lived considerably longer than mice injected with virus, peptide or medium only. Also, PeptiENV HSV-1 hinders tumour growth distinctly more than virus only and slightly more than SIINFEKL only, but unfortunately it did not have an evident impact on the survival time. In both experiments, the PeptiENV treatment elicits the largest proportional amount of SIINFEKL-specific CD8+ T cells. In other words, PeptiENV engenders a tumour-specific immune response. In the PeptiENV VACV study the difference to control treatments is clearer than in the PeptiENV HSV-1 study. At present, the PeptiENV platforms performs better with VACV than HSV-1. With further investigations however, the results can be verified and improved. All in all, the results are encouraging. The PeptiENV platform shows great promise for being a part of personalized cancer immunotherapy developments in the future.

The usage of polymer conjugation to modulate the biopharmaceutical behavior of both protein drugs as well as small molecule drugs is discussed. Emphasis has been given to polyethylene glycol (PEG) and poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) but also other polymers are looked into. Drug products on the market as well as drug candidates in clinical trials are used as examples when reviewing different polymers. The material is looked upon from a biopharmaceutical point of view. In the experimental part a polymer-drug conjugate for the treatment of ovarian cancer is synthesized and characterized. The conjugate has a HPMA polymer backbone with the anticancer drug gemcitabine attached through enzymatically labile Gly-Phe-Leu-Gly linkers. The conjugate is expected to target passively and actively to cancer tissue. The enhanced permeation and retention effect is responsible for the passive targeting, while Fab' fragments of OV-TL16 monoclonal antibodies provide the active targeting of the copolymer conjugate. In vitro cytotoxicity studies of a PHPMA-gemcitabine conjugate (without active targeting) was carried out on two ovarian cancer cell lines, A2780S and A2780AD. The IC50 values of the conjugate was shown to be 50.6 nM and 14.3 nM for A2780S and A2780AD, respectively. The corresponding IC50 values for free gemcitabine were 7.0 nM for the A2780S cell line and 3.9 nM for A2780AD cells. A preliminary in vivo efficacy study in mice with subcutaneous A2780AD tumor xenografts showed that a PHMA-gemcitabine conjugate given at a dose of 15 mg/kg (gemcitabine equivalence) was able to shrink the tumor volume by 50 % while only inducing minor body weight loss.

Reverse-phase protein microarray, RPMA, is a novel and promising technology for proteomic profiling. The low sample consumption, high-throughput format, high sensitivity and good precision make RPMA attractive tool for clinical use. In RPMA, cellular lysates obtained from various sources (e.g. clinical samples, cell lines) are arrayed onto a substratum as a small spots such that an array is comprised of hundreds to thousands of different samples. The array is incubated with a capture molecule (e.g. antibody) that is validated to recognize the analyte of interest. Signal is created by labelled secondary antibody and the signal is detected by colorimetry, chemiluminescence or fluorescence methods. The literature part introduces the RPMA technology and its applications. RPMA have been utilized in versatile applications for example in cell signal pathway profiling, drug discovery and discovery and validation of biomarkers. In the future, it is hoped to allow individual therapy regimes and the evaluation of treatment efficacy. The aim of the experimental part was to culture various cell lines and prepare lysates for RPMA. The lysates were prepared of ARPE-19, HepG2, Hepa-RG, SKOV-3-ip1, SKOV-3, Caco-2, hCMEC/D3, HCE and D-407 cell cultures. The lysates were stored in -80 °C for subsequent use in RPMAs. The purpose was to optimize the method and based on the optimization studies, to print one RPMA. Cell lysates were arrayed onto nitrocellulose coated glass slide using Nano-Plotter (Gesim)-device which allows automated sample printing. β-actin and α-tubulin proteins were assessed from the samples. To create the signal, fluorescence dye was used, and detected at the visible wavelengths. Based on this study, more optimization is required. The detection method used in the RPMA was not optimal, but the experiment showed promising potential. By taking into account the development issues, the method performance can be significantly improved. Of these issues, perhaps the most important is to use infrared region for the signal detection instead of visible wavelengths.

Cellulose has already been used as an industrial raw material for over a century. However, during recent years the nanostructural features of the naturally occurring biopolymer have been fully investigated and characterized through different processing methods as nanofibrillar cellulose (NFC). This has led to a rapid development of novel cellulose based nanoscale materials and advancements in the field of composite materials. NFC offers interesting specific properties that differ from many other natural and synthetic polymers, such as self-renewable raw materials, semi-crystalline morphology, broad chemical modification capacity, biocompatibility and biodegradability. Biocompatibility and the biomimetic aspects of NFC have enabled the fabrication of nanoporous membranes and scaffolds that can function as medical devices (e.g. tissue engineering, wound healing, novel active implants). In this study, the properties of plant-derived NFC, as potential injectable drug releasing hydrogel "implants" were investigated. Three different sized candidate molecules were selected (123I-NaI, 123I-β-CIT and 99mTc-HSA, from small to large respectively) and investigated with the use of a small animal SPECT/CT molecular imaging device. Study compounds were mixed with the NFC biomaterial and injected into the pelvic region of mice. Drug release was observed for a period of 24 hours and the results were compared to saline/study compound control injections. In addition, 99mTc labeled NFC hydrogels were prepared for dual label tracing to observe the hydrogel positioning during the SPECT/CT acquisitions. For the smaller compounds (123I-NaI, 123I-β-CIT), no differences were found in the drug release or absorption in between the NFC biomaterial and saline injections. However, a clear difference was found with the large compound (99mTc-HSA). In the NFC hydrogel, the rate of release was slower and the distribution of 99mTc-HSA was more concentrated around the area of injection. In addition, the NFC hydrogel did not migrate from, or disintegrate, at the site of injection, suggesting a robust enough structural integrity to withstand normal movement and activity. In conclusion, the labeling of NFC was found to be a reliable and simple method. NFC hydrogels have the potential use as drug releasing medical devices with larger compounds. NFC matrix did not have any controlled release effect on the studied small molecules. Therefore further studies are required for more specific conclusions.