Case report: Avoiding intolerance to antipsychotics through a personalized treatment approach based on pharmacogenetics.

Introduction: The standard approach to treatment in psychiatry is known as "treatment as usual" (TAU), in which the same types of treatment are administered to a group of patients. TAU often requires numerous dose adjustments and medication changes due to ineffectiveness and/or the occurrence of adverse drug reactions (ADRs). This process is not only time-consuming but also costly. Antipsychotic medications are commonly used to treat various psychiatric disorders such as schizophrenia and mood disorders. Some of the inter-individual differences in efficacy and ADRs observed in psychopharmacotherapy can be explained by genetic variability in the pharmacokinetics and pharmacodynamics of antipsychotics. A better understanding of (in)efficacy and possible ADRs can be achieved by pharmacogenetic analysis of genes involved in the metabolism of antipsychotics. Most psychotropic drugs are metabolized by genetically variable CYP2D6, CYP1A2, CYP3A4, and CYP2C19 enzymes. To demonstrate the utility of pharmacogenetic testing for tailoring antipsychotic treatment, in this paper, we present the case of a patient in whom a pharmacogenetic approach remarkably altered an otherwise intolerant or ineffective conventional TAU with antipsychotics. Methods: In this case report, we present a 60-year-old patient with psychotic symptoms who suffered from severe extrapyramidal symptoms and a malignant neuroleptic syndrome during treatment with risperidone, fluphenazine, aripiprazole, brexpiprazole, and olanzapine. Therefore, we performed a pharmacogenetic analysis by genotyping common functional variants in genes involved in the pharmacokinetic pathways of prescribed antipsychotics, namely, CYP2D6, CYP3A4, CYP3A5, CYP1A2, ABCB1, and ABCG2. Treatment recommendations for drug-gene pairs were made according to available evidence-based pharmacogenetic recommendations from the Dutch Pharmacogenetics Working Group (DPWG) or Clinical Pharmacogenetics Implementation Consortium (CPIC). Results: Pharmacogenetic testing revealed a specific metabolic profile and pharmacokinetic phenotype of the patient, which in retrospect provided possible explanations for the observed ADRs. Based on the pharmacogenetic results, the choice of an effective and safe medication proved to be much easier. The psychotic symptoms disappeared after treatment, while the negative symptoms persisted to a lesser extent. Conclusion: With the case presented, we have shown that taking into account the pharmacogenetic characteristics of the patient can explain the response to antipsychotic treatment and associated side effects. In addition, pharmacogenetic testing enabled an informed choice of the most appropriate drug and optimal dose adjustment. This approach makes it possible to avoid or minimize potentially serious dose-related ADRs and treatment ineffectiveness. However, due to the complexity of psychopathology and the polypharmacy used in this field, it is of great importance to conduct further pharmacokinetic and pharmacogenetic studies to better assess gene-drug and gene-gene-drug interactions.

Telomere length and TERT polymorphisms as biomarkers in asbestos-related diseases.

BACKGROUND: Asbestos exposure has been proposed as a risk factor for shorter telomere length. The aim of our study was to investigate whether telomere length in leukocytes and hTERT genetic polymorphisms may serve as potential biomarkers for the risk of developing asbestos-related diseases and as biomarkers of progression and chemotherapy response rate in malignant mesothelioma (MM). SUBJECTS AND METHODS: We conducted two retrospective studies. In the first study, a case-control study, telomere length and hTERT polymorphisms were determined in patients with MM, subjects with pleural plaques and controls without the asbestos related disease, who were occupationally exposed to asbestos. In the second study, a longitudinal observational study, telomere length was also determined in samples from MM patients before and after chemotherapy. Telomere length was determined by monochromatic multiplex quantitative polymerase chain reaction (PCR), while competitive allele-specific PCR was used to genotype hTERT rs10069690, rs2736100 and rs2736098. Logistic regression and survival analysis were used in statistical analysis. RESULTS: Patients with MM had shorter telomere length than subjects with pleural plaques (p < 0.001). After adjustment for age, rs2736098 CT, and rs10069690 TT and CT+TT genotypes were significantly associated with a higher risk of MM (padj = 0.023; padj = 0.026 and padj = 0.017), while rs2736100 AA and CA+AA genotypes conferred to a lower risk for MM compared to all other subjects (padj = 0.017, and padj = 0.026). Telomere length was not associated with a response to chemotherapy (p > 0.05) or time to disease progression (p > 0.05). Carriers of one or two polymorphic rs10069690 T alleles had a good response to chemotherapy (p = 0.039, and p = 0.048), these associations remained statistically significant after adjustment for age (padj = 0.019; padj = 0.017). Carriers of two polymorphic rs2736100 A alleles had a longer time to disease progression (p = 0.038). CONCLUSIONS: Shorter telomere length and hTERT polymorphisms may serve as a biomarker for the risk of developing MM. Additionally, rs10069690 and rs2736100 polymorphisms, but not telomere length, were associated with a chemotherapy response or MM progression.

Case report: application of pharmacogenetics in the personalized treatment of an elderly patient with a major depressive episode.

Background: Pharmacogenetic analyses can predict interpersonal differences in response to psychopharmacotherapy, which greatly facilitates the selection of the most effective medication at optimal doses. By personalizing therapy in this way, we can minimize adverse drug reactions (ADR) and prevent polypharmacy. Most psychotropic medications are metabolized by the cytochrome P450 enzymes CYP2D6, CYP2C19, and CYPA3A4, which influence drug metabolism and concentration, affecting both efficacy and the occurrence of ADR. The relationships between genetic variations and enzymatic activity allow pharmacogenetic analysis to provide important data for optimal drug selection. The following case report illustrates the impact of pharmacogenetic analysis on the course of pharmacologic treatment in an elderly patient with a major depressive episode. Methods: We present a case of a 79-year-old patient treated for severe depression with psychotic symptoms. We collected data on treatment selection and response to treatment before and after pharmacogenetic analysis. For pharmacogenetic analysis, common functional variants in CYP1A2, CYP3A4, CYP2B6, CYP2C19, and CYP2D6 were genotyped, and corresponding evidence-based treatment recommendations were prepared. Results: The patient suffered from lack of efficacy and serious ADR of several medications, resulting in worsening depression and treatment resistance over the course of several months of treatment. Pharmacogenetic analysis provided important insights into the patient's pharmacokinetic phenotype and allowed us to personalize treatment and achieve remission of the depressive episode. Conclusion: In the case presented, we have shown how consideration of pharmacogenetic characteristics in an individual patient can improve treatment outcome and patient well-being. Knowledge of the patient's pharmacogenetic characteristics helped us to personalize treatment, resulting in complete remission of psychopathology. Due to the complexity of psychiatric disorders, the efficacy of combinations of different medications, which are often required in individual patients, cannot be clearly explained. Therefore, it is of great importance to conduct further pharmacokinetic and pharmacogenetic studies to better assess gene-drug interactions in psychopharmacotherapy.

Inflammation and Oxidative Stress Gene Variability in Retinal Detachment Patients with and without Proliferative Vitreoretinopathy.

This study investigated the association between certain genetic variations and the risk of developing proliferative vitreoretinopathy (PVR) after surgery. The study was conducted on 192 patients with primary rhegmatogenous retinal detachment (RRD) who underwent 3-port pars plana vitrectomy (PPV). The distribution of single nucleotide polymorphisms (SNPs) located in genes involved in inflammation and oxidative stress associated with PVR pathways were analyzed among patients with and without postoperative PVR grade C1 or higher. A total of 7 defined SNPs of 5 genes were selected for genotyping: rs4880 (SOD2); rs1001179 (CAT); rs1050450 (GPX1); rs1143623, rs16944, rs1071676 (IL1B); rs2910164 (MIR146A) using competitive allele-specific polymerase chain reaction. The association of SNPs with PVR risk was evaluated using logistic regression. Furthermore, the possible association of SNPs with postoperative clinical parameters was evaluated using non-parametric tests. The difference between two genotype frequencies between patients with or without PVR grade C1 or higher was found to be statistically significant: SOD2 rs4880 and IL1B rs1071676. Carriers of at least one polymorphic IL1B rs1071676 GG allele appeared to have better postoperative best-corrected visual acuity only in patients without PVR (p = 0.070). Our study suggests that certain genetic variations may play a role in the development of PVR after surgery. These findings may have important implications for identifying patients at higher risk for PVR and developing new treatments.

Atmospheric pressure plasma jet-mouse skin interaction: Mitigation of damages by liquid interface and gas flow control.

The possible benefits of an atmospheric pressure plasma jet skin treatment have been tested in vivo on mouse skin. Many studies have been conducted in vitro on mouse skin cells, but only a few in vivo where, due to the complexity of the biological system, plasma can cause severe damages. For this reason, we investigated how kHz plasma generated in a jet that is known to inflict skin damage interacts with mouse skin and explored how we can reduce the skin damage. First, the focus was on exploring plasma effects on skin damage formation with different plasma gases and jet inclinations. The results pointed to the perpendicular orientation of a He plasma jet as the most promising condition with the least skin damage. Then, the skin damage caused by a He plasma jet was explored, focusing on damage mitigation with different liquid interfaces applied to the treatment site, adding N2 to the gas mixture, or alternating the gas flow dynamics by elongating the jet's glass orifice with a funnel. All these mitigations proved highly efficient, but the utmost benefits for skin damage reduction were connected to skin temperature reduction, the reduction in reactive oxygen species (ROS), and the increase in reactive nitrogen species (RNS).

Electrotransfer parameters as a tool for controlled and targeted gene expression in skin.

Skin is an attractive target for gene electrotransfer. It consists of different cell types that can be transfected, leading to various responses to gene electrotransfer. We demonstrate that these responses could be controlled by selecting the appropriate electrotransfer parameters. Specifically, the application of low or high electric pulses, applied by multi-electrode array, provided the possibility to control the depth of the transfection in the skin, the duration and the level of gene expression, as well as the local or systemic distribution of the transgene. The influence of electric pulse type was first studied using a plasmid encoding a reporter gene (DsRed). Then, plasmids encoding therapeutic genes (IL-12, shRNA against endoglin, shRNA against melanoma cell adhesion molecule) were used, and their effects on wound healing and cutaneous B16F10 melanoma tumors were investigated. The high-voltage pulses resulted in gene expression that was restricted to superficial skin layers and induced a local response. In contrast, the low-voltage electric pulses promoted transfection into the deeper skin layers, resulting in prolonged gene expression and higher transgene production, possibly with systemic distribution. Therefore, in the translation into the clinics, it will be of the utmost importance to adjust the electrotransfer parameters for different therapeutic approaches and specific mode of action of the therapeutic gene.

Endoglin silencing has significant antitumor effect on murine mammary adenocarcinoma mediated by vascular targeted effect.

New targets and therapeutic approaches for vascular targeted strategies in oncology are continuously explored. Endoglin, a co-receptor of TGF-ß, is a known target, however, its silencing with vector-based RNA interference technology has not been evaluated yet. Therefore, in our study, we assembled plasmid DNA coding for shRNA against endoglin, and used gene electrotransfer as a delivery method to determine its antitumor and vascular targeted effects. In vitro and in vivo data provide evidence of vascular targeted effects of endoglin silencing. The vascular targeted action of endoglin silencing could be described as a result of two separated effect; antiangiogenic and vascular disrupting effect. This was first supported by in vitro data; predominantly by reduction of proliferation and tube formation of endothelial cells. In the TS/A murine mammary carcinoma model, in which the tumor cells do not express endoglin, reduced tumor growth and number of vessels were observed. Quick destruction of existing activated blood vessels at the site of tumor cells' injection and sustained growth of tumors afterwards was observed in tumors that were growing in dorsal window chamber by intravital microscopy. This observation supports both vascular disrupting and antiangiogenic action. In conclusion, the results of our study provide evidence of endoglin as a valid target for cancer therapy and support further development of plasmid shRNA delivery, which have prolonged antitumor effect, especially in combined schedules.

In situ monitoring of electric field distribution in mouse tumor during electroporation.

PURPOSE: To investigate the feasibility of magnetic resonance (MR) electric impedance tomography ( EIT electric impedance tomography ) technique for in situ monitoring of electric field distribution during in vivo electroporation of mouse tumors to predict reversibly electroporated tumor areas. MATERIALS AND METHODS: All experiments received institutional animal care and use committee approval. Group 1 consisted of eight tumors that were used for determination of predicted area of reversibly electroporated tumor cells with MR EIT electric impedance tomography by using a 2.35-T MR imager. In addition, T1-weighted images of tumors were acquired to determine entrapment of contrast agent within the reversibly electroporated area. A correlation between predicted reversible electroporated tumor areas as determined with MR EIT electric impedance tomography and areas of entrapped MR contrast agent was evaluated to verify the accuracy of the prediction. Group 2 consisted of seven tumors that were used for validation of radiologic imaging with histopathologic staining. Histologic analysis results were then compared with predicted reversible electroporated tumor areas from group 1. Results were analyzed with Pearson correlation analysis and one-way analysis of variance. RESULTS: Mean coverage ± standard deviation of tumors with electric field that leads to reversible electroporation of tumor cells obtained with MR EIT electric impedance tomography (38% ± 9) and mean fraction of tumors with entrapped MR contrast agent (41% ± 13) were correlated (Pearson analysis, r = 0.956, P = .005) and were not statistically different (analysis of variance, P = .11) from mean fraction of tumors from group 2 with entrapped fluorescent dye (39% ± 12). CONCLUSION: MR EIT electric impedance tomography can be used for determining electric field distribution in situ during electroporation of tissue. Implementation of MR EIT electric impedance tomography in electroporation-based applications, such as electrochemotherapy and irreversible electroporation tissue ablation, would enable corrective interventions before the end of the procedure and would additionally improve the treatment outcome.

A cell-based biosensor system HepG2CDKN1A-DsRed for rapid and simple detection of genotoxic agents.

The regulatory requirements for genotoxicity testing rely on a battery of genotoxicity tests, which generally consist of bacterial and mammalian cell assays for detection of gene mutations and chromosomal aberrations. However, for rapid screening, these methods are not appropriate. We have developed a new cell-based biosensor system that provides rapid and simple detection of genotoxic substances. This is based on stable transfection of human hepatoma HepG2 cells with a plasmid that encodes the red fluorescent protein DsRed2 under the control of the CDKN1A promoter (HepG2CDKN1A-DsRed cells). As the major downstream target gene of activated TP53, the tumour-suppressor gene CDKN1A is responsible for cell-cycle arrest following DNA damage, and it has been shown to be specifically up-regulated by genotoxic carcinogens. The assay is optimised for a 96-well microplate format and spectrofluorimetric quantification of induced DsRed expression. The assay was evaluated by testing direct-acting and indirect-acting genotoxic compounds with different mechanisms of action, along with non-genotoxic compounds. Out of 25 compounds that are known to be genotoxic in vitro and in vivo, 21 (84%) are detected as positive at non-cytotoxic doses, whereas of 12 compounds not considered genotoxic, 11 (92%) are negative. These data indicate the high sensitivity and specificity of our biosensor system. Based on its simplicity and sensitivity, this biosensor developed with HepG2CDKN1A-DsRed cells has the potential to become a valuable tool for genotoxicity screening for chemical safety evaluation, as well as for environmental and occupational monitoring of exposure to genotoxic agents and their complex mixtures.

In vivo real-time monitoring system of electroporation mediated control of transdermal and topical drug delivery.

Electroporation (EP) is a physical method for the delivery of molecules into cells and tissues, including the skin. In this study, in order to control the degree of transdermal and topical drug delivery, EP at different amplitudes of electric pulses was evaluated. A new in vivo real-time monitoring system based on fluorescently labeled molecules was developed, for the quantification of transdermal and topical drug delivery. EP of the mouse skin was performed with new non-invasive multi-array electrodes, delivering different amplitudes of electric pulses ranging from 70 to 570 V, between the electrode pin pairs. Patches, soaked with 4 kDa fluorescein-isothiocyanate labeled dextran (FD), doxorubicin (DOX) or fentanyl (FEN), were applied to the skin before and after EP. The new monitoring system was developed based on the delivery of FD to and through the skin. FD relative quantity was determined with fluorescence microscopy imaging, in the treated region of the skin for topical delivery and in a segment of the mouse tail for transdermal delivery. The application of electric pulses for FD delivery resulted in enhanced transdermal delivery. Depending on the amplitude of electric pulses, it increased up to the amplitude of 360 V, and decreased at higher amplitudes (460 and 570 V). Topical delivery steadily enhanced with increasing the amplitude of the delivered electric pulses, being even higher than after tape stripping used as a positive control. The non-invasive monitoring of the delivery of DOX, a fluorescent chemotherapeutic drug, qualitatively and quantitatively confirmed the effects of EP at 360 and 570 V pulse amplitudes on topical and transdermal drug delivery. Delivery of FEN at 360 and 570 V pulse amplitudes verified the observed effects as obtained with FD and DOX, by the measured physiological responses of the mice as well as FEN plasma concentration. This study demonstrates that with the newly developed non-invasive multi-array electrodes and with the varying electric pulse amplitude, the amount of topical and transdermal drug delivery to the skin can be controlled. Furthermore, the newly developed monitoring system provides a tool for rapid real-time determination of both, transdermal and topical delivery, when the delivered molecule is fluorescent.

Biological properties of melanoma and endothelial cells after plasmid AMEP gene electrotransfer depend on integrin quantity on cells.

The data on the biological responsiveness of melanoma and endothelial cells that are targeted by Antiangiogenic MEtargidin Peptide (AMEP) are limited; therefore, the antiproliferative, antimetastatic and antiangiogenic effects of AMEP were investigated in murine melanoma and human endothelial cells after plasmid AMEP gene electrotransfer into the cells in vitro. Plasmid AMEP, a plasmid coding for the disintegrin domain of metargidin targeting specific integrins, had cytotoxic and antiproliferative effects on murine melanoma and human endothelial cells. Among the metastatic properties of cells, migration, invasion and adhesion were investigated. Plasmid AMEP strongly affected the migration of murine melanoma and human endothelial cell lines and also affected the invasion of highly metastatic murine melanoma B16F10 and human endothelial cell lines. There was no effect on cell adhesion on Matrigel(TM) or fibronectin in all cell lines. The antiangiogenic effect was shown with tube formation assay, where human microvascular endothelial cell line (HMEC-1) proved to be more sensitive to plasmid AMEP gene electrotransfer than the human umbilical vein endothelial cell line (HUVEC). The study indicates that antiproliferative and antimetastatic biological responses to gene electrotransfer of plasmid AMEP in murine melanoma cells were dependent on the integrin quantity on melanoma cells and not on the expression level of AMEP. The strong antiangiogenic effect expressed in human endothelial cell lines was only partly dependent on the quantity of integrins and seemed to be plasmid AMEP dose dependent.

Potentiation of electrochemotherapy by intramuscular IL-12 gene electrotransfer in murine sarcoma and carcinoma with different immunogenicity.

BACKGROUND.: Electrochemotherapy provides good local tumor control but requires adjuvant treatment for increased local response and action on distant metastasis. In relation to this, intramuscular interleukin-12 (IL-12) gene electro-transfer, which provides systemic shedding of IL-12, was combined with local electrochemotherapy with cisplatin. Furthermore, the dependence on tumor immunogenicity and immunocompetence of the host on combined treatment response was evaluated. MATERIALS AND METHODS.: Sensitivity of SA-1 sarcoma and TS/A carcinoma cells to electrochemotherapy with cisplatin was tested in vitro. In vivo, intratumoral electrochemotherapy with cisplatin (day 1) was combined with a single (day 0) or multiple (days 0, 2, 4) intramuscular murine IL-12 (mIL-12) gene electrotransfer. The antitumor effectiveness of combined treatment was evaluated on immunogenic murine SA-1 sarcoma in A/J mice and moderately immunogenic murine TS/A carcinoma, in immunocompetent BALB/c and immunodeficient SCID mice. RESULTS.: Electrochemotherapy in vitro resulted in a similar IC(50) values for both sarcoma and carcinoma cell lines. However, in vivo electrochemotherapy was more effective in the treatment of sarcoma, the more immunogenic of the tumors, resulting in a higher log cell kill, longer specific tumor growth delay, and also 17% tumor cures compared to carcinoma where no tumor cures were observed. Adjuvant intramuscular mIL-12 gene electrotransfer increased the log cell kill in both tumor models, potentiating the specific tumor growth delay by a factor of 1.8-2 and increasing tumor cure rate by approximately 20%. In sarcoma tumors, the potentiation of the response by intramuscular mIL-12 gene electrotransfer was dose-dependent and also resulted in a faster onset of tumor cures. Comparison of the carcinoma response to the combined treatment modality in immunocompetent and immunodeficient mice demonstrated that the immune system is needed both for increased cell kill and for attaining tumor cures. CONCLUSIONS.: Based on the comparison of the antitumor effectiveness of electrochemotherapy to intratumoral cisplatin administration, we can conclude that the fraction of cells killed and the tumor cure rate are higher in immunogenic sarcoma tumor compared to moderately immunogenic carcinoma tumor. The tumor cell kill and cure rate depend on the immune response elicited by the destroyed tumor cells, which might depend on the tumor immunogenicity. The effect of adjuvant intramuscular mIL-12 gene electrotransfer is dependent on the amount of IL-12 in the system and the immune competence of the host, as demonstrated by the dose-dependent increase in the cure rate of SA-1 tumors after multiple intramuscular mIL-12 gene electrotransfer and in the differential cure rate of TS/A tumors growing in immunocompetent and immunodeficient mice.