Mode Of Drug Action Research Articles

Serum Metabolites as Potential Markers and Predictors of Depression-like Behavior and Effective Fluoxetine Treatment in Chronically Socially Isolated Rats.

Metabolic perturbation has been associated with depression. An untargeted metabolomics approach using liquid chromatography-high resolution mass spectrometry was employed to detect and measure the rat serum metabolic changes following chronic social isolation (CSIS), an animal model of depression, and effective antidepressant fluoxetine (Flx) treatment. Univariate and multivariate statistics were used for metabolic data analysis and differentially expressed metabolites (DEMs) determination. Potential markers and predictive metabolites of CSIS-induced depressive-like behavior and Flx efficacy in CSIS were evaluated by the receiver operating characteristic (ROC) curve, and machine learning (ML) algorithms, such as support vector machine with linear kernel (SVM-LK) and random forest (RF). Upregulated choline following CSIS may represent a potential marker of depressive-like behavior. Succinate, stachydrine, guanidinoacetate, kynurenic acid, and 7-methylguanine were revealed as potential markers of effective Flx treatment in CSIS rats. RF yielded better accuracy than SVM-LK (98.50% vs. 85.70%, respectively) in predicting Flx efficacy in CSIS vs. CSIS, however, it performed almost identically in classifying CSIS vs. control (75.83% and 75%, respectively). Obtained DEMs combined with ROC curve and ML algorithms provide a research strategy for assessing potential markers or predictive metabolites for the designation or classification of stress-induced depressive phenotype and mode of drug action.

Quantification of chemotherapy contributions to chemoradiotherapy

Chemoradiotherapy (CRT) treatments are those which use chemotherapy drugs to improve the tumor control obtainable from radiotherapy schedules. Such additions are known to be beneficial and are widely prescribed. However, the methodologies employed for defining chemotherapy and radiation doses are entirely different, meaning that quantitative elucidation of the chemotherapy contribution to a CRT schedule remains difficult. One solution is to express the chemotherapy effect in terms of its radiation-equivalent and several authors have examined ways of doing this. This article outlines an approach which uses radiation-alone dose-response data as a platform against which to derive estimates of the drug contribution to CRT treatments. Such a methodology is potentially advantageous as radiation-alone parameters derived from observations of tumor response over a range of doses provide a realistic representation of whole-population behaviour and do not require prior assumptions to be made concerning radiosensitivity variations, etc. Although essentially a theoretical investigation. this approach provides some insight into how derived CRT dose-response curves can vary according to the possible modes of drug action, four of which are considered here, i.e. independent cell kill, dose sensitisation, reduced repopulation or combinations of each. It is shown that, for the moderate drug-induced TCP improvements usually observed in practice, the chemotherapy dose-equivalents derived from individual observations are a reasonable predictor of those operating at other levels of response, irrespective of the underlying drug-radiation synergy.

A novel series of putative Brugia malayi histone demethylase inhibitors as potential anti-filarial drugs.

Filariasis, caused by a family of parasitic nematodes, affects millions of individuals throughout the tropics and is a major cause of acute and chronic morbidity. Current drugs are largely used in mass drug administration programs aimed at controlling the spread of disease by killing microfilariae, larval forms of the parasite responsible for transmission from humans to humans through insect vectors with limited efficacy against adult parasites. Although these drugs are effective, in some cases there are toxic liabilities. In case of loiasis which is caused by the parasitic eyeworm Loa loa, mass drug administration is contraindicative due to severe side effects of microfilariae killing, which can be life threatening. Our screening program and medicinal chemistry efforts have led to the identification of a novel series of compounds with potent killing activity against adult filarial parasites and minimal activity against microfilariae. A structural comparison search of our compounds demonstrated a close structural similarity to a recently described histone demethylase inhibitor, GSKJ1/4 which also exhibits selective adult parasite killing. We demonstrated a modification of histone methylation in Brugia malayi parasites treated with our compounds which might indicate that the mode of drug action is at the level of histone methylation. Our results indicate that targeting B. malayi and other filarial parasite demethylases may offer a novel approach for the development of a new class of macrofilaricidal therapeutics.

Laboratory Selection of Trypanosomatid Pathogens for Drug Resistance.

The selection of parasites for drug resistance in the laboratory is an approach frequently used to investigate the mode of drug action, estimate the risk of emergence of drug resistance, or develop molecular markers for drug resistance. Here, we focused on the How rather than the Why of laboratory selection, discussing different experimental set-ups based on research examples with Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. The trypanosomatids are particularly well-suited to illustrate different strategies of selecting for drug resistance, since it was with African trypanosomes that Paul Ehrlich performed such an experiment for the first time, more than a century ago. While breakthroughs in reverse genetics and genome editing have greatly facilitated the identification and validation of candidate resistance mutations in the trypanosomatids, the forward selection of drug-resistant mutants still relies on standard in vivo models and in vitro culture systems. Critical questions are: is selection for drug resistance performed in vivo or in vitro? With the mammalian or with the insect stages of the parasites? Under steady pressure or by sudden shock? Is a mutagen used? While there is no bona fide best approach, we think that a methodical consideration of these questions provides a helpful framework for selection of parasites for drug resistance in the laboratory.

Orphan Medicinal Products for the Treatment of Pancreatic Cancer: Lessons Learned From Two Decades of Orphan Designation.

Pancreatic cancer has a dismal prognosis and only a few treatment options are available. In the European Union, pancreatic cancer classifies as a rare disease, allowing drug developers to apply for orphan medicinal product (OMP) designation. The aim of this study was to provide more detail on OMPs for pancreatic cancer. All applications for OMP designation submitted to the EMA between 2000 and 2019 were identified. For each medicinal product that received an OMP designation, the mode of drug action, use of protocol assistance, and current life cycle status was determined. Fifty-two medicinal products received an OMP designation. At the time of submission, eighteen OMPs were at the non-clinical and 34 OMPs were at the clinical stage of development. At least fourteen kinds of mode of action were explored in the condition. For eighteen out of 52 OMPs protocol assistance was sought. At the time of data analysis, one OMP received marketing authorisation and 24 OMPs were ongoing in development. Many medicinal products for pancreatic cancer received an OMP designation and the majority of these products was already in the clinical stage of development. Nonetheless, the success rate of OMPs for pancreatic cancer that reach the market is low, and increasing this rate is something to aspire. Fortunately, development is still ongoing for a part of the OMPs, and a few developers are planning to submit a marketing authorisation application in the near future. This however does not guarantee success, as pancreatic cancer remains a difficult disease to treat. Developers are advised to make optimal use of incentives such as protocol assistance, establishing (early) dialogue between regulators and drug developers and to agree on important topics such as clinical trial design.

Asymmetric Interplay Between K+ and Blocker and Atomistic Parameters From Physiological Experiments Quantify K+ Channel Blocker Release.

Modulating the activity of ion channels by blockers yields information on both the mode of drug action and on the biophysics of ion transport. Here we investigate the interplay between ions in the selectivity filter (SF) of K+ channels and the release kinetics of the blocker tetrapropylammonium in the model channel KcvNTS. A quantitative expression calculates blocker release rate constants directly from voltage-dependent ion occupation probabilities in the SF. The latter are obtained by a kinetic model of single-channel currents recorded in the absence of the blocker. The resulting model contains only two adjustable parameters of ion-blocker interaction and holds for both symmetric and asymmetric ionic conditions. This data-derived model is corroborated by 3D reference interaction site model (3D RISM) calculations on several model systems, which show that the K+ occupation probability is unaffected by the blocker, a direct consequence of the strength of the ion-carbonyl attraction in the SF, independent of the specific protein background. Hence, KcvNTS channel blocker release kinetics can be reduced to a small number of system-specific parameters. The pore-independent asymmetric interplay between K+ and blocker ions potentially allows for generalizing these results to similar potassium channels.

Lensoside Aβ as an Adjuvant to the Anti-Glioma Potential of Sorafenib.

Simple SummaryFlavonoids are plant polyphenolic secondary metabolites, commonly consumed in the human diet. Lensoside Aβ is a quercetin glycoside isolated from the aerial parts of lentil (Lens culinaris) organs. The activity of this secondary metabolite, especially in terms of its anticancer potential, has been poorly studied. Currently, there are no published data about the effect of this flavonoid on gliomas, which are so-far incurable, aggressive neoplasms of the central nervous system with a highly infiltrative character. In this study, we found that lensoside Aβ itself exhibits poor anti-glioma properties but exerts a strongly potentiated effect in combination with sorafenib (inhibitor of Raf kinase) on apoptosis induction in cancer cells. Our results have shown that sorafenib with lensoside Aβ seems to be a promising combination that might be useful in glioma therapy. Additionally, the former observation, pointing to the key role of flavonoids as adjuvants in chemotherapy, is confirmed.Aim: The anti-glioma effect of lensoside Aβ alone and in combination with sorafenib (pro-survival Raf kinase inhibitor) was evaluated for the first time in terms of programmed cell death induction in anaplastic astrocytoma and glioblastoma multiforme cell lines as an experimental model. Apoptosis, autophagy, and necrosis were identified microscopically (fluorescence and scanning microscopes) and confirmed by flow cytometry (mitochondrial membrane potential MMP and cell death). The expression of apoptotic (caspase 3) and autophagic markers (beclin 1) as well as Raf kinase were estimated by immunoblotting. The FTIR method was used to determine the interaction of the studied drugs with lipid and protein groups within cells, while the modes of drug action within the cells were assessed with the FLIM technique. Results: Lensoside Aβ itself does not exhibit anti-glioma activity but significantly enhances the anti-cancer potential of sorafenib, initiating mainly apoptosis of up to 90% of cells. It was correlated with an increased level of active caspase 3, a reduced MMP value, and a lower level of Raf kinase. The interaction with membrane structures led to morphological changes typical of programmed death. Conclusions: Our results indicate that lensoside Aβ plays an important role as an adjuvant in chemotherapy with sorafenib and may be a potential candidate in anti-glioma combination therapy.

DDIS-35. RelA-ACTIVITY IS ESSENTIAL FOR CANNABIDIOL-MEDIATED CYTOTOXICITY IN AN IDENTIFIED SUBSET OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) therapy could strongly profit from simplified procedures for personalized medicine. Cannabidiol (CBD) is explored for GBM-patients, but the mode of drug-action is largely unknown and stratification markers are missing. We investigated CBD-induced cytotoxicity in a panel of human primary GBM cells as well as transgenic mouse gliomas and in different orthotopic or transgenic in vivo models. We observed therapeutic efficiency of CBD in a subset of GBM, obtained genetic markers indicating CBD-sensitive GBM and found that the p53 status segregated cell-death modes. Genome wide loss of function screening, transcription factor binding studies and knockout-models indicated a central for role for NFkB (RelA) in CBD-initiated GBM-death. CBD mediated nuclear import of RelA lacking an essential post-translational modification and reduced RelA-activity. CBD altered redox levels specifically in drug-sensitive GBM and ROS-levels in unstimulated GBM predicted therapeutic response. Hence, quantifying ROS in GBM represents a simple bioassay to identify individuals potentially profiting from CBD-application in neurooncology.

Bromodomain and extraterminal inhibitors block the Epstein-Barr virus lytic cycle at two distinct steps

Lytic infection by the Epstein-Barr virus (EBV) poses numerous health risks, such as infectious mononucleosis and lymphoproliferative disorder. Proteins in the bromodomain and extraterminal (BET) family regulate multiple stages of viral life cycles and provide promising intervention targets. Synthetic small molecules can bind to the bromodomains and disrupt function by preventing recognition of acetylated lysine substrates. We demonstrate that JQ1 and other BET inhibitors block two different steps in the sequential cascade of the EBV lytic cycle. BET inhibitors prevent expression of the viral immediate-early protein BZLF1. JQ1 alters transcription of genes controlled by the host protein BACH1, and BACH1 knockdown reduces BZLF1 expression. BET proteins also localize to the lytic origin of replication (OriLyt) genetic elements, and BET inhibitors prevent viral late gene expression. There JQ1 reduces BRD4 recruitment during reactivation to preclude replication initiation. This represents a rarely observed dual mode of action for drugs.

Fludioxonil Induces Drk1, a Fungal Group III Hybrid Histidine Kinase, To Dephosphorylate Its Downstream Target, Ypd1.

Novel antifungal drugs and targets are urgently needed. Group III hybrid histidine kinases (HHKs) represent an appealing new therapeutic drug target because they are widely expressed in fungi but absent from humans. We investigated the mode of action of the widely utilized, effective fungicide fludioxonil. The drug acts in an HHK-dependent manner by constitutive activation of the HOG (high-osmolarity glycerol) pathway, but its mechanism of action is poorly understood. Here, we report a new mode of drug action that entails conversion of the HHK from a kinase into a phosphatase. We expressed Drk1 (dimorphism-regulating kinase), which is an intracellular group III HHK from the fungal pathogen Blastomyces dermatitidis, in Saccharomyces cerevisiae Drk1 engendered drug sensitivity in B. dermatitidis and conferred sensitivity upon S. cerevisiae In response to fludioxonil, Drk1 behaved as a phosphatase rather than as a kinase, leading to dephosphorylation of its downstream target, Ypd1, constitutive activation of the HOG pathway, and yeast cell death. Aspartic acid residue 1140 in the Drk1 receiver domain was required for in vivo phosphatase activity on Ypd1, and Hog1 was required for drug effect, indicating fidelity in HHK-dependent drug action. In in vitro assays with purified protein, intact Drk1 demonstrated intrinsic kinase activity, and the Drk1 receiver domain exhibited intrinsic phosphatase activity. However, fludioxonil failed to induce intact Drk1 to dephosphorylate Ypd1. We conclude that fludioxonil treatment in vivo likely acts on an upstream target that triggers HHK to become a phosphatase, which dephosphorylates its downstream target, Ypd1.

Pharmacological Treatment of Drug-Resistant Epilepsy in Adults: a Practical Guide.

More than 30% of adults with epilepsy do not fully control on the currently available antiepileptic drugs (AEDs). For these and many other patients, combinations of agents, often possessing different mechanisms of actions, are employed with the aim of achieving seizure freedom or the best available prognosis in terms of reduced seizure numbers and severity. This review discusses my own approach to optimising outcomes in as many of these patients as possible by adjusting the drug burden using a combination of two, three or sometimes four or more AEDs. Modes of drug action are reviewed and practical strategies for treating different patients with drug-resistant epilepsy have been explored. Only for sodium valproate with lamotrigine is there good evidence of synergism. The final part of this practical paper consists of six individual illustrative cases with appropriate comments.

Cogena, a novel tool for co-expressed gene-set enrichment analysis, applied to drug repositioning and drug mode of action discovery.

BackgroundDrug repositioning, finding new indications for existing drugs, has gained much recent attention as a potentially efficient and economical strategy for accelerating new therapies into the clinic. Although improvement in the sensitivity of computational drug repositioning methods has identified numerous credible repositioning opportunities, few have been progressed. Arguably the “black box” nature of drug action in a new indication is one of the main blocks to progression, highlighting the need for methods that inform on the broader target mechanism in the disease context.ResultsWe demonstrate that the analysis of co-expressed genes may be a critical first step towards illumination of both disease pathology and mode of drug action. We achieve this using a novel framework, co-expressed gene-set enrichment analysis (cogena) for co-expression analysis of gene expression signatures and gene set enrichment analysis of co-expressed genes. The cogena framework enables simultaneous, pathway driven, disease and drug repositioning analysis. Cogena can be used to illuminate coordinated changes within disease transcriptomes and identify drugs acting mechanistically within this framework. We illustrate this using a psoriatic skin transcriptome, as an exemplar, and recover two widely used Psoriasis drugs (Methotrexate and Ciclosporin) with distinct modes of action. Cogena out-performs the results of Connectivity Map and NFFinder webservers in similar disease transcriptome analyses. Furthermore, we investigated the literature support for the other top-ranked compounds to treat psoriasis and showed how the outputs of cogena analysis can contribute new insight to support the progression of drugs into the clinic. We have made cogena freely available within Bioconductor or https://github.com/zhilongjia/cogena.ConclusionsIn conclusion, by targeting co-expressed genes within disease transcriptomes, cogena offers novel biological insight, which can be effectively harnessed for drug discovery and repositioning, allowing the grouping and prioritisation of drug repositioning candidates on the basis of putative mode of action.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2737-8) contains supplementary material, which is available to authorized users.

The structure-dependent self-association of five phenolic acids in aqueous solution.

Weak self-interaction plays an important role in interpreting the biomechanisms and modes of drug action. The structure-dependent self-association of five phenolic acids with various bioactivities, including danshensu (DSS), caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SA), was investigated by (1)H NMR. These phenolic acids have similar condensed structures, with a CA moiety and varying numbers of DSS moieties. The strengths of the self-association constants are in the order DSS < CA < RA < LA < SA, which corresponds to the increasing molecular size of these phenolic acids and roughly corresponds to the increasing number of DSS moieties. The binding site for the self-aggregation of these phenolic acids has been identified to be on the CA moiety, rather than on the DSS moiety, as a result of CA's stronger aromatic π-π interactions, which cause larger chemical shift variations. The thermodynamic parameters for the self-association of these phenolic acids show that the self-association is spontaneous and enthalpically favorable at room temperature in all cases. It was inferred that π-π interactions and intermolecular hydrogen bonding stabilize the stacking structures of the phenolic acids. Knowledge of self-association processes will enable us to quantitatively assess the possible effects of self-aggregation on the interaction between drug and protein.

Large scale integration of drug-target information reveals poly-pharmacological drug action mechanisms in tumor cell line growth inhibition assays

Understanding therapeutic mechanisms of drug anticancer cytotoxicity represents a key challenge in preclinical testing. Here we have performed a meta-analysis of publicly available tumor cell line growth inhibition assays (~ 70 assays from 6 independent experimental groups covering ~ 500 000 molecules) with the primary goal of understanding molecular therapeutic mechanisms of cancer cytotoxicity. To implement this we have collected currently available information on protein targets for molecules that were tested in the assays. We used a statistical methodology to identify protein targets overrepresented among molecules exhibiting cancer cytotoxicity with the particular focus of identifying overrepresented patterns consisting of several proteins (i.e. proteins "A" and "B" and "C"). Our analysis demonstrates that targeting individual proteins can result in a significant increase (up to 50-fold) of the observed odds for a molecule to be an efficient inhibitor of tumour cell line growth. However, further insight into potential molecular mechanisms reveals a multi-target mode of action: targeting a pattern of several proteins drastically increases the observed odds (up to 500-fold) for a molecule to be tumour cytotoxic. In contrast, molecules targeting only one protein but not targeting an additional set of proteins tend to be nontoxic. Our findings support a poly-pharmacology drug discovery paradigm, demonstrating that anticancer cytotoxicity is a product, in most cases, of multi-target mode of drug action.

Advanced intravital subcellular imaging reveals vital three‐dimensional signalling events driving cancer cell behaviour and drug responses in live tissue

The integration of signal transduction pathways plays a fundamental role in governing disease initiation, progression and outcome. It is therefore necessary to understand disease at the signalling level to enable effective treatment and to intervene in its progression. The recent extension of in vitro subcellular image-based analysis to live in vivo modelling of disease is providing a more complete picture of real-time, dynamic signalling processes or drug responses in live tissue. Intravital imaging offers alternative strategies for studying disease and embraces the biological complexities that govern disease progression. In the present review, we highlight how three-dimensional or live intravital imaging has uncovered novel insights into biological mechanisms or modes of drug action. Furthermore, we offer a prospective view of how imaging applications may be integrated further with the aim of understanding disease in a more physiological and functional manner within the framework of the drug discovery process.

Determination of antiprotozoal drug mechanisms by metabolomics approaches

The discovery, development and optimal utilization of pharmaceuticals can be greatly enhanced by knowledge of their modes of action. However, many drugs currently on the market act by unknown mechanisms. Untargeted metabolomics offers the potential to discover modes of action for drugs that perturb cellular metabolism. Development of high resolution LC-MS methods and improved data analysis software now allows rapid detection of drug-induced changes to cellular metabolism in an untargeted manner. Several studies have demonstrated the ability of untargeted metabolomics to provide unbiased target discovery for antimicrobial drugs, in particular for antiprotozoal agents. Furthermore, the utilization of targeted metabolomics techniques has enabled validation of existing hypotheses regarding antiprotozoal drug mechanisms. Metabolomics approaches are likely to assist with optimization of new drug candidates by identification of drug targets, and by allowing detailed characterization of modes of action and resistance of existing and novel antiprotozoal drugs.

Effects of treatment on IgE responses against parasite allergen-like proteins and immunity to reinfection in childhood schistosome and hookworm coinfections.

Naturally occurring human immunity to both schistosomiasis and hookworm infection has been associated with IgE responses against parasite allergen-like proteins. Since the two helminths frequently coinfect the same individuals, there is growing advocacy for their concurrent treatment. However, both helminths are known to exert strong immunomodulatory effects; therefore, coinfected individuals could have immune responses different from those characteristically seen in monoinfected individuals. In this study, we measured changes in IgE, IgG1, and IgG4 responses to schistosome and hookworm antigens, including the allergen-like proteins Schistosoma mansoni tegumental-allergen-like 1 protein (SmTAL1), SmTAL2, and Necator americanus Ancylostoma-secreted protein-2 (Na-ASP-2), following concurrent treatment of schoolchildren coinfected with Schistosoma mansoni and hookworm. Antibody responses to schistosome egg (soluble egg antigen and SmTAL2) or somatic adult hookworm (AHW) antigens either decreased after treatment or were unchanged, whereas those to schistosome worm antigens (soluble worm antigen and SmTAL1) increased. The observed different effects of treatment likely reflect the different modes of drug action and sites of infection for these two helminths. Importantly, there was no evidence that the simultaneous treatment of coinfected children with praziquantel and albendazole affected schistosome- and hookworm-specific humoral responses differently from those characteristic of populations in which only one organism is endemic; schistosome- and hookworm-specific responses were not associated, and there was no evidence for cross-regulation. Posttreatment increases in the levels of IgE to schistosome worm antigens were associated with lower Schistosoma mansoni reinfection intensity, while no associations between humoral responses to AHW antigen and protection from hookworm reinfection were observed in this sample of school-aged children.

Analysis of exposure–response of CI-945 in patients with epilepsy: application of novel mixed hidden Markov modeling methodology

We propose to describe exposure-response relationship of an antiepileptic agent, using mixed hidden Markov modeling methodology, to reveal additional insights in the mode of the drug action which the novel approach offers. Daily seizure frequency data from six clinical studies including patients who received gabapentin were available for the analysis. In the model, seizure frequencies are governed by underlying unobserved disease activity states. Individual neighbouring states are dependent, like in reality and they exhibit their own dynamics with patients transitioning between low and high disease states, according to a set of transition probabilities. Our methodology enables estimation of unobserved disease dynamics and daily seizure frequencies in all disease states. Additional modes of drug action are achievable: gabapentin may influence both daily seizure frequencies and disease state dynamics. Gabapentin significantly reduced seizure frequencies in both disease activity states; however it did not significatively affect disease dynamics. Mixed hidden Markov modeling is able to mimic dynamics of seizure frequencies very well. It offers novel insights into understanding disease dynamics in epilepsy and gabapentin mode of action.

HIV-1 Antiretroviral Drug Therapy

The most significant advance in the medical management of HIV-1 infection has been the treatment of patients with antiviral drugs, which can suppress HIV-1 replication to undetectable levels. The discovery of HIV-1 as the causative agent of AIDS together with an ever-increasing understanding of the virus replication cycle have been instrumental in this effort by providing researchers with the knowledge and tools required to prosecute drug discovery efforts focused on targeted inhibition with specific pharmacological agents. To date, an arsenal of 24 Food and Drug Administration (FDA)-approved drugs are available for treatment of HIV-1 infections. These drugs are distributed into six distinct classes based on their molecular mechanism and resistance profiles: (1) nucleoside-analog reverse transcriptase inhibitors (NNRTIs), (2) non-nucleoside reverse transcriptase inhibitors (NNRTIs), (3) integrase inhibitors, (4) protease inhibitors (PIs), (5) fusion inhibitors, and (6) coreceptor antagonists. In this article, we will review the basic principles of antiretroviral drug therapy, the mode of drug action, and the factors leading to treatment failure (i.e., drug resistance).

The role of the protein-binding on the mode of drug action as well the interactions with other drugs

Drug transport and disposition are influenced by a non-specific and reversible drug binding to plasma and tissues proteins. Albumin and al acid glycoprotein are the most important transport proteins of the blood. Albumin possesses specific sites for acidic and basic drug binding and can interact with them in the plasma since a third site is trapped only by digoxin. Diseases and stress conditions induce conformational changes either in plasma or in tissue proteins by the synthesis of endogenous substances which can strong interfere with the amount of the free pharmacological effective drug ratio. This may affect the binding of drugs in target molecules inducing significant pharmacokinetic alterations. Stress conditions are associated with FFA increase in serum playing an antagonistic role with other acidic molecules (e.g. ampicillin) to the same binding site. The bounded drug is displaced and freer ratio is available to interact with various organ receptors leading to pharmacological effect enhancement and therefore to side effects manifestation such as seizures. Furthermore conjunctive tissues diseases, ageing, prolonged bleeding, starvation or diseases affecting protein profile, characterized by reduced total plasma proteins, followed by albumin decrease and lessen binding sites lead to more free drug availability enhancing its pharmacological effect. Increased a1-acid glycoprotein the acute phase protein as by heart infraction or liver morbidities (e.g CCl4 intoxication) mainly occupied from basic substances, in the case of cationic drug treatment resulted to the enhancement of them and consequently to pronounced effectiveness. In addition, renal failure reduced free fractions of many acidic drugs. It may be concluded that by narrowed therapeutic index of a medicine, and when drug/drug or drug/disease interactions are anticipated, drug monitoring seems to be necessary for its dosage adjustment.