LIPOSOME-ENABLED SYNERGISTIC INTERACTION OF ANTIMICROBIAL AGENTS

ABSTRACT

Antimicrobial agents may interact synergistically when both drugs are present at the infected site for an adequate period of time at sufficient concentrations. Generally speaking, the agents in the combination show different tissue distributions and pharmacokinetics. By co-encapsulation of the drugs in a drug carrier, like liposomes, parallel tissue distributions of both drugs may be ensured and drug concentrations at the site of infection may be increased. In this presentation therapeutic efficacy of liposome-co-encapsulated gentamicin (GN) and ceftazidime (CZ) will be shown in a GN-CZ-susceptible and GN-CZ-resistant Klebsiella pneumoniae-pneumonia in rats.     

Pharmacokinetic requirements for successful site-directed targeting of drugs

Targeted drugs can be defined as those in which features of the molecule, additional to those required for receptor interaction, substantially improve the concentration ratio of active substance at the site of action compared to the site where side-effects occur.These requirements for successful targeting of systemically administered drugs can be determined by pharmacokinetic modeling. The requirements depend on the mechanism of targeting and on whether targeting is to be achieved for continuous therapy or for acute treatment. For continuous therapy (lasting several days) the success of targeting using a prodrug which is locally activated and has linear pharmacokinetics is proportional to the clearance of active drug from the body and inversely proportional the rate constant for leaving the site of action and to the volume of the tissue. The relationship can be mapped graphically and typical values for these parameters are considered so that situations where targeting can be successful can be identified. The prodrug must also have properties which result in sufficient concentration of drug being formed at the site of action. These properties can also be described by simple proportionality relationships and the conditions for success illustrated graphically.Kinetic considerations are also important for targeting. For continuous therapy it is desirable that steady drug concentrations should be reached rapidly. This is best achieved by using molecules which rapidly exchange between body compartments and have low tissue binding. For acute therapy the rules for targeting can be quite different and an example is given where binding can be responsible for a form of targeting.The results emphasise the need for careful consideration of the properties of targeted system taking into account transport, binding and clearance of both prodrug and drug. Specific details of the disease are also critical both in terms of local tissue properties and the desired time course of drug action.     

Concentration of Antibacterial Agents in Interstitial Tissue Fluid

The concentration of antibacterial agents in the interstitial tissue fluid has been studied in an experimental model using implanted perforated Silastic capsules (tissue cages). Tissue fluid concentrations were always lower than the initial peak concentration in the serum, but for those drugs that were more slowly excreted the tissue fluid was similar to the serum concentration after six hours. In contrast the concentration in the tissue fluid for those drugs that are excreted rapidly was unpredictable, being either negligible or maintained at concentrations significantly better than in the serum. There was no evidence of accumulation in tissue fluid with regular dosage. These results indicate that the tissues may never be exposed to an adequate concentration of some antibacterial agents. This may be of clinical importance in tissue infections. Antibiotics which are rapidly excreted should be given in large enough doses and often enough for adequate blood levels to be maintained, so allowing equilibrium with tissue fluid to be achieved.     

Triple combination antiviral drug (TCAD) composed of amantadine, oseltamivir, and ribavirin impedes the selection of drug-resistant influenza A virus

Widespread resistance among circulating influenza A strains to at least one of the anti-influenza drugs is a major public health concern. A triple combination antiviral drug (TCAD) regimen comprised of amantadine, oseltamivir, and ribavirin has been shown to have synergistic and broad spectrum activity against influenza A strains, including drug resistant strains. Here, we used mathematical modeling along with three different experimental approaches to understand the effects of single agents, double combinations, and the TCAD regimen on resistance in influenza in vitro, including: 1) serial passage at constant drug concentrations, 2) serial passage at escalating drug concentrations, and 3) evaluation of the contribution of each component of the TCAD regimen to the suppression of resistance. Consistent with the modeling which demonstrated that three drugs were required to suppress the emergence of resistance in influenza A, treatment with the TCAD regimen resulted in the sustained suppression of drug resistant viruses, whereas treatment with amantadine alone or the amantadine-oseltamivir double combination led to the rapid selection of resistant variants which comprised ～100% of the population. Furthermore, the TCAD regimen imposed a high genetic barrier to resistance, requiring multiple mutations in order to escape the effects of all the drugs in the regimen. Finally, we demonstrate that each drug in the TCAD regimen made a significant contribution to the suppression of virus breakthrough and resistance at clinically achievable concentrations. Taken together, these data demonstrate that the TCAD regimen was superior to double combinations and single agents at suppressing resistance, and that three drugs at a minimum were required to impede the selection of drug resistant variants in influenza A virus. The use of mathematical modeling with multiple experimental designs and molecular readouts to evaluate and optimize combination drug regimens for the suppression of resistance may be broadly applicable to other infectious diseases.     

Effects of various antimicrobial agents on multi-directional differentiation potential of bone marrow-derived mesenchymal stem cells

Antimicrobial drugs of several classes play an important role in the treatment of bone and joint infections. In addition to fighting pathogenic microorganisms, the effects of drugs on local tissues and cells are also related to the course and prognosis of bone and joint infections. The multi-directional differentiation potential of bone marrow-derived mesenchymal stem cells (MSCs) is essential for tissue repair after local injury, which is directly related to the recovery of bone, cartilage, and medullary adipose tissue. Our previous studies and the literature indicate that certain antimicrobial agents can regulate the differentiation potential of bone marrow-derived MSCs. Here, in order to systematically analyze the effects of various antimicrobial drugs on local tissue regeneration, we comprehensively review the studies on the effects of these drugs on MSC differentiation, and classify them according to the three differentiation directions (osteogenesis, chondrogenesis, and adipogenesis). Our review demonstrates the specific effects of different antimicrobial agents on bone marrow-derived MSCs and the range of concentrations at which they work, and provides a basis for drug selection at different sites of infection.     

Biogenesis of mitochondria

Summary The action of ethidium bromide and berenil on the mitochondrial genome of Saccharomyces cerevisiae has been compared in three types of study: (i) early kinetics (up to 4 h) of petite induction by the drugs in the presence or absence of sodium dodecyl sulphate; (ii) genetic consequences of long-term (8 cell generations) exposure to the drugs; (iii) inhibition of mitochondrial DNA replication, both in whole cells and in isolated mitochondria.The results have been interpreted as follows. Firstly, the early events in petite induction differ markedly for the two drugs, as indicated by differences in the short-term kinetics. After some stage a common pathway is apparently followed because the composition of the population of petite cells induced after long-term exposure are very similar for both ethidium bromide and berenil. Secondly, both drugs probably act at the same site to inhibit mitochondrial DNA replication, in view of the fact that a petite strain known to be resistant to ethidium bromide inhibition of mitochondrial DNA replication was found to have simultaneously acquired resistance to berenil. From consideration of the drug concentrations needed to inhibit mitochondrial DNA replication in vivo and in vitro it is suggested that in vivo permeability barriers impede the access of ethidium bromide to the site of inhibition of mitochondrial DNA replication, whilst access of berenil to this site is facilitated. The site at which the drugs act to inhibit mitochondrial DNA replication may be different from the site(s) involved in early petite induction. Binding of the drugs at the latter site(s) is considered to initiate a series of events leading to the fragmentation of yeast mitochondrial DNA and petite induction.     

Pharmacogenetically hazardous drugs: a proposed new scoring method.

Similar doses of a drug given to different individuals can result in widely disparate plasma concentrations and hence effects. Beside intraindividual differences also inter-ethnic differences of drug response must be taken into consideration. Both inter-individual and inter-ethnic variations of drug response are mostly related to genetic factors (polymorphism) involved in drug metabolism and kinetics. The farmacogenetic disorders involved clinically result in pharmacogenetic side effects. In order to avoid pharmacogenetic side effects, beside phenotyping of the patients, selection of drugs subjected to different pharmacogenetic disorders may be of great clinical importance. Therefore, a scoring method was carried out for the selection of pharmacogenetically hazardous drugs. With regard to both genetic and environmental factors influencing the drug response, 140 suspicious drugs were studied and classified with the method. Eighteen was the maximum point value for genetic and 12 for contributing factors involved, so 30 was the maximum point number in each drug studied. Out of 140 substances 50 drugs (qualified with 20 points or more) proved to be hazardous in different pharmacogenetic disorders, among them several widely used agents, e.g. Diazepam, Isoniazid, Phenytoin, Warfarin, Quinidine, Tolbutamide, etc. The article sums up the findings in a Table and comments them. This scoring method may be useful in drug safety and preventive medicine.     

Arachidonic acid metabolism in the murine eosinophil. III. Effect of nonsteroid anti-inflammatory drugs on lymphokine-directed eosinophil migration in vivo

In vitro studies of murine eosinophils indicated that lymphokine-stimulated metabolism of arachidonic acid by a lipoxygenase pathway was required for a migration response. In this study we tested the effects of drugs that affect arachidonic acid metabolism on lymphokine-dependent eosinophil accumulation in vivo by the use of 111In-labeled eosinophils. Indomethacin at different dosages either stimulated (1 mg/kg) or partially inhibited (5 to 25 mg/kg) eosinophil accumulation. Aspirin had no significant effects in doses up to 50 mg/kg. BW755C, a drug that inhibits both cyclooxygenase and lipoxygenase, dramatically inhibited eosinophil accumulation at 25 to 125 mg/kg. Pretreatment in vitro of 111In-labeled eosinophils with some drugs known to inhibit lipoxygenase reduced their subsequent accumulation at a lymphokine-injected site in vivo, but the high concentrations required for inhibition suggested that the effects of the drugs were at least partially reversible. Pretreatment with indomethacin did not inhibit the ability of 111In-labeled eosinophils to accumulate at the site of lymphokine injection in vivo. It may be anticipated from these results that drugs inhibiting lipoxygenase will have critical effects on manifestations of immunologic reactions that recruit eosinophils.     

Evidence of a common site of action for the antitumor agents, hydroxyurea and guanazole

Hydroxyurea and guanazole were used as selective agents in tissue culture to obtain independent Chinese hamster ovary cell lines resistant to the cytotoxic effects of hydroxyurea or guanazole. In all cases tested a cell line selected for resistance to one of the antitumor agents exhibited resistance to both drugs. This result supports the view that these two drugs act at a common site.     

Conceptualizing Cancer Drugs as Classifiers

Cancer and healthy cells have distinct distributions of molecular properties and thus respond differently to drugs. Cancer drugs ideally kill cancer cells while limiting harm to healthy cells. However, the inherent variance among cells in both cancer and healthy cell populations increases the difficulty of selective drug action. Here we formalize a classification framework based on the idea that an ideal cancer drug should maximally discriminate between cancer and healthy cells. More specifically, this discrimination should be performed on the basis of measurable cell markers. We divide the problem into three parts which we explore with examples. First, molecular markers should discriminate cancer cells from healthy cells at the single-cell level. Second, the effects of drugs should be statistically predicted by these molecular markers. Third, drugs should be optimized for classification performance. We find that expression levels of a handful of genes suffice to discriminate well between individual cells in cancer and healthy tissue. We also find that gene expression predicts the efficacy of some cancer drugs, suggesting that these cancer drugs act as suboptimal classifiers using gene profiles. Finally, we formulate a framework that defines an optimal drug, and predicts drug cocktails that may target cancer more accurately than the individual drugs alone. Conceptualizing cancer drugs as solving a discrimination problem in the high-dimensional space of molecular markers promises to inform the design of new cancer drugs and drug cocktails.     

The Oxidative Inactivation of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) by Hypochlorous Acid (HOCl) is Suppressed by Anti-Rheumatic Drugs

Tissue inhibitors of metalloproteinases (TIMPs) prevent uncontrolled connective tissue destruction by limiting the activity of matrix metalloproteinases (MMPs). That TIMPs should be susceptible to oxidative inactivation is suggested by their complex tertiary structure which is dependent upon 6 disulphide bonds. We examined the oxidative inactivation of human recombinant TIMP-1 (hr TIMP-1) by HOCl and the inhibition of this process by anti-rheumatic agents.

TIMP-1 was exposed to HOCl in the presence of a variety of disease modifying anti-rheumatic drugs. TIMP-1 activity was measured by its ability to inhibit BC1 collagenase activity as measured by a fluorimetric assay using the synthetic pEptide substrate (DNP-Pro-Leu-Ala-Leu-Trp-Ala-Arg), best cleaved by MMP-1.

The neutrophil derived oxidant HOCl, but not the derived oxidant N-chlorotaurine, can inactivate TIMP-1 at concentrations achieved at sites of inflammation. Anti-rheumatic drugs have the ability to protect hrTIMP-1 from inactivation by HOCl. For D-penicil-lamine, this effect occurs at plasma levels achieved with patients taking the drug but for other anti-rheumatic drugs tested this occurs at relatively high concentrations that are unlikely to be achieved in vivo, except possibly in a microenvironment. These results are in keeping with the concept that biologically derived oxidants can potentiate tissue damage by inactivating key but susceptible protein inhibitors such as TIMP-1 which form the major local defence against MMP induced tissue breakdown.     

Effect of cisplatin upon expression of in vivo immune tumor resistance

The major intent of cancer treatment with cytotoxic drugs is direct tumor cell damage, but some of these drugs have been shown to be immunomodulatory. Cisplatin is a widely used cytotoxic drug that has been combined with biological response modifiers in recent clinical trials. To evaluate further whether cisplatin may independently alter the level of host resistance against tumor growth, the drug was tested in the Mc7 sarcoma rat tumor model. The expression of in vivo tumor resistance against Mc7 sarcoma in syngeneic Wistar rats is mediated by circulating non-cytotoxic T lymphocytes. These cells interact specifically with tumor cells to generate cytotoxic effectors locally at the site of a tumor challenge. Activities of these components of expression of tumor resistance were measured in vivo after administration of cisplatin and dose-dependent effects were found. Low-dose cisplatin (0.3 mg/kg) increased the activity of the circulating lymphocytes that mediate tumor resistance, and high-dose cisplatin (9 mg/kg) suppressed both mediator lymphocyte activity and the generation of antitumor effector mechanisms. These studies suggest that low-dose cisplatin may be immunomodulatory and combining it with biological response modifiers might be a useful strategy. However, high-dose cisplatin given with biological response modifiers may negate potential immunomodulatory activities of such agents.     

In silico optimization of atrial fibrillation-selective sodium channel blocker pharmacodynamics

Atrial fibrillation (AF) is the most common type of clinical arrhythmia. Currently available anti-AF drugs are limited by only moderate efficacy and an unfavorable safety profile. Thus, there is a recognized need for improved antiarrhythmic agents with actions that are selective for the fibrillating atrium. State-dependent Na(+)-channel blockade potentially allows for the development of drugs with maximal actions on fibrillating atrial tissue and minimal actions on ventricular tissue at resting heart rates. In this study, we applied a mathematical model of state-dependent Na(+)-channel blocking (class I antiarrhythmic drug) action, along with mathematical models of canine atrial and ventricular cardiomyocyte action potentials, AF, and ventricular proarrhythmia, to determine the relationship between their pharmacodynamic properties and atrial-selectivity, AF-selectivity (atrial Na(+)-channel block at AF rates versus ventricular block at resting rates), AF-termination effectiveness, and ventricular proarrhythmic properties. We found that drugs that target inactivated channels are AF-selective, whereas drugs that target activated channels are not. The most AF-selective drugs were associated with minimal ventricular proarrhythmic potential and terminated AF in 33% of simulations; slightly fewer AF-selective agents achieved termination rates of 100% with low ventricular proarrhythmic potential. Our results define properties associated with AF-selective actions of class-I antiarrhythmic drugs and support the idea that it may be possible to develop class I antiarrhythmic agents with optimized pharmacodynamic properties for AF treatment.     

Tumor Xenograft Response to Redox-Active Therapies Assessed by Magnetic Resonance Imaging Using a Thiol-Bearing DOTA Complex of Gadolinium

Gd-LC6-SH is a thiol-bearing DOTA complex of gadolinium designed to bind plasma albumin at the conserved Cys(34) site. The binding of Gd-LC6-SH shows sensitivity to the presence of competing thiols. We hypothesized that Gd-LC6-SH could provide magnetic resonance imaging (MRI) enhancement that is sensitive to tumor redox state and that the prolonged retention of albumin-bound Gd-LC6-SH in vivo can be exploited to identify a saturating dose above which the shortening of MRI longitudinal relaxation time (T(1)) of tissue is insensitive to the injected gadolinium dose. In the Mia-PaCa-2 pancreatic tumor xenograft model in SCID mice, both the small-molecule Gd-DTPA-BMA and the macromolecule Galbumin MRI contrast agents produced dose-dependent decreases in tumor T(1). By contrast, the decreases in tumor T(1) provided by Gd-LC6-SH at 0.05 and 0.1 mmol/kg were not significantly different at longer times after injection. SCID mice bearing Mia-PaCa-2 or NCI-N87 tumor xenografts were treated with either the glutathione synthesis inhibitor buthionine sulfoximine or the thiol-oxidizing anticancer drug Imexon, respectively. In both models, there was a significantly greater increase in tumor R(1) (=1/T(1)) 60 minutes after injection of Gd-LC6-SH in drug-treated animals relative to saline-treated controls. In addition, Mercury Orange staining for nonprotein sulfhydryls was significantly decreased by drug treatment relative to controls in both tumor models. In summary, these studies show that thiol-bearing complexes of gadolinium such as Gd-LC6-SH can serve as redox-sensitive MRI contrast agents for detecting differences in tumor redox status and can be used to evaluate the effects of redox-active drugs.     

Evaluation of the antioxidant properties of the angiotensin-converting enzyme inhibitor,captopril and the nucleotide enhancing agent,acadesine

The angiotensin-converting enzyme inhibitor, captopril and the nucleotide enhancing agent, acadesine, protect myocardial tissue from ischaemia/reperfusion-induced injury. Although both drugs have well established, independent mechanisms of cardiac protection, they may also have antioxidant activity which could contribute to their beneficial action. In this study we have examined the antioxidant activity of captopril and acadesine by examining their ability to scavenge ABTS radicals, formed from the interaction of ferryl metmyoglobin with phenothiazine in the presence of hydrogen peroxide. For comparison, we compared these results to those obtained for a range of other drugs commonly used for the treatment of cardiovascular disorders. These included verapamil (arrhythmia), isosorbide dinitrate (angina), atenolol (hypertension) and enalapril (congestive heart failure). The antioxidant properties of these drugs were then compared to the well characterised antioxidants, Trolox (a water soluble vitamin E analogue), ascorbate and glutathione. Captopril and acadesine were both shown to be efficient scavengers of ABTS radicals, importantly at drug concentrations expected to be found in vivo. These data confirm that the antioxidant potential of captopril and acadesine may be an important component of their mechanism of action, with both drugs probably protecting the myocardium against oxygen derived free radicals during ischaemia/reperfusion.     

Fitness landscapes emerging from pharmacodynamic functions in the evolution of multidrug resistance

Adaptive evolution often involves beneficial mutations at more than one locus. In this case, the trajectory and rate of adaptation is determined by the underlying fitness landscape, that is, the fitness values and mutational connectivity of all genotypes under consideration. Drug resistance, especially resistance to multiple drugs simultaneously, is also often conferred by mutations at several loci so that the concept of fitness landscapes becomes important. However, fitness landscapes underlying drug resistance are not static but dependent on drug concentrations, which means they are influenced by the pharmacodynamics of the drugs administered. Here, I present a mathematical framework for fitness landscapes of multidrug resistance based on Hill functions describing how drug concentrations affect fitness. I demonstrate that these ‘pharmacodynamic fitness landscapes’ are characterized by pervasive epistasis that arises through (i) fitness costs of resistance (even when these costs are additive), (ii) nonspecificity of resistance mutations to drugs, in particular cross‐resistance, and (iii) drug interactions (both synergistic and antagonistic). In the latter case, reciprocal drug suppression may even lead to reciprocal sign epistasis, so that the doubly resistant genotype occupies a local fitness peak that may be difficult to access by evolution. Simulations exploring the evolutionary dynamics on some pharmacodynamic fitness landscapes with both constant and changing drug concentrations confirm the crucial role of epistasis in determining the rate of multidrug resistance evolution.     

Modulation of heme and myristate binding to human serum albumin by anti-HIV drugs. An optical and NMR spectroscopic study

Human serum albumin (HSA) has an extraordinary ligand-binding capacity, and transports Fe(III)heme and medium- and long-chain fatty acids. In human immunodeficiency virus-infected patients the administered drugs bind to HSA and act as allosteric effectors. Here, the binding of Fe(III)heme to HSA in the presence of three representative anti-HIV drugs and myristate is investigated. Values of the dissociation equilibrium constant K(d) for Fe(III)heme binding to HSA were determined at different myristate concentrations, in the absence and presence of anti-HIV drugs. Nuclear magnetic relaxation dispersion profiles of HSA-Fe(III)heme were measured, at different myristate concentrations, in the absence and presence of anti-HIV drugs. Structural bases for anti-HIV drug binding to HSA are provided by automatic docking simulation. Abacavir and nevirapine bind to HSA with K(d) values of 1 x 10(-6) and 2 x 10(-6) M, respectively. Therefore, at concentrations used in therapy (in the 1-5 x 10(-6) M range) abacavir and nevirapine bind to HSA and increase the affinity of heme for HSA. In the presence of abacavir or nevirapine, the affinity is not lowered by myristate. FA7 should therefore be intended as a secondary binding site for abacavir and nevirapine. Binding of atazanavir is limited by the large size of the drug, although preferential binding may be envisaged to a site positively coupled with FA1 and FA2, and negatively coupled to FA7. As a whole, these results provide a foundation for the comprehension of the complex network of links modulating HSA-binding properties.     

Activation of prodrugs by antibody-enzyme conjugates: a new approach to cancer therapy

A new strategy for the delivery of cytotoxic agents to solid tumors is described in which monoclonal antibodies are used as carriers for enzymes to tumor cell surfaces. The enzymes are chosen for their abilities to convert relatively noncytotoxic drug precursors (pro-drugs) into active anticancer drugs. The drugs thus formed can then penetrate into nearby tumor cells, resulting in cell death. A number of prodrugs have been developed that can be transformed into active anti-cancer drugs by enzymes of both mammalian and non-mammalian origin. The enzymes have been shown to localize into tumors when linked to monoclonal antibodies that bind to tumor-associated antigens. In vivo studies indicate that MAb-enzyme/prodrug combinations can result in antitumor activities significantly greater than those of the prodrugs or drugs given alone. This is most likely due to the generation of large amounts of active drug at the tumor site.     

Distribution of topoisomerase II cleavage sites in simian virus 40 DNA and the effects of drugs.

The distributions of DNA cleavage sites induced by topoisomerase II in the presence or absence of specific drugs were mapped in the simian virus 40 genome. The drugs studied were 5-iminodaunorubicin, amsacrine (m-AMSA), teniposide (VM-26) and 2-methyl-9-hydroxyellipticinium; each produced a distinctive pattern of enhanced cleavage. Consistently intense cleavage, both in the presence and in the absence of drugs, occurred in the nuclear matrix-associated region. Since topoisomerase II is a major constituent of the nuclear matrix, and cleavage complexes include a covalent link between topoisomerase II and DNA, the findings suggest that topoisomerase II may function to attach DNA to the nuclear matrix. Cleavage usually occurred on both DNA strands with the expected four base-pair 5' stagger, and strong sites tended to occur within A/T runs such as have been associated with binding to the nuclear scaffold. Intense cleavage was present also in the replication termination region, but was absent from the vicinity of the replication origin. Cleavage intensities were found to change with time in a manner that depended both on the site and on the drug, suggesting that topoisomerase II can move along the DNA from a kinetically preferred site to a thermodynamically preferred site.     

EFFECTS OF PSYCHOTROPIC DRUGS ON NEUROTUBULE ASSEMBLY

Abstract— The interaction of psychotropic drugs with brain microtubules was assessed by viscometry, [³H]colchicine binding studies and neurite growth in neuroblastoma cells in tissue culture. Of the thirteen drug- tested, only d-amphetamine, chlorpromazine and reserpine inhibited the in vitro repoly-merization of microtubules. However, the maximal drug effects observed in these studies occurred at concentrations in excess of the pharmacologically theraputic range. These results suggest: (1) the affinity of these drugs for tubulin appears to be similar to, but clearly less than, that observed for colchicine; (2) the toxic side effects of these drugs may be mediated, in part, by the microtubule system; (3) these drugs may inhibit microtubule assembly by disrupting secondary processes rather than by a direct interaction with tubulin.