Mechanisms of in situ activation for peroxidic antimalarials

Abstract

This review describes mechanisms of action of artemisinin-related antimalarials, emphasizing the site and target of activation, pathways of generating reactive species, and possible targets of free radicals with implications for antimalarial peroxide drug design. It also presents a useful link between the mode of action of artemisinin and that of chloroquine, and highlights redox cycles involved in the interaction between the drug and vital biomolecules.     

A Characterization of Children's Intuitive Theories of Drug Action

In an attempt to devise a methodology for characterizing children's intuitive theories of drug action, 217 children in Grades 1 to 6 were interviewed about how two substances, alcohol and cocaine, cause behavioral changes in their users. Measures tapped both structure (Piagetian complexity of causal reasoning, coherence, and construction of a causal explanatory framework with nodes, links, and causal mechanisms) and content (a relevant biological ontology, mastery of propositions in a scientifically correct theory of drug action, and reliance on alternative theoretical models). Scores on all measures increased with age, especially between first and second grade and third and fourth grade. Growth between third/fourth and fifth/sixth grades was more evident for cocaine, the less familiar of the two substances. Measures of structure and content were correlated. Overall, elementary school age children appear capable of framing causal explanations of drug action and possess relevant biological knowledge, particularly about the central role of the brain in mediating the effects of drugs on behavior. It remains to be seen whether programs guided by an intuitive theories perspective and designed to increase children's understandings of how drugs alter behavior can contribute meaningfully to drug prevention efforts.     

Measurement of fasciculations as motor nerve ending discharges in the rat: a dose related effect of neostigmine

The muscle fasciculations caused by neostigmine and similar agents are the result of a primary drug action on motor nerve endings. Asynchronous, repetitive firing of action potentials are evoked at motor nerve endings which are then transmitted to muscle. A dose-response relationship between neostigmine dose and the rate of/or total neural activity has been established in the rat. This fasciculatory response to neostigmine can serve as an index of motor nerve ending excitability and may be useful in assessing the effects of certain pathological states or drug actions at the neuromuscular junction.     

山莨菪碱与酸性磷脂脂质体的亲水与疏水作用——激光拉曼光谱及差光谱的研究

本文从分子水平上研究了山茛菪碱与酸性磷脂脂质体的亲水与疏水相互作用.通过测定在不同pH条件下,酸性磷脂头部基因PO_3~(2-)的特征拉曼光谱振模的变化,证明药物与酸性磷脂头部的负电基团有较强的静电相互作用,并且这种作用与药物引起脂肪酸链构象改变有关.采用拉曼差光谱的方法,进一步研究了药物的苯环与酸性磷脂的疏水相互作用.观察到反映苯环两种不同振动的特征振模在药物同磷脂作用过程中发生的不同变化,从而获得较直接的证据说明药物的苯环插入脂双层.     

Effect of colour of drugs: systematic review of perceived effect of drugs and of their effectiveness.

OBJECTIVE: To assess the impact of the colour of a drug''s formulation on its perceived effect and its effectiveness and to examine whether antidepressant drugs available in the Netherlands are different in colour from hypnotic, sedative, and anxiolytic drugs. DESIGN: Systematic review of 12 published studies. Six studies examined the perceived action of different coloured drugs and six the influence of the colour of a drug on its effectiveness. The colours of samples of 49 drugs affecting the central nervous system were assessed using a colour atlas. MAIN OUTCOME MEASURES: Perceived stimulant action versus perceived depressant action of colour of drugs; the trials that assessed the effect of drugs in different colours were done in patients with different diseases and had different outcome measures. RESULTS: The studies on perceived action of coloured drugs showed that red, yellow, and orange are associated with a stimulant effect, while blue and green are related to a tranquillising effect. The trials that assessed the impact of the colour of drugs on their effectiveness showed inconsistent differences between colours. The quality of the methods of these trials was variable. Hypnotic, sedative, and anxiolytic drugs were more likely than antidepressants to be green, blue, or purple. CONCLUSIONS: Colours affect the perceived action of a drug and seem to influence the effectiveness of a drug. Moreover, a relation exists between the colouring of drugs that affect the central nervous system and the indications for which they are used. Research contributing to a better understanding of the effect of the colour of drugs is warranted.     

In situ biochemical demonstration that P-glycoprotein is a drug efflux pump with broad specificity

While P-glycoprotein (Pgp) is the most studied protein involved in resistance to anti-cancer drugs, its mechanism of action is still under debate. Studies of Pgp have used cell lines selected with chemotherapeutics which may have developed many mechanisms of resistance. To eliminate the confounding effects of drug selection on understanding the action of Pgp, we studied cells transiently transfected with a Pgp-green fluorescent protein (GFP) fusion protein. This method generated a mixed population of unselected cells with a wide range of Pgp-GFP expression levels and allowed simultaneous measurements of Pgp level and drug accumulation in living cells. The results showed that Pgp-GFP expression was inversely related to the accumulation of chemotherapeutic drugs. The reduction in drug concentration was reversed by agents that block multiple drug resistance (MDR) and by the UIC2 anti-Pgp antibody. Quantitative analysis revealed an inverse linear relationship between the fluorescence of Pgp-GFP and MDR dyes. This suggests that Pgp levels alone limit drug accumulation by active efflux; cooperativity between enzyme, substrate, or inhibitor molecules is not required. Additionally, Pgp-GFP expression did not change cellular pH. Our study demonstrates the value of using GFP fusion proteins for quantitative biochemistry in living cells.     

A two-drug model for etoposide action against human topoisomerase IIalpha

The widely used anticancer drug etoposide kills cells by increasing levels of topoisomerase II-mediated DNA breaks. While it is known that the drug acts by inhibiting the ability of topoisomerase II to ligate cleaved DNA molecules, the precise mechanism by which it accomplishes this action is not well understood. Because there are two scissile bonds per enzyme-mediated double-stranded DNA break, it has been assumed that there are two sites for etoposide in every cleavage complex. However, it is not known whether the action of etoposide at only one scissile bond is sufficient to stabilize a double-stranded DNA break or whether both drug sites need to be occupied. An oligonucleotide system was utilized to address this important issue. Results of DNA cleavage and ligation assays support a two-drug model for the action of etoposide against human topoisomerase IIalpha. This model postulates that drug interactions at both scissile bonds are required in order to increase enzyme-mediated double-stranded DNA breaks. Etoposide actions at either of the two scissile bonds appear to be independent of one another, with each individual drug molecule stabilizing a strand-specific nick rather than a double-stranded DNA break. This finding suggests (at least in the presence of drug) that there is little or no communication between the two promoter active sites of topoisomerase II. The two-drug model has implications for cancer chemotherapy, the cellular processing of etoposide-stabilized enzyme-DNA cleavage complexes, and the catalytic mechanism of eukaryotic topoisomerase II.     

Evaluation of 3,6-dibutanoylmorphine as an analgesic in vivo: comparison with morphine and 3,6-diacetylmorphine

Dibutanoylmorphine (DBM), a synthetic diester of morphine, was compared with morphine (M) and diacetylmorphine (DAM) for analgesic efficacy, potency and duration of action following I.V. administration in rats. Analgesia was assessed in groups of eight animals using both tail-flick and hot-plate testing methods following random administration of five different doses of each drug. DBM was found to be substantially more potent than M, but less potent than DAM in both tail-Flick and hot-plate tests of nociception. Similarly, assessment of duration of action at the ED50 for each drug revealed that DBM has a duration of analgesia which is intermediate between the durations of M and DAM. Thus, in rats in vivo, DBM is an effective analgesic and has a reasonable duration of action release to other opioids.     

Measuring drug-related receptor occupancy with positron emission tomography

Several techniques can be used to measure indirectly the effect of drugs (e.g., EEG, fMRI) in healthy volunteers and in patients. Although each technique has its merits, a direct link between drug efficacy and site of action in vivo usually cannot be established. In addition, when the specific mode of action of a drug has been determined from preclinical studies, it is often not known whether the administered dose is optimal for humans. Both industry and academia are becoming more and more interested in determining the dose-related occupancy of specific targets caused by administration of drugs under test. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are noninvasive imaging techniques that can give insight into the relationship between target occupancy and drug efficacy, provided a suitable radioligand is available. Although SPECT has certain advantages (e.g., a long half-life of the radionuclides), the spatial and temporal resolution as well as the labeling possibilities of this technique are limited. This review focuses on PET methodology for conducting drug occupancy studies in humans.     

New targets for an old drug

Methotrexate has been a clinical agent used in cancer, immunosuppression, rheumatoid arthritis and other highly proliferative diseases for many years, yet its underlying molecular mechanism of action in these therapeutic areas is still unclear. We present a chemical proteomics approach that uses ultra-sensitive mass spectrometry coupled to an inverse protein-ligand docking computational technique to unravel the mechanism of action of this drug. Using methotrexate tethered to a solid support we were able to isolate a signficant number of proteins. We effectively captured a large portion of the de novo purine metaolome and propose a pathway architecture similar to that seen in signaling pathways and consistent with substrate channeling. More importantly, we were able to capture protein targets that could potentially provide new insights into the mechanism of action of methotrexate in rheumatoid arthritis and immunosuppression. The application of this approach to other drugs and drug candidates may facilitate the prediction of unknown and secondary therapeutic target proteins and those related to the side effects and toxicity. These results demonstrate that this proteomics technology could play an important role in drug discovery and development since it allows monitoring of the interactions between a drug and the protein content of a cell.     

Interaction of thiostrepton with an RNA fragment derived from the plastid-encoded ribosomal RNA of the malaria parasite.

Although eukaryotes are not generally sensitive to thiostrepton, growth of the human malaria parasite Plasmodium falciparum is severely inhibited by the drug. The proposed target in P. falciparum is the ribosome of the plastid-like organelle (35 kb circular genome) of unknown function. Positive identification of the drug target would confirm that the organelle is essential for blood-stage development of Plasmodium and help clarify the plastid's biological role. The action of thiostrepton as an antibiotic relates to its affinity for a conserved domain of eubacterial rRNA. Its effect on organelles is unknown. Because a number of different point mutations within the Escherichia coli domain abrogates thiostrepton binding, extensive sequence differences between eubacterial and plastid domains brings into question the site of drug action. We have examined temperature-dependent hyperchromicity profiles of synthetic RNAs corresponding to domains in the plastid and cytoplasmic RNAs of P. falciparum. Thiostrepton induces a tertiary structure in the plastid-like fragment similar to that seen in eubacterial rRNA, even though the two share only about 60% sequence identity. A single point mutation in the plastid-like fragment removes thiostrepton-dependent tertiary structure formation. Thus, the plastid and eubacterial RNAs share a stabilized tertiary structure induced by the drug. This direct indicator of drug sensitivity in eubacteria suggests that the plastid-encoded ribosome is similarly sensitive to thiostrepton and that the plastid is the site of drug action. Correlation of thiostrepton-sensitive and -resistant phenotypes with physical parameters suggests thiostrepton resistance as a selectable marker for plastid transformation.     

Ultrastructural disorders of the mitotic apparatus during cytochalasin B action

A correlation between the number of chromosome sets and the number of centrioles (8n--8 centrioles) was observed in polyploid metaphase cells, during cytochalasin B treatment on the cultured Chinese hamster cells. There is no correlation between the number of chromosome sets and the centriole number after stopping the action of the drug in many cells, but a great variation is observed in maintenance of chromosomes and centrioles (up 6 to 25 n and up 4 to 22 centrioles). In multipolar mitosis, either during the drug action or after its stopping, different numbers of chromosomes are directed towards the poles not depending on the number of centrioles in the poles. During the cytochalasin B treatment, either in bipolar or multipolar metaphases, there are destructions in the ultrastructure of the mitotic apparatus: there are no astral microtubules; in the poles there are diplosomes and duplex of centrioles with fibrillar material around both centrioles; kinetochores are of prometaphase type. After stopping the drug action the astral microtubules appear, but no other patterns of normalization in the mitotic apparatus occur. Desynchronization of three cycles (chromosomal, centriolar and centrosomal) is discussed as a factor of abnormal development of the mitotic apparatus and as a factor of stabilization of aneuploidy in the cell culture.     

Analysis of bisdioxopiperazine dexrazoxane binding to human DNA topoisomerase II alpha: decreased binding as a mechanism of drug resistance

Topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double stranded DNA segment and transporting it through another DNA molecule. Despite the extensive use of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, much remains unknown concerning the interactions between these agents and topoisomerase II. To identify the interaction of the bisdioxopiperazine dexrazoxane (ICRF-187) with topoisomerase II, we developed a rapid gel-filtration assay and characterized the binding of ((3)H)-dexrazoxane to human topoisomerase II alpha. Dexrazoxane binds to human topoisomerase II alpha in the presence of DNA and ATP with an apparent K(d) of 23 microM and a stoichiometry of 1 drug molecule per enzyme dimer. Various N-terminal single amino acid substitutions in human topoisomerase II alpha that were previously shown to confer specific bisdioxopiperazine resistance either totally abolished drug binding or resulted in less efficient binding. The effect of the various mutations on drug binding correlated well with their effect on drug resistance in vivo and in vitro. Interestingly, an altered active site tyrosine mutant of human topoisomerase II alpha, which is incapable of carrying out DNA strand passage, was unable to bind dexrazoxane, which agrees with the drug's proposed mechanism of action late in the topoisomerase II catalytic cycle. The direct correlation between the level of drug binding and dexrazoxane resistance is consistent with a decreased drug binding mechanism of action for these dexrazoxane resistance conferring mutations.     

Population based models of cortical drug response: insights from anaesthesia

A great explanatory gap lies between the molecular pharmacology of psychoactive agents and the neurophysiological changes they induce, as recorded by neuroimaging modalities. Causally relating the cellular actions of psychoactive compounds to their influence on population activity is experimentally challenging. Recent developments in the dynamical modelling of neural tissue have attempted to span this explanatory gap between microscopic targets and their macroscopic neurophysiological effects via a range of biologically plausible dynamical models of cortical tissue. Such theoretical models allow exploration of neural dynamics, in particular their modification by drug action. The ability to theoretically bridge scales is due to a biologically plausible averaging of cortical tissue properties. In the resulting macroscopic neural field, individual neurons need not be explicitly represented (as in neural networks). The following paper aims to provide a non-technical introduction to the mean field population modelling of drug action and its recent successes in modelling anaesthesia.     

Quinolones inhibit DNA religation mediated by Staphylococcus aureus topoisomerase IV. Changes in drug mechanism across evolutionary boundaries

Quinolones are the most active oral antibacterials in clinical use and act by increasing DNA cleavage mediated by prokaryotic type II topoisomerases. Although topoisomerase IV appears to be the primary cytotoxic target for most quinolones in Gram-positive bacteria, interactions between the enzyme and these drugs are poorly understood. Therefore, the effects of ciprofloxacin on the DNA cleavage and religation reactions of Staphylococcus aureus topoisomerase IV were characterized. Ciprofloxacin doubled DNA scission at 150 nM drug and increased cleavage approximately 9-fold at 5 microM. Furthermore, it dramatically inhibited rates of DNA religation mediated by S. aureus topoisomerase IV. This inhibition of religation is in marked contrast to the effects of antineoplastic quinolones on eukaryotic topoisomerase II, and suggests that the mechanistic basis for quinolone action against type II topoisomerases has not been maintained across evolutionary boundaries. The apparent change in quinolone mechanism was not caused by an overt difference in the drug interaction domain on topoisomerase IV. Therefore, we propose that the mechanistic basis for quinolone action is regulated by subtle changes in drug orientation within the enzyme.drug.DNA ternary complex rather than gross differences in the site of drug binding.     

Sulphasalazine in rheumatoid arthritis.

Seventy-four patients with rheumatoid arthritis were treated with sulphasalazine. There was a significant improvement in clinical score, with substantial falls in serum C-reactive protein concentrations and erythrocyte sedimentation rate four weeks after starting the drug. Improvement was maintained in the 38 patients who remained on the durg for one year. The mean Rose-Waaler titre did not change. There was little difference between the results in seropositive and seronegative patients. The commonest adverse effect was dyspepsia, but five patients developed a megaloblastic anaemia and one patient neutropenia; all made a complete recovery. The results suggest that the drug has a disease-modifying action not attributable to its "salicylate" content. The mode of action might be by an antibacterial effect on gut flora.     

Insight into the strong inhibitory action of salt on activity of neocarzinostatin

Enediyne anticancer drugs belong to one of the most potent category in inducing DNA damage. We report 85 ± 5% inhibition on activity of neocarzinostatin by salt. As high sodium ion concentration is a known tumor cell feature, we explored the dynamic mechanism of inhibition. Using various analytical tools, we examined parameters involved in the four consecutive steps of the drug action, namely, drug releasing from carrier protein, drug–DNA binding, drug activating, and DNA damaging. Neither protein stability, nor drug release rate, was altered by salt. The salt inhibition level was similar in between the protein-bound and unbound enediyne chromophore. Salt did not quench the thiol-induced drug activation. The inhibition was independent of DNA lesion types and irrelevant with thiol structures. Collectively, no salt interaction was found in the releasing, activating, and DNA damaging step of the drug action. However, binding with DNA decreased linearly with salt and corresponded well with the salt-induced inhibition on the drug activity. Salt interference on the affinity of DNA binding was the main and sole cause of the severe salt inhibition. The inhibition factor should be carefully considered for all agents with similar DNA binding mode.     

Progesterone attenuates impulsive action in a Go/No-Go task for sucrose pellets in female and male rats

Impulsivity, or a tendency to act without anticipation of future consequences, is associated with drug abuse. Impulsivity is typically separated into two main measures, impulsive action and impulsive choice. Given the association of impulsivity and drug abuse, treatments that reduce impulsivity have been proposed as an effective method for countering drug addiction. Progesterone has emerged as a promising treatment, as it is associated with decreased addiction-related behaviors and impulsive action. The goal of the present study was to determine the effects of progesterone (PRO) on impulsive action for food: a Go/No-Go task. Female and male rats responded for sucrose pellets during a Go component when lever pressing was reinforced on a variable-interval 30-s schedule. During the alternate No-Go component, withholding a lever press was reinforced on a differential reinforcement of other (DRO) behavior 30-s schedule, where a lever press reset the DRO timer. Impulsive action was operationally defined as the inability to withhold a response during the No-Go component (i.e. the number of DRO resets). Once Go/No-Go behavior was stable, responding between rats treated with PRO (0.5 mg/kg) or vehicle was examined. Progesterone significantly decreased the total number of DRO resets in both males and females, but it did not affect VI responding for sucrose pellets. This suggests that PRO decreases motor impulsivity for sucrose pellets without affecting motivation for food. Thus, PRO may reduce motor impulsivity, a behavior underlying drug addiction.     

Kinetic analysis of drug-induced G2 block in vitro

The data on cell-cycle effects of two prospective antitumour agents, (+)-1,2,-bis(3,5-dioxopiperazine-1-yl)propane (soluble ICRF; NSC 169780) and 1,4-bis(2'chloroethyl)-1,4-diazabicyclo [2.2.1] heptane diperchlorate (CBH; NSC 57198) were used to determine whether a modified stathmokinetic experiment could predict the effects of continuous, long-term (0-48 hr) drug exposure in an in vitro L1210 murine leukaemia cell system. Generally, continuous drug exposure of exponentially growing cells does not provide sufficient quantitative information concerning cell-cycle-phase-specific mechanisms of drug action. Alternatively, stathmokinetic experiments, which are usually limited to some fraction of one cell doubling time, provide little information about long-term drug effects. By using mathematical models constructed for this purpose, however, stathmokinetic data can predict the overall proportion of cells affected by a drug though failing to discern between various kinds of drug action (e.g. reversible v. irreversible block, blocking v. killing action, etc.), especially when it occurs in G2 phase. In addition, it can be shown that for at least one of the drugs (soluble ICRF) the stathmokinetic experiment fails to predict 'after-effects' of drug treatment which extend into the following cell cycle(s). It also becomes clear that the degradation of exponential growth characteristics of quickly dividing cells during long-term, continuous drug exposure makes prediction of cell-cycle kinetic perturbations uncertain when derived from short-duration stathmokinetic experiments. However, with care, the joint application of 'short term' (e.g. stathmokinesis) and 'long term' (e.g. continuous exposure) techniques allow adequate quantitative insight into drug-perturbed cell-cycle kinetics. The applicability of modelling techniques is discussed: in the present instance it is limited to lower drug concentrations. For higher drug concentrations, effects like increased ploidy, ineffective division, etc., make it impossible in the present study to obtain a clear picture of the kinetics.     

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.