DNA topoisomerases as targets for antiprotozoal therapy.

Diseases caused by parasitic protozoa present a health problem of immense magnitude, and there is an urgent need for safe and effective new therapies. DNA topoisomerases are clinically relevant targets for anti-cancer and anti-bacterial agents. Inhibitor studies on parasite topoisomerases have revealed that these enzymes have great promise as molecular targets for anti-parasitics, and have helped to dissect the basic biology of DNA topoisomerases in these organisms. This review provides a brief introduction to DNA topoisomerases and anti-topoisomerase drugs, and an overview of studies on protozoal DNA topoisomerases and their inhibitors.

Immune mimicry in malaria: Plasmodium falciparum secretes a functional histamine-releasing factor homolog in vitro and in vivo.

The Plasmodium falciparum translationally controlled tumor protein (TCTP) is a homolog of the mammalian histamine-releasing factor (HRF), which causes histamine release from human basophils and IL-8 secretion from eosinophils. Histamine, IL-8, and eosinophils have been reported to be elevated in patients with malaria. This study was undertaken to determine whether malarial TCTP is found in the plasma of malaria-infected patients and to determine whether it has HRF biologic activity. Malarial TCTP was found in lightly infected human volunteers and in heavily infected Malawian children, but not in uninfected patients. Recombinant malarial TCTP, like HRF, stimulated histamine release from basophils and IL-8 secretion from eosinophils in vitro. Whereas malarial TCTP was less active than HRF, the concentrations that were effective in vitro could be achievable in vivo. These data suggest that malarial TCTP, present in human plasma during a malarial illness, may affect host immune responses in vivo.

Antimalarial simplified 3-aryltrioxanes: synthesis and preclinical efficacy/toxicity testing in rodents.

A streamlined five-step chemical synthesis of rationally designed, simplified 3-aryltrioxane 8a is described. A noteworthy feature of this synthetic scheme is use of air rather than expensive molecular oxygen as the source of the pharmacologically critical peroxide unit in trioxane 8a. This simplified acetal trioxane carboxylic acid 8a is thermally stable, and it is hydrolytically stable in water even at 40 degrees C and pH 7.4 for at least 7 days. Preclinical evaluation of this water-soluble synthetic trioxane 8a in rodents shows it to have at least as good a therapeutic index (efficacy/toxicity) as that of water-soluble semisynthetic trioxane artelinic acid (5).

Chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in humans.

Broccoli sprouts are a rich source of glucosinolates and isothiocyanates that induce phase 2 detoxication enzymes, boost antioxidant status, and protect animals against chemically induced cancer. Glucosinolates are hydrolyzed by myrosinase (an enzyme found in plants and bowel microflora) to form isothiocyanates. In vivo, isothiocyanates are conjugated with glutathione and then sequentially metabolized to mercapturic acids. These metabolites are collectively designated dithiocarbamates. We studied the disposition of broccoli sprout glucosinolates and isothiocyanates in healthy volunteers. Broccoli sprouts were grown, processed, and analyzed for (a) inducer potency; (b) glucosinolate and isothiocyanate concentrations; (c) glucosinolate profiles; and (d) myrosinase activity. Dosing preparations included uncooked fresh sprouts (with active myrosinase) as well as homogenates of boiled sprouts that were devoid of myrosinase activity and contained either glucosinolates only or isothiocyanates only. In a crossover study, urinary dithiocarbamate excretion increased sharply after administration of broccoli sprout glucosinolates or isothiocyanates. Cumulative excretion of dithiocarbamates following 111-micromol doses of isothiocyanates was greater than that after glucosinolates (88.9 +/- 5.5 and 13.1 +/- 1.9 micromol, respectively; P < 0.0003). In subjects fed four repeated 50-micromol doses of isothiocyanates, the intra- and intersubject variation in dithiocarbamate excretion was very small (coefficient of variation, 9%), and after escalating doses, excretion was linear over a 25- to 200-micromol dose range. Dithiocarbamate excretion was higher when intact sprouts were chewed thoroughly rather than swallowed whole (42.4 +/- 7.5 and 28.8 +/- 2.6 micromol; P = 0.049). These studies indicate that isothiocyanates are about six times more bioavailable than glucosinolates, which must first be hydrolyzed. Thorough chewing of fresh sprouts exposes the glucosinolates to plant myrosinase and significantly increases dithiocarbamate excretion. These findings will assist in the design of dosing regimens for clinical studies of broccoli sprout efficacy.

Antimalarial sulfide, sulfone, and sulfonamide trioxanes.

A series of trioxanes featuring sulfide, sulfone, and sulfonamide substituents in diverse positions has been prepared. Structure-activity relationship (SAR) generalizations highlight two major factors controlling the antimalarial potency of these new chemical entities: (1) the proximity of the sulfur-containing substituent to the crucial peroxide bond and (2) the oxidation state of the sulfur-containing substituent. Generally, sulfones are more antimalarially potent than the corresponding sulfides.

Antimalarial, antiproliferative, and antitumor activities of artemisinin-derived, chemically robust, trioxane dimers.

Nine C-10 non-acetal derivatives of the natural trioxane artemisinin (1) were prepared as dimers using some novel chemistry. As designed, each dimer was stable chemically. C-10 Olefinic dimers 7 and C-10 saturated dimers 8-13 all showed good to excellent antimalarial and antiproliferative activities in vitro. Dimers 8, 10, and 12 were especially potent and selective at inhibiting growth of some human cancer cell lines in the NCI in vitro 60-cell line assay.

Antitrypanosomal activity of fluoroquinolones.

Six fluoroquinolones presently in clinical use and four investigational tetracyclic fluoroquinolones were tested for in vitro activity against bloodstream-form Trypanosoma brucei brucei. All compounds had measurable activity, but the tetracyclic analogs were most potent, with 50% effective concentrations in the low micromolar range. In general, trypanosomes were more susceptible than L1210 leukemia cells. Consistent with the notion that they target type II topoisomerase in trypanosomes, the fluoroquinolones promote the formation of protein-DNA covalent complexes.

Prophylactic activity of atovaquone against Plasmodium falciparum in humans.

The prophylactic antimalarial activity of atovaquone was determined in a randomized, double-blind, placebo-controlled study of healthy volunteers who were challenged by the bite of Plasmodium falciparum-infected Anopheles stephensi. Subjects were randomly assigned to one of three groups: six received seven daily doses of 750 mg of atovaquone, starting the day before challenge; six received a single dose of 250 mg of atovaquone the day before challenge; and four received placebo. Polymerase chain reaction- and culture-confirmed parasitemia developed in all four placebo recipients, but in none of the drug recipients, indicating that either of the atovaquone regimens provides effective prophylaxis (P = 0.005). However, in low-dose recipients, the drug levels by day 6.5 were profoundly subtherapeutic, indicating that parasites were eliminated prior to the establishment of erythrocytic infection. Atovaquone thus protects non-immune subjects against mosquito-transmitted falciparum malaria, and has causal prophylactic activity.

Orally active, hydrolytically stable, semisynthetic, antimalarial trioxanes in the artemisinin family.

In only three chemical operations, natural trioxane lactone artemisinin (1) was converted into a series of C-10 carbon-substituted 10-deoxoartemisinin compounds 4-9. The three steps involved lactone reduction, replacement of the anomeric lactol OH by F using diethylaminosulfur trifluoride, and finally boron trifluoride-promoted substitution of F by aryl, heteroaryl, and acetylide nucleophiles. All of these C-10 nonacetal, chemically robust, enantiomerically pure compounds 4-9 have high antimalarial potencies in vitro against Plasmodium falciparum malaria parasites, and furans 5a and 5b and pyrrole 7a are antimalarially potent also in vivo even when administered to rodents orally.

Atovaquone and proguanil hydrochloride for prophylaxis of malaria.

BACKGROUND: The spread of drug-resistant malaria and appreciation of side effects associated with existing antimalarial drugs emphasize the need for new drugs to prevent malaria. The combination of atovaquone and proguanil hydrochloride was previously shown to be safe and highly effective for treatment of malaria, including multi-drug-resistant Plasmodium falciparum. METHODS: We reviewed results of clinical trials that evaluated either a fixed-dose combination of atovaquone and proguanil hydrochloride for malaria prophylaxis or atovaquone alone for causal prophylactic activity against P. falciparum. RESULTS: In three placebo-controlled trials, 331 subjects received 250 mg atovaquone and 100 mg proguanil hydrochloride (or an equivalent dose based on body weight in children) once daily for 10 to 12 weeks. The overall efficacy for preventing parasitemia was 98%. Among 175 nonimmune volunteers taking the same dose of atovaquone/proguanil once daily for 10 weeks while temporarily residing in a malaria-endemic area, malaria developed in one patient who was noncompliant with therapy. Results of volunteer challenge studies indicate that both atovaquone and proguanil have causal prophylactic activity directed against the liver stages of P. falciparum. Adverse events occurred with similar or lower frequencies in subjects treated with atovaquone/proguanil compared to placebo. Less than 1% of patients discontinued from these studies due to a treatment-related adverse event. CONCLUSION: A fixed-dose combination of atovaquone and proguanil hydrocloride is a promising new alternative for malaria prophylaxis.

Human metabolism and excretion of cancer chemoprotective glucosinolates and isothiocyanates of cruciferous vegetables.

Isothiocyanates and their naturally occurring glucosinolate precursors are widely consumed as part of a diet rich in cruciferous vegetables. When plant cells are damaged, glucosinolates are released and converted to isothiocyanates by the enzyme myrosinase. Many isothiocyanates inhibit the neoplastic effects of various carcinogens at a number of organ sites. Consequently, these agents are attracting attention as potential chemoprotectors against cancer. As a prerequisite to understanding the mechanism of the protective effects of these compounds, which is thought to involve the modulation of carcinogen metabolism by the induction of phase 2 detoxication enzymes and the inhibition of phase 1 carcinogen-activating enzymes, we examined the fate of ingested isothiocyanates and glucosinolates in humans. Recently developed novel methods for quantifying isothiocyanates (and glucosinolates after their quantitative conversion to isothiocyanates by purified myrosinase) and their urinary metabolites (largely dithiocarbamates) have made possible a detailed examination of the fates of isothiocyanates and glucosinolates of dietary crucifers. In a series of studies in normal volunteers, we made these findings. First, in nonsmokers, urinary dithiocarbamates were detected only after the consumption of cruciferous vegetables and condiments rich in isothiocyanates and/or glucosinolates. In sharp contrast, the consumption of noncrucifers (corn, tomatoes, green beans, and carrots) did not lead to the excretion of dithiocarbamates. Moreover, the quantities of dithiocarbamates excreted were related to the glucosinolate/isothiocyanate profiles of the cruciferous vegetables administered (kale, broccoli, green cabbage, and turnip roots). Second, eating prepared horseradish containing graded doses of isothiocyanates (12.3-74 micromol; mostly allyl isothiocyanate) led to a rapid excretion of proportionate amounts (42-44%) of urinary dithiocarbamates with first-order kinetics. The ingestion of broccoli in which myrosinase had been heat-inactivated also led to proportionate but low (10-20%) recoveries of urinary dithiocarbamates. Broccoli samples subsequently treated with myrosinase to produce the cognate isothiocyanates were much more completely (47%) converted to dithiocarbamates. Finally, when bowel microflora were reduced by mechanical cleansing and antibiotics, the conversion of glucosinolates became negligible. These results establish that humans convert substantial amounts of isothiocyanates and glucosinolates to urinary dithiocarbamates that can be easily quantified, thus paving the way for meaningful studies of phase 2 enzyme induction in humans.

DNA topoisomerases: a new twist for antiparasitic chemotherapy?

The parasitic protozoa are notorious for their bizarre cellular structures and metabolic pathways, a characteristic also true for their nucleic acids. Despite these florid differences from mammalian cells, however, it has proven surprisingly difficult to devise novel chemotherapy against these pathogens. In recent years, the DNA topoisomerases from parasites have been the focus of considerable study, not only because they are intrinsically interesting, but also because they may provide a target for much-needed new antiparasitic chemotherapy.

Antimalarial cyclic peroxy ketals.

Over 20 new, cyclic, peroxy ketals have been prepared via a two-step protocol starting with readily available aryl methyl ketones. Structure-activity correlations using in vitro antimalarial data as a guide for optimization of potency have led to the design and synthesis of seven new peroxides that have IC50 values of 31-85 nM (artemisinin IC50 = 8.4 nM). Some SAR generalizations are discussed.

Orally active antimalarial 3-substituted trioxanes: new synthetic methodology and biological evaluation.

On the basis of a mechanistic understanding of the mode of action of artemisinin-like antimalarials, a series of structurally simple 3-aryl-1,2,4-trioxanes 5 was designed and was prepared in three to five operations from commercial reactants. The 3-aryl group was attached in each case as a nucleophile. In an electronically complementary fashion, 3-(fluoroalkyl)-trioxanes 6 were prepared via attachment of electrophilic fluoroalkyl esters. Both in vitro and in vivo antimalarial evaluations of these new trioxanes showed 12 beta-methoxy-3-aryltrioxanes 5g, 5j, 5k, and 51 to be highly potent, with crystalline fluorobenzyl ether trioxane 5k especially potent even when administered to rodents orally. As shown by rearrangement of hexamethyl Dewar benzene into hexamethylbenzene, iron-induced degradation of some of these 3-aryltrioxanes 5 involves generation of high-valent iron oxo species that might kill malaria parasites.

Design, synthesis, derivatization, and structure-activity relationships of simplified, tricyclic, 1,2,4-trioxane alcohol analogues of the antimalarial artemisinin.

Novel C4-(hydroxyalkyl)trioxanes 5d and 5e were designed and synthesized based on an understanding of the molecular mechanism of action of similar 1,2,4-trioxanes structurally related to the antimalarial natural product artemisinin (1). In vitro efficacies of these two new pairs of C4-diastereomers against chloroquine-sensitive Plasmodium falciparum support conclusions about the importance to antimalarial activity of formation of a C4 radical by a 1,5-hydrogen atom abstraction. Derivatives 6, 7, and 21 of C4 beta-substituted trioxane alcohols 4a, 5d, and 5e were prepared, each in a single-step, high-yielding transformation. Four of these new analogues, 6a-c and 7, are potent in vitro antimalarials, having 140 to 50% of the efficacy of the natural trioxane artemisinin (1).

Effects of camptothecin, a topoisomerase I inhibitor, on Plasmodium falciparum.

Currently, the treatment of falciparum malaria is seriously compromised by spreading drug resistance. We studied the effects of camptothecin, a potent and specific topoisomerase I inhibitor, on erythrocytic malaria parasites in vitro. In Plasmodium falciparum, camptothecin trapped protein-DNA complexes, inhibited nucleic acid biosynthesis, and was cytotoxic. These results provide proof for the concept that topoisomerase I is a vulnerable target for new antimalarial drug development.

Synthesis and in vitro antimalarial activity of sulfone endoperoxides.

A series of 4,8-dimethyl-4-phenylsulfonylmethyl-2,3-dioxabicyclo[3.3.1]+ ++nonanes, carrying a variety of substituents at position-8 (4) were prepared by a short and efficient method from R-(+)-limonene. Key reactions include thiol oxygen cooxidation, and alkylation and acylation of a sterically hindered tertiary alcohol compatible with the endoperoxy functionality. Some of compounds 4, which are structurally related to yingzhaosu A (2), were found to exhibit in vitro antimalarial activity comparable to that of artemisinin (1) and superior to that of arteflene (3).

Biomechanical properties of human tibias in long-term spinal cord injury.

Long-term spinal cord injury (SCI) profoundly alters skeletal structure and function. In this study, the biomechanical properties of tibias from persons with SCI and from individuals closely matched in age and size but without SCI were quantified at both the structural and material levels. Nondestructive torsion tests were performed to determine apparent shear moduli for the tibia. The cortical thicknesses and polar moment of inertia were determined numerically. Four-point bending tests were performed to determine flexural modulus of elasticity on cortical bone specimens of the tibia. The apparent shear moduli of the SCI tibias were found to be lower than the non-SCI tibias (p < 0.05). The cortical thicknesses of the SCI tibias were significantly thinner than the control tibias (p < 0.05), while the polar moment of inertia showed no significant differences between control and SCI tibial cross sections (p > 0.05). The flexural modulus of elasticity of the cortical bone specimens were lower in the SCI tibias than the controls (p < 0.05). These differences suggest that tibias may undergo micro-structural changes as well as structural adaptation following SCI, which alter their mechanical properties.

Trioxane dimers have potent antimalarial, antiproliferative and antitumor activities in vitro.

A series of tetracyclic and tricyclic trioxane dimers has been prepared with ether and ester tethers of varying length and flexibility. Several of these trioxane dimers have been found to have potent and potentially therapeutically valuable antimalarial, antiproliferative, and antitumor activities in vitro.