Jon C. Mirsalis

Scaleable manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component

To prevent sexually transmitted HIV, the most desirable active ingredients of microbicides are antiretrovirals (ARVs) that directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals, which are costly to manufacture and deliver to resource-poor areas where effective microbicides are urgently needed. Here, we report a manufacturing breakthrough for griffithsin (GRFT), one of the most potent HIV entry inhibitors. This red algal protein was produced in multigram quantities after extraction from Nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing GRFT. Plant-produced GRFT (GRFT-P) was shown as active against HIV at picomolar concentrations, directly virucidal via binding to HIV envelope glycoproteins, and capable of blocking cell-to-cell HIV transmission. GRFT-P has broad-spectrum activity against HIV clades A, B, and C, with utility as a microbicide component for HIV prevention in established epidemics in sub-Saharan Africa, South Asia, China, and the industrialized West. Cognizant of the imperative that microbicides not induce epithelial damage or inflammatory responses, we also show that GRFT-P is nonirritating and noninflammatory in human cervical explants and in vivo in the rabbit vaginal irritation model. Moreover, GRFT-P is potently active in preventing infection of cervical explants by HIV-1 and has no mitogenic activity on cultured human lymphocytes.

A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria

The antimalarial drug DSM265 displays activity against blood and liver stages of Plasmodium falciparum and has a long predicted half-life in humans. Long-acting new treatment for drug-resistant malaria Malaria kills 0.6 million people annually, yet current malaria drugs are no longer fully effective because the parasite that causes malaria is becoming resistant to these agents. Phillips et al. have identified a new drug that kills both drug-sensitive and drug-resistant malaria parasites by targeting the ability of the parasite to synthesize the nucleotide precursors required for synthesis of DNA and RNA. This drug kills parasites in both the blood and liver and is sufficiently long-acting that it is expected to cure malaria after a single dose or to be effective if dosed weekly for chemoprevention. Malaria is one of the most significant causes of childhood mortality, but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in combating malaria requires development of new, easy to administer drug combinations with broad-ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective toward DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200 to 400 mg. DSM265 was well tolerated in repeat-dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood- and liver-stage activity, and predicted long half-life in humans position DSM265 as a new potential drug combination partner for either single-dose treatment or once-weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive against the parasite liver stage.

In vivo transgenic mutation assays.

Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage‐based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of ∼3 × 10−5 mutants/locus and 125,000–300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two‐thirds the MTD, and one‐third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose‐response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control. Environ. Mol. Mutagen. 35:253–259, 2000

A protocol and guide for the in vitro rat hepatocyte DNA-repair assay.

The in vitro rat-hepatocyte DNA-repair assay is a valuable tool in assessing the genotoxic activity of chemical agents. An advantage of the assay is that the target cells themselves are metabolically competent, so that the patterns of metabolic activation and detoxification closely reflect those in the whole animal. This article provides a typical procedure and guidelines for conducting the rat in vitro hepatocyte DNA-repair assay.

(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium

Significance Useful antimalarial drugs must be rapidly acting, highly efficacious, and have low potential for developing resistance. (+)-SJ733 targets a Plasmodium cation-transporting ATPase, ATP4. (+)-SJ733 cleared parasites in vivo as quickly as artesunate by specifically inducing eryptosis/senescence in infected, treated erythrocytes. Although in vitro selection of pfatp4 mutants with (+)-SJ733 proceeded with moderate frequency, during in vivo selection of pbatp4 mutants, resistance emerged slowly and produced marginally resistant mutants with poor fitness. In addition, (+)-SJ733 met all other criteria for a clinical candidate, including high oral bioavailability, a high safety margin, and transmission blocking activity. These results demonstrate that targeting ATP4 has great potential to deliver useful drugs for malaria eradication. Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na+ levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na+ homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.

Transgenic Animal Models for Measuring Mutations In Vivo

Transgenic animal models for measuring mutations provide a powerful tool for rapidly assessing tissue-specific mutations following in vivo treatment. These models are based on the insertion into the rodent genome of the Escherichia coli lacI (lac repressor) or lacZ (beta-galactosidase) genes that serve as targets for mutations. Following in vivo treatment of animals, genomic DNA is isolated from various tissues and the target gene is packaged into lambda-phage heads; the lambda-phage are used to infect E. coli in order to produce plaques. Mutations in the target gene are then detected using colorimetric or selective procedures. In this review methods are discussed for producing these transgenic models, the target genes used, gene rescue techniques, sequencing of isolated mutants, and parameters that affect dosing regimens and design of studies. We also present a summary of data published to date with these systems and present our conclusions and proposed directions for future research.

Micronucleated erythrocyte frequency in peripheral blood of B6C3F1 mice from short‐term, prechronic, and chronic studies of the NTP carcinogenesis bioassay program

The mouse peripheral blood micronucleus (MN) test was performed on samples collected from 20 short‐term, 67 subchronic, and 5 chronic toxicity and carcinogenicity studies conducted by the National Toxicology Program (NTP). Data are presented for studies not previously published. Aspects of protocol that distinguish this test from conventional short‐term bone marrow MN tests are duration of exposure, and absence of repeat tests and concurrent positive controls. Furthermore, in contrast to short‐term bone marrow MN tests where scoring is limited to polychromatic erythrocytes (PCE), longer term studies using peripheral blood may evaluate MN in both, or either, the normochromatic (NCE) or PCE populations. The incidence of MN‐PCE provides an index of damage induced within 72 hr of sampling, whereas the incidence of MN in the NCE population at steady state provides an index of average damage during the 30‐day period preceding sampling. The mouse peripheral blood MN test has been proposed as a useful adjunct to rodent toxicity tests and has been effectively incorporated as a routine part of overall toxicity testing by the NTP. Data derived from peripheral blood MN analyses of dosed animals provide a useful indication of the in vivo potential for induced genetic damage and supply an important piece of evidence to be considered in the overall assessment of toxicity and health risk of a particular chemical. Although results indicate that the test has low sensitivity for prediction of carcinogenicity, a convincingly positive result in this assay appears to be highly predictive of rodent carcinogenicity. Environ. Mol. Mutagen. 36:163–194, 2000

Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with dinitrotoluene.

Abstract The purpose of this study was to examine the induction of unscheduled DNA synthesis (UDS) by the potent hepatocarcinogen technical grade dinitrotoluene (tgDNT; 76% 2, 4-DNT, 19% 2, 6-DNT) using the in vivo-in vitro hepatocyte DNA repair assay. Male Fischer-344 rats were treated by gavage and hepatocytes were isolated by liver perfusion and cultured with [ 3 H]thymidine. UDS was measured by quantitative autoradiography as net grains/nucleus (NG); ≥5 NG was considered positive. Controls consistently had − 3 to − 6 NG. A dose-related increase in UDS was observed 12 h after treatment, with 200 mg/kg tgDNT producing 26 NG. A 50-fold increase in the number of cells in S-phase was observed at 48 h after treatment. This increase in S-phase cells could be suppressed in the presence of 10–20 mM hydroxyurea (HU), while the same levels of HU did not affect the level of UDS at 12 h after treatment. 2, 4-DNT produced only a weak response, in contrast to 2, 6-DNT which was a potent inducer of UDS. Treatment of female rats with tgDNT yielded only modest increases in UDS and DNA replication relative to males. These results are consistent with the carcinogenicity studies and indicate that tgDNT is a potent genotoxic agent, with 2, 6-DNT contributing the major portion of the effect.

Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice.

Ionizing radiation induces gene mutations (point mutations, deletions and insertions) as well as chromosome damage in mammalian cells. Although these effects have been studied extensively in cells in culture, until recently it has not been possible to analyze the mutagenic potential of ionizing radiation in vivo, especially at the molecular level. The development of transgenic mutagenesis systems has now made it possible to study the effects of ionizing radiation at both the molecular and chromosomal levels in the same animal. In this report we present preliminary data on the response of Big Blue lacI transgenic mice to ionizing radiation as measured by lacI mutations and micronuclei. C57Bl/6 transgenic mice were irradiated with 137Cs gamma-rays at doses ranging from 0.1 to 14 Gy, and expression times ranging from 2 to 14 days. Dose-related increases in the mutant frequency were observed after irradiations with longer expression times. Mutant plaques were analyzed by restriction enzyme digestion to detect large structural changes in the target sequence. Of 34 gamma-ray-induced mutations analyzed, 4 were large-scale rearrangements. 3 of these rearrangements were deletions within the lacI gene characterized by the presence of short regions of homology at the breakpoint junctions. The fourth rearrangement was a deletion that extended from within the alpha lacZ gene into downstream sequences and that had 43 bp of homology at the junction. These data indicate that the Big Blue lacI transgenic mouse system in sensitive to the types of mutations induced by ionizing radiation. To determine whether the presence of the transgene affects micronucleus induction we compared the response of nontransgenic to hemizygous transgenic B6C3F1 mice and the response of nontransgenic to hemizygous and homozygous transgenic C57Bl/6 mice. The presence or absence of the lacI transgene had no effect on spontaneous micronucleus frequencies for either strain. However, radiation-induced micronucleus frequencies were significantly higher in hemizygous lacI B6C3F1 mice than in nontransgenic litter mates; the converse was true in C57Bl/6 mice. These data suggest that the lacI transgene does not cause chromosome instability as measured by spontaneous micronucleus levels. However, the response of these transgenic mice to a variety of clastogenic agents needs to be investigated before they are integrated into standard in vivo assays for chromosome damage.

Gene Expression Analysis Reveals New Possible Mechanisms of Vancomycin-Induced Nephrotoxicity and Identifies Gene Markers Candidates

Vancomycin, one of few effective treatments against methicillin-resistant Staphylococcus aureus, is nephrotoxic. The goals of this study were to (1) gain insights into molecular mechanisms of nephrotoxicity at the genomic level, (2) evaluate gene markers of vancomycin-induced kidney injury, and (3) compare gene expression responses after iv and ip administration. Groups of six female BALB/c mice were treated with seven daily iv or ip doses of vancomycin (50, 200, and 400 mg/kg) or saline, and sacrificed on day 8. Clinical chemistry and histopathology demonstrated kidney injury at 400 mg/kg only. Hierarchical clustering analysis revealed that kidney gene expression profiles of all mice treated at 400 mg/kg clustered with those of mice administered 200 mg/kg iv. Transcriptional profiling might thus be more sensitive than current clinical markers for detecting kidney damage, though the profiles can differ with the route of administration. Analysis of transcripts whose expression was changed by at least twofold compared with vehicle saline after high iv and ip doses of vancomycin suggested the possibility of oxidative stress and mitochondrial damage in vancomycin-induced toxicity. In addition, our data showed changes in expression of several transcripts from the complement and inflammatory pathways. Such expression changes were confirmed by relative real-time reverse transcription–polymerase chain reaction. Finally, our results further substantiate the use of gene markers of kidney toxicity such as KIM-1/Havcr1, as indicators of renal injury.