Preclinical studies on the pharmacokinetics, safety, and toxicology of oxfendazole: toward first in human studies.

A 2-week study in rats identified target organs of oxfendazole toxicity to be bone marrow, epididymis, liver, spleen, testis, and thymus. Female rats had greater oxfendazole exposure and exhibited toxicities at lower doses than did males. Decreased white blood cell levels, a class effect of benzimidazole anthelmintics, returned to normal during the recovery period. The no observed adverse effect level was determined to be >5 but <25 mg/kg/d and the maximum tolerated dose 100 mg/kg/d. The highest dose, 200 mg/kg/d, resulted in significant toxicity and mortality, leading to euthanization of the main study animals in this group after 7 days. Oxfendazole did not exhibit genetic toxicology signals in standard Ames bacterial, mouse lymphoma, or rat micronucleus assays nor did it provoke safety concerns when evaluated for behavioral effects in rats or cardiovascular safety effects in dogs. These results support the transition of oxfendazole to First in Human safety studies preliminary to its evaluation in human helminth diseases.

Genotoxicity of the cancer chemopreventive drug candidates CP-31398, SHetA2, and phospho-ibuprofen.

The genotoxic activities of three cancer chemopreventive drug candidates, CP-31398 (a cell permeable styrylquinazoline p53 modulator), SHetA2 (a flexible heteroarotinoid), and phospho-ibuprofen (PI, a derivative of ibuprofen) were tested. None of the compounds were mutagenic in the Salmonella/Escherichia coli/microsome plate incorporation test. CP-31398 and SHetA2 did not induce chromosomal aberrations (CA) in Chinese hamster ovary (CHO) cells, either in the presence or absence of rat hepatic S9 (S9). PI induced CA in CHO cells, but only in the presence of S9. PI, its parent compound ibuprofen, and its moiety diethoxyphosphoryloxybutyl alcohol (DEPBA) were tested for CA and micronuclei (MN) in CHO cells in the presence of S9. PI induced CA as well as MN, both kinetochore-positive (Kin+) and -negative (Kin-), in the presence of S9 at ≤100µg/ml. Ibuprofen was negative for CA, positive for MN with Kin+ at 250µg/ml, and positive for MN with Kin- at 125 and 250µg/ml. DEPBA induced neither CA nor MN at ≤5000µg/ml. The induction of chromosomal damage in PI-treated CHO cells in the presence of S9 may be due to its metabolites. None of the compounds were genotoxic, in the presence or absence of S9, in the GADD45α-GFP Human GreenScreen assay and none induced MN in mouse bone marrow erythrocytes.

Evaluation of chemopreventive agents for genotoxic activity.

We conducted genetic toxicity evaluations of 11 candidate chemopreventive agents with the potential for inhibiting carcinogenesis in humans at increased risk of cancer. The compounds were evaluated for bacterial mutagenesis in the Salmonella-E. coli assay, for mammalian mutagenesis in mouse lymphoma cells, for chromosome aberrations in Chinese Hamster Ovary (CHO) cells, and for micronucleus induction in mouse bone marrow. Tested agents were indole 3-carbinol (I3C), bowman-birk inhibitor concentrate (BBIC), black tea polyphenols (BTP), farnesol, geraniol, l-Se-methylselenocysteine (SeMC), 5,6-dihydro-4H-cyclopenta[1,2]-dithiol-3-thione(DC-D3T), 4'-bromoflavone, 2,5,7,8-tetramethyl-(2R-[4R,8R,12-trimethyltridecyl] chroman-6-yloxy) acetic acid (alpha-TEA), SR13668 (2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo[2,3-b] carbazole and SR16157 (3-O-sulfamoyloxy-7alpha-methyl-21-(2-N,N-diethylaminoethoxy)-19-norpregna-1,3,5(10)-triene). All these agents, except I3C and BTP, were negative in the Salmonella-E. coli assay in the presence and absence of metabolic activation (S9). I3C and BTP induced a weak mutagenic response in the presence and absence of S9 with strains TA100 and TA98, respectively. Of the three compounds tested in the mouse lymphoma assay (I3C, BBIC, and BTP), only BTP was mutagenic in the presence of S9. In the chromosomal aberration assay, of the 8 compounds that were tested, 4'-bromoflavone elicited a positive response in the absence of S9 only, while SR16157 was positive in the presence of S9. The results with geraniol remain inconclusive. I3C, BBIC and BTP were not tested in the chromosomal aberration assay. None of the 11 agents induced micronuclei in mouse bone marrow erythrocytes.

Mouse lymphoma thymidine kinase gene mutation assay: meeting of the International Workshop on Genotoxicity Testing, San Francisco, 2005, recommendations for 24-h treatment.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (IWGT), comprised of experts from Japan, Europe and the United States, met on September 9, 2005, in San Francisco, CA, USA. This meeting of the MLA Workgroup was devoted to reaching a consensus on issues involved with 24-h treatment. Recommendations were made concerning the acceptable values for the negative/solvent control (mutant frequency, cloning efficiency and suspension growth) and the criteria to define an acceptable positive control response. Consensus was also reached concerning the use of the global evaluation factor (GEF) and appropriate statistical trend analysis to define positive and negative responses for the 24-h treatment. The Workgroup agreed to continue their support of the International Committee on Harmonization (ICH) recommendation that the MLA assay should include a 24-h treatment (without S-9) in those situations where the short treatment (3-4 h) gives negative results.

Preliminary safety evaluation of the putative cancer chemopreventive agent tricin, a naturally occurring flavone.

PURPOSE: Naturally occurring flavonoids such as quercetin and genistein possess cancer chemopreventive properties in experimental models. However, adverse effects such as their mutagenicity confound their potential clinical usefulness. Furthermore in leukaemia cells some flavonoids cleave the breakpoint cluster region of the mixed lineage leukaemia (MLL) gene as a consequence of inhibition of topoisomerase II. The choice of flavonoids to be developed as cancer chemopreventive agents depends crucially on their safety. Here, we explored safety aspects of the novel flavone tricin, a constituent of rice bran and other grass species, which has recently been found to interfere with murine gastrointestinal carcinogenesis. METHODS: Evidence of pathological or morphological changes in liver, lung, heart, spleen, kidney, adrenal gland, pancreas or thymus tissues was studied in mice which received tricin, genistein or quercetin 1,000 mg/kg daily by the oral route on five consecutive days. The ability of tricin (50 microM) to cleave the MLL gene was studied in human leukaemia cells by Southern blotting, and its effect on human topoisomerase II activity was investigated in incubations with supercoiled DNA. The mutagenicity of tricin was assessed in the Salmonella/Escherichia coli assay, and its clastogenicity was adjudged by chromosomal aberrations in Chinese hamster ovary cells and occurrence of micronuclei in bone marrow erythrocytes in Swiss-Webster mice. RESULTS: Neither tricin, quercetin, or genistein caused pathological or morphological changes in any of the murine tissues studied. Tricin (50 microM) failed to cause MLL gene breakage, and it inhibited topoisomerase II only at 500 microM, but not at 10, 50 or 100 microM. Tricin lacked genotoxic properties in the systems studied here. CONCLUSION: The results tentatively suggest that tricin may be considered safe enough for clinical development as a cancer chemopreventive agent.

Low-calcemic, efficacious, 1alpha,25-dihydroxyvitamin D3 analog QW-1624F2-2: calcemic dose-response determination, preclinical genotoxicity testing, and revision of A-ring stereochemistry.

Based on an X-ray crystal structure determination, the A-ring stereochemistry of hybrid analog QW-1624F2-2 (1alpha-hydroxymethyl-16-ene-24,24-difluoro-25-hydroxy-26,27-bis-homovitamin D3) is revised to be 1alpha-CH2OH-3beta-OH. This analog is shown to be approximately 80-100 times less calciuric than the natural hormone 1alpha,25-dihydoxyvitamin D3. This analog is shown also to be non-genotoxic in three different standard assays.

Dose-dependent efficacy and safety toxicology of hydroxypyridinonate actinide decorporation agents in rodents: towards a safe and effective human dosing regimen.

Two hydroxypyridinone-containing actinide decorporation agents, 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO), are being developed for the treatment of internal actinide contamination by chelation therapy. Dose-response efficacy profiles in mice were established for the removal of intravenously injected (238)Pu and (241)Am after parenteral and oral treatment with these chelators. In both cases, presumed efficacious doses promoted substantially greater actinide elimination rates than the currently approved agent, diethylenetriamine-pentaacetic acid, considering two different interspecies scaling methods for the conversion of human doses to equivalent rodent dose levels. In addition, genotoxicity of both ligands was assessed using the Salmonella/ Escherichia coli /microsome plate incorporation test and the Chinese hamster ovary cell chromosome aberration assay, showing that neither ligand is genotoxic, in the presence and absence of metabolic activation. Finally, maximum tolerated dose studies were performed in rats for seven consecutive daily oral administrations with the chelators, confirming the safety of the presumed efficacious doses for 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO). The results of these studies add to the growing body of evidence that both decorporation agents have remarkable decorporation efficacy properties and promising safety toxicology profiles. These results are necessary components of the regulatory approval process and will help determine the optimal human dosing regimens for the treatment of internal radionuclide contamination.

Discovery and optimization of benzotriazine di-N-oxides targeting replicating and nonreplicating Mycobacterium tuberculosis.

Compounds bactericidal against both replicating and nonreplicating Mtb may shorten the length of TB treatment regimens by eliminating infections more rapidly. Screening of a panel of antimicrobial and anticancer drug classes that are bioreduced into cytotoxic species revealed that 1,2,4-benzotriazine di-N-oxides (BTOs) are potently bactericidal against replicating and nonreplicating Mtb. Medicinal chemistry optimization, guided by semiempirical molecular orbital calculations, identified a new lead compound (20q) from this series with an MIC of 0.31 µg/mL against H37Rv and a cytotoxicity (CC(50)) against Vero cells of 25 µg/mL. 20q also had equivalent potency against a panel of single-drug resistant strains of Mtb and remarkably selective activity for Mtb over a panel of other pathogenic bacterial strains. 20q was also negative in a L5178Y MOLY assay, indicating low potential for genetic toxicity. These data along with measurements of the physiochemical properties and pharmacokinetic profile demonstrate that BTOs have the potential to be developed into a new class of antitubercular drugs.

Evaluation of the impact of shielding materials in radiation protection in transgenic animals.

We are using a plasmid-based transgenic mouse mutation model system to evaluate the effectiveness of aluminum or low-density polyethylene (LDPE) shielding after 250 MeV/u protons or 1 GeV/u iron ion irradiation. Transgenic mice, with multiple copies of the plasmid pUR288 lacZ transgene integrated into the genome of every cell of the animal, were either irradiated or sham-treated. Multiple endpoints, including early cytogenetic damage in erythrocytes at 48 h after exposure, chromosome aberrations in bone marrow lymphocytes, and lacZ mutant frequencies (MF) in brain and spleen tissues were measured in the same animals. The frequency of total circulating reticulocytes (fRET) dropped precipitously at 48 h after 2 Gy of proton irradiation. The average level of micronucleated reticulocytes (fMN-RET) was fivefold higher in the irradiated samples relative to the controls at the same time point. There was an increase in total chromosome aberrations in bone marrow lymphocytes at 8 weeks after proton irradiation but this increase was not statistically significant relative to the controls. Evaluation of the lacZ MF in the brain and spleen tissues showed that proton irradiation induced a twofold increase in MF in each tissue. Similar samples were collected from animals that were shielded from the proton beam by aluminum. Compared to the unshielded treatment group, we noted no difference in fRET, fMN-RET, chromosome aberrations in lymphocytes and lacZ MF in brain and spleen tissues obtained from these animals. In a separate study, animals were exposed to high-energy iron ions with or without 10 or 15 cm LDPE. Using the same approach, we noted a precipitous drop in fRET, and an elevation in fMN-RET within 48 h after 1 Gy of iron ions. Total chromosome aberrations in bone marrow lymphocytes were slightly elevated but not significant at 8 weeks after iron ion exposure. Shielding animals with 10 or 15 cm of polyethylene appeared to have no effect on the level of RET, MN-RET or chromosome aberrations in these animals. LacZ MF in brain and spleen tissues increased 1.5-2-fold above control levels after 1 Gy iron ions at 8 weeks after treatment. On the other hand, MF in tissues harvested from shielded animals appeared to be lower than their unshielded litermates, suggesting the polyethylene shielding was effective in reducing the iron-induced genomic damage in tissues. Although shielding may be effective, in some cases, in reducing the physical dose of particle radiation, our cytogenetic results showed that the biological impact of the particle beam remain unchanged. On the other hand, reduction in transgene MF in tissues from LDPE-shielded animals but not in the aluminum-shielded animals strongly suggests that careful consideration of the biological endpoints used is necessary in the evaluation of the efficacy of the selected shielding material.