Complementary and alternative groups contemplate the need for effectiveness, safety and cost-effectiveness research.

OBJECTIVE: To examine the views of complementary and alternative medicine (CAM) groups on the need to demonstrate the effectiveness, safety and cost-effectiveness of their therapies and practices. DESIGN: Qualitative interviews were conducted with 22 representatives of three CAM groups (chiropractic, homeopathy and Reiki). Qualitative content analysis was used to identify similarities and differences among and across groups. SETTING: Ontario, Canada. RESULTS: There were striking differences in the views of the three sets of respondents. The chiropractors agreed that it was essential for their group to provide scientific evidence that their interventions work, are safe and cost-effective. The leaders of the homeopathic group were divided on these points and the Reiki respondents showed virtually no interest in undertaking such research. CONCLUSIONS: CAM groups that are more formally organized are most likely to recognize the importance of scientific research on their practices and therapies.

Structure-activity studies of antitumor agent irofulven (hydroxymethylacylfulvene) and analogues.

Many analogues of the antitumor agent irofulven have been readily prepared by replacing the allylic hydroxyl with a variety of nucleophiles. Analogues of acylfulvene (the precursor to irofulven) were also prepared by Michael reaction with acrolein. The toxicity of the analogues was determined, as well as preclinical antitumor activity. Several analogues exhibited good activity in mouse xenografts. Structural requirements for activity are discussed.

Sesquiterpenes from the basidiomycete Omphalotus illudens.

A new sesquiterpene, omphadiol (4), has been isolated from cultures of Omphalotus illudens. Several known compounds, including illudosin (1), were also obtained. Structures were determined using MS, NMR, and X-ray techniques.

Preparation and biological activity of amino acid and peptide conjugates of antitumor hydroxymethylacylfulvene.

The primary hydroxyl group in hydroxymethylacylfulvene, a potent antitumor drug, is readily replaced by thiols including cysteine, N-acetylcysteine, homocysteine, and glutathione. Best yields are obtained when reaction is carried out in the presence of dilute sulfuric acid. A variety of sulfur-containing analogues have been prepared, and their toxicity to tumor cells was examined.

Efficacy of MGI 114 (HMAF) against the MRP+ metastatic MV522 lung carcinoma xenograft.

This study is part of an effort to evaluate efficacy of the novel agent MGI 114 (HMAF) against tumors resistant to conventional chemotherapeutic agents. MGI 114 is a novel semisynthetic anticancer agent currently in chemotherapeutic phase II trials to evaluate activity against various solid tumors. Previous studies indicate MGI 114 was active against human MDR1/gp170+ solid tumor xenografts. Recent evidence suggests overexpression of the MRP protein may also be clinically relevant to development of drug resistance in solid tumors. We evaluated the efficacy of MGI 114 against a human MRP+ lung carcinoma xenograft. Parent MV522 lung carcinoma cells were transfected with a MRP cDNA expression vector and resistant cells selected by exposure to vinblastine (30-fold resistance). Analysis of resistant clones indicated 20- to 40-fold increases in expression of both MRP mRNA and MRP protein. Administration of MGI 114 at the maximum tolerated dose (7 mg/kg, 5 x/week for 3 weeks) to MRP tumor-bearing mice demonstrated this novel agent was active against MRP+ tumors and significantly extended their lifespan (p<0.001). In contrast, other cytotoxic agents had minimal activity against this MRP+ xenograft. These results indicate MGI 114 should retain activity in vivo against MRP+ tumor types. The development of this MRP+ xenograft model, in conjunction with the parent MV522 and MDR1/gp170+ xenograft models, will be useful for screening new classes of agents for activity against multidrug-resistant tumors.

Structural organization of the human gastrointestinal glutathione peroxidase (GPX2) promoter and 3'-nontranscribed region: transcriptional response to exogenous redox agents.

The flanking upstream and downstream regions of the human GPX270%). The human GPX2 promoter region was not G-C rich (<50% G+C) and classical TATA/CCAAT elements were not present. The ubiquitous SP1 and AP elements were present. Several GATA elements as well as liver-specific sites (HNF series) were present. Despite the unique intestinal specific expression of GPX2, classical intestine-specific sites were not detected in the flanking 5' or 3' regions. The ability of the GPX2 promoter to direct transcription was confirmed. Exogenous agents capable of producing oxidative stress, such as paraquat, could induce the transcriptional activity of the GPX2 promoter. Analysis of three previously reported polymorphism sites revealed that they represented the most common polymorphisms. Surprisingly, the human GPX2 promoter could direct transcription and respond to oxidative stress in the murine NIH3T3 fibroblast cell line, which is devoid of the ability to bind to a variety of intestinal specific elements. This finding suggests that the unique intestinal specific expression of GPX2 may be due to elements in the intron, the flanking 3'-nontranslated region, or to elements existing even farther upstream. The ability of GPX2 to respond transcriptionally to redox stress is likely to be more physiologically relevant than post-transcriptional regulation which is dependent upon selenium availability.

Structural organization of the microsomal glutathione S-transferase gene (MGST1) on chromosome 12p13.1-13.2. Identification of the correct promoter region and demonstration of transcriptional regulation in response to oxidative stress.

The structure and regulation of the microsomal glutathione S-transferase gene (MGST1) are considerably more complex than originally perceived to be. The MGST1 gene has two alternative first exons and is located in the 12p13.1-13.2 region. Two other potential first exons were determined to be nonfunctional. The region between the functional first exons cannot direct transcription. Thus, one common promoter element directing transcription exists, and RNA splicing occurs such that only one of the first exons (containing only untranslated mRNA) is incorporated into each mRNA species with common downstream exons. MGST1 expression and regulation are therefore similar to those of other hepatic xenobiotic handling enzymes, which also produce mRNA species differing only in the 5'-untranslated regions to yield identical proteins. MGST1 was previously considered a "housekeeping" gene, as non-oxidant inducers had little effect on activity. However, the promoter region immediately upstream of the dominant first exon transcriptionally responds to oxidative stress. In this respect, MGST1 is similar to glutathione peroxidases that also transcriptionally respond to oxidative stress. The discovery that MGST1 utilizes alternative first exon splicing eliminates a problem with the first description of MGST1 cDNA in that it appeared that MGST1 expression was in violation of the ribosomal scanning model. The identification that the first exon originally noted is in fact a minor alternative first exon far downstream of the primary first exon eliminates this conundrum.

Structural organization of the human glutathione reductase gene: determination of correct cDNA sequence and identification of a mitochondrial leader sequence.

The primary structure of human glutathione reductase gene (GSR) was determined by genomic cloning. The gene structure of human GSR spans 50 kb, consists of 13 exons, and was found to be highly similar to the mouse GSR gene. The coding sequence of human GSR resides on all 13 exons. An N-terminal arginine-rich mitochondrial leader sequence was present, with high homology to the murine leader sequence, between two in-frame start codons in the first exon. The 5' and 3' intron/exon splice junctions, with one exception, followed the general consensus sequences for intron spliced donor and acceptance sites.

Anti-leukemic action of the novel agent MGI 114 (HMAF) and synergistic action with topotecan.

The illudin derivative MGI 114 (6-hydroxymethylacylfulvene or HMAF) is currently in phase II chemotherapeutic clinical trials for a variety of solid tumors. The illudins were originally thought to be potentially useful agents for myeloid leukemias, because hematopoietic tumor cells were markedly sensitive whereas normal bone marrow progenitors were relatively resistant to the cytotoxic effects of illudins. Due to the marked preclinical efficacy of MGI 114 against a variety of solid tumor xenografts, the current phase II human trials are restricted to solid tumor (breast, lung, colon, ovarian, pancreas, prostate, etc) malignancies. The present studies were undertaken to evaluate the efficacy of MGI 114 in the HL60/MRI myeloid leukemia xenograft. In addition, because of the reported synergistic cytotoxic activity between MGI 114 and the topoisomerase I inhibitor topotecan towards pediatric human tumor cell lines, we tested the activity of MGI 114 and topotecan combinations against HL60 cells in vitro and the HL60/MRI myelocytic xenograft. Our results indicate that MGI 114 at maximum tolerated doses (MTD) of 7 mg/kg, five times per week for 3 weeks does display anti-myeloid leukemic properties in the HL60/MRI xenograft model which exceeds activity noted with other conventional agents (TGI > 70%). A marked therapeutic synergistic action was observed with MGI 114 and topotecan combinations of (1/2) MTD of each agent producing complete tumor remission in 50% of animals, without development of excessive or additive toxicity in animals. These results support further in vitro and clinical investigation into both the anti-myeloid leukemic activity of MGI-114, and the cooperative pharmacologic interaction noted between MGI-114 and topoisomerase I inhibitors. Leukemia (2000) 14, 136-141.

Characterization of MGI 114 (HMAF) histiospecific toxicity in human tumor cell lines.

PURPOSE: The acylfulvenes are a class of antitumor agents derived from the fungal toxin illudin S. One acylfulvene derivative, MGI 114 (HMAF), demonstrates marked efficacy in xenograft carcinoma models when compared to the parent acylfulvene or related illudin compounds. The maximum tolerated dose (MTD) of the two analogs in animals, however, is similar. To help elucidate the basis of the increased therapeutic efficacy of MGI 114, we determined the in vitro cytotoxicity, cellular accumulation and DNA incorporation of this drug and compared the results with those from the parent acylfulvene analog. METHODS: The cytotoxicity of acylfulvene analogs was tested in vitro against a variety of tumor cell lines. Radiolabeled MGI 114 was used for cellular accumulation and DNA incorporation studies. RESULTS: MGI 114 retained relative histiospecific toxicity towards myeloid leukemia and various carcinoma cell lines previously noted with the parent acylfulvene compound. Markedly fewer intracellular molecules of MGI 114 were required to kill human tumor cells in vitro as compared to the parent acylfulvene, indicating that MGI 114 was markedly more toxic on a cellular level. At equitoxic concentrations, however, the incorporation of MGI 114 into genomic tumor cell DNA was equivalent to that of acylfulvene. Analysis of cellular accumulation of MGI 114 into tumor cells revealed a lower Vmax for tumor cells, and a markedly lower Vd for diffusion accumulation as compared to acylfulvene. CONCLUSIONS: The addition of a single methylhydroxyl group to acylfulvene to produce MGI 114 results in a marked increase in cytotoxicity in vitro towards tumor cells as demonstrated by the reduction in IC50 values. There was a corresponding decrease in the number of intracellular molecules of MGI 114 required to kill tumor cells, but no quantitative alteration in covalent binding of the drugs to DNA at equitoxic concentrations. This indicates that cellular metabolism plays a role in the in vitro cytotoxicity of MGI 114. The equivalent incorporation into genomic DNA at equitoxic doses suggests that DNA damage produced by acylfulvene and MGI 114 is equivalent in regard to cellular toxicity and ability to repair DNA. This increased cellular toxicity, together with the decrease in diffusion rate, may explain the increased therapeutic efficacy of MGI 114 as compared to the parent acylfulvene analog.

Metabolism of antitumor hydroxymethylacylfulvene by rat liver cytosol.

Acylfulvenes are a potent class of antitumor agents derived from illudin S, a fungal sesquiterpene. Illudin S possesses antitumor activity but has a poor therapeutic index. Acylfulvene is 100-fold less toxic against human lung adenocarcinoma cells than illudin S, but inhibits tumor growth in human xenografts, opposite to illudin S. An analog of acylfulvene, MGI 114 (hydroxymethylacylfulvene), shows much greater efficacy, producing complete tumor regression in xenograft models. MGI 114 is currently in phase II clinical trials. Cytotoxicity of MGI 114, like that of illudin S, is believed to involve both chemical reaction and enzymatic reduction. Enzymatic reduction by a cytosolic NADPH-dependent enzyme (from rat liver) produced an aromatic metabolite similar to that formed from illudin S. However, the reaction occurred more slowly. In addition, four new metabolites were isolated, two hydroxylated derivatives and two in which the primary allylic hydroxyl was replaced by hydride. All retained the reactive centers of the parent MGI 114.

Distribution of microsomal glutathione transferase 1 in mammalian tissues. A predominant alternate first exon in human tissues.

An extensive Northern blot analysis of microsomal glutathione transferase 1 in human and rat tissues was performed. When normalized against the glyceraldehyde-3-phosphate dehydrogenase or actin expression it was evident that the predominant expression occurs in liver and pancreas. An ontogenetic, as well as a functional, basis for the high levels in these two organs is possible. The relative expression levels in man ranged from: liver and pancreas (100%), to kidney, prostate, colon (30-40%), heart, brain, lung, testis, ovary, small intestine (10-20%), placenta, skeletal muscle, spleen, thymus and peripheral blood leucocytes (1-10%). Liver-enriched expression was detected in human fetal tissues with lung and kidney displaying lower levels (10-20%). No transcripts could be detected in fetal brain or heart. When comparing the expression levels between rat and man it is apparent that human extrahepatic mRNA levels are much higher relative to liver. Rat microsomal glutathione transferase mRNA expression ranges from 0.2 to 10% that of liver, with adrenal, uterus, ovary and stomach displaying the highest levels of the organs tested. Based on these observations, and the fact that the enzyme is encoded by a highly conserved single-copy gene, it is suggested that microsomal glutathione transferase 1 performs essential functions vital to most mammalian cell types. We suggest that protection against oxidative stress constitutes one such function. Human expressed sequence tag (EST) characterization yielded four alternate mRNA transcripts with different 5'-ends (four alternate noncoding exons 1). The predominant exon (based on the observed EST frequency) revealed a tissue distribution similar to that obtained using the reading frame as probe. Thus, it appears that one exon preferentially gives rise to mature mRNA in the human tissues examined. This exon is different from the one reported in the original cDNA characterized.

Metabolism of antitumor acylfulvene by rat liver cytosol.

Illudins are novel compounds from which a potent class of antitumor agents, called acylfulvenes, have been synthesized. The model illudin, illudin S, has marked in vitro and in vivo toxicity but displays a poor therapeutic index. The toxicity of illudin S is believed to involve a combination of enzymatic reduction and chemical reaction. Enzymatic reduction by a cytosolic NADPH-dependent enzyme produces an aromatic metabolite, as does reaction with thiols. Acylfulvene is formed from illudin S by reverse Prins reaction. Acylfulvene is 100-fold less toxic in vitro and in vivo than illudin S but possesses marked antitumor efficacy in vivo, thus displaying opposite properties from illudin S. For this reason we investigated the in vitro metabolism of acylfulvene. Incubation of acylfulvene with NADPH and rat liver cytosol yielded two metabolites. One metabolite, the aromatic product, is similar to that obtained with illudin S in this in vitro system and was anticipated. The other metabolite, the hydroxylated product, was not expected and no corresponding metabolite for illudin S could be detected. The production of this hydroxylated metabolite from acylfulvene may explain, in part, the increased antitumor activity of novel acylfulvenes as compared with the illudins.

Efficacy of MGI 114 (6-hydroxymethylacylfulvene, HMAF) against the mdr1/gp170 metastatic MV522 lung carcinoma xenograft.

Illudins are a novel class of agents with a chemical structure entirely different from current chemotherapeutic agents. A new semisynthetic derivative, MGI 114 (NSC 683,863, 6-hydroxymethyl-acylfulvene, HMAF), is markedly effective in a variety of lung, breast and colon carcinoma xenograft models. This analogue, MGI 114, is currently in phase I human clinical trials, and is scheduled for two different phase II trials. To determine if MGI 114 could be effective in vivo against mdr tumour cells, we generated an mdr1/gp170-positive clone of the metastatic MV522 human lung carcinoma line by transfecting a eukaryotic expression vector containing the cDNA encoding for the human gp170 protein. This MV522/mdr1 daughter line retained the metastatic ability of parental cells. The parental MV522 xenograft is mildly responsive in vivo to mitomycin C and paclitaxel, as evidenced by partial tumour growth inhibition and a small increase in life span, whereas MV522/mdr1 xenografts were resistant to these agents. In contrast to mitomycin C and paclitaxel, MGI 114 produced xenograft tumour regressions in 32 of 32 animals and completely eliminated tumours in more than 30% of MV522/mdr1 tumour-bearing mice. Thus, MGI 114 should be effective in vivo against mdr1/gp170-positive tumours.

Structural organization of the human selenium-dependent phospholipid hydroperoxide glutathione peroxidase gene (GPX4): chromosomal localization to 19p13.3.

The primary structure of human selenium-dependent phospholipid hydroperoxide glutathione peroxidase (GPX4) was determined by genomic cloning. The gene structure of GPX4 spans only 2.8 kb and consists of 7 exons. The coding sequence resides on all 7 exons, and the mitochondrial leader sequence is contained entirely within the first exon. The selenocysteine coding nucleotide resides on the third exon. The introns all commenced with the consensus nucleotide sequence GTR and ended with the consensus nucleotide sequence YAG. Analysis of the GPX4 gene sequence identified a potential alternative tissue-specific first exon. Chromosomal FISH studies placed the GPX4 gene at 19p13.3 location, and downstream of the 23 k-Da polypeptide DNA-directed RNA polymerase gene.

Characterization of acylfulvene histiospecific toxicity in human tumor cell lines.

PURPOSE: Acylfulvene derivatives demonstrate marked efficacy in xenograft carcinoma models as compared with the parent illudin compounds. To elucidate the increased therapeutic efficacy of acylfulvene analogs, we compared them with the illudin compounds in terms of their in vitro cytotoxicity, cellular accumulation and DNA incorporation. METHODS: The cytotoxicity of various acylfulvene analogs was tested in vitro against a variety of tumor cell lines. Radiolabelled acylfulvene analog was prepared and used for cellular accumulation and DNA incorporation studies. RESULTS: The prototype acylfulvene analog retained selective histiospecific toxicity towards myeloid leukemia and various carcinoma cell lines. In vitro killing of tumor cells by acylfulvene required up to a 30-fold increase in molecules per cell, as compared with illudin S, indicating that acylfulvene was less toxic on a cellular level. At equitoxic concentrations, acylfulvene incorporation into genomic tumor cell DNA was equivalent to illudin S suggesting that cellular metabolism has a role in acylfulvene cytotoxicity. Analysis of cellular accumulation of acylfulvene into tumor cells revealed a markedly higher Vmax for tumor cells, and a lower Vd for diffusion accumulation into other cells. CONCLUSIONS: The combination of higher Vmax and lower Vd may explain the increased in vivo efficacy of acylfulvene.

Preclinical antitumor activity of 6-hydroxymethylacylfulvene, a semisynthetic derivative of the mushroom toxin illudin S.

6-Hydroxymethylacylfulvene (HMAF; MGI 114) is a novel semisynthetic antitumor agent derived from the sesquiterpene mushroom toxin illudin S. In vitro cytotoxicity determinations produced IC50 concentrations (concentrations required for 50% inhibition of growth) ranging from 160 nM in sensitive MCF-7 human mammary carcinoma cells to 17 microM in relatively insensitive murine B16 melanoma cells. In vivo antitumor activity was consistent with in vitro sensitivity. HMAF was very effective in human tumor xenograft models, including MX-1 breast carcinoma, MV522 lung adenocarcinoma, and HT-29 colon carcinoma, but not murine B16 melanoma or P388 leukemia. Excellent responses were observed in animals bearing MX-1 tumors administered i.v. or i.p. doses of 3-7.5 mg/kg daily for 5 days, with complete regression recorded in 29 of 30 animals administered i.v. HMAF. Extensive tumor shrinkage was also observed with MV522, and significant tumor growth inhibition was obtained with HT-29 when animals received 5 daily i.p. doses ranging from 3.75 to 7.5 mg/kg. Complete regressions were also observed in individual animals with MV522 and HT-29. The excellent activity of HMAF in several human solid tumor xenografts, including the more refractory MV522 and HT-29 models, warrants the further investigation of this novel agent in clinical trials.

Heterologous expression of carbonyl reductase: demonstration of prostaglandin 9-ketoreductase activity and paraquat resistance.

Transfection of murine NIH3T3 fibroblasts with a pSV2-derived eukaryotic expression vector for human cytosolic carbonyl reductase (E.C. 1.1.1.141) resulted in clones with increased carbonyl reductase activity as demonstrated by an elevation in cellular NADPH-dependent alcohol (menadione) reductase activity. Prostaglandin 9-ketoreductase (9KR) activity, previously noted only in purified enzyme preparations, was also elevated. Although the cellular molar capacity of 9KR activity was less than menadione reductase activity (picomoles versus nanomoles per mg of protein), when compared to endogenous activity there was a greater relative increase in 9KR activity as compared to menadione activity (10 fold increase versus 3 fold). Thus, the 9KR properties of carbonyl reductase may have a physiologic role in prostaglandin regulation. Most transgenic clones lost their enhanced carbonyl reductase activity despite continuous selection, but two clones retained enhanced enzyme activity. RNA analysis indicated that these two murine clones expressed human carbonyl reductase mRNA. These two clones overexpressing carbonyl reductase did not display resistance to menadione, in agreement with a previous report. There was, however, a demonstrable increase in resistance to paraquat of a magnitude similar to that previously noted with transgenic cell lines overexpressing manganese superoxide dismutase.