Application of the CFU-GM assay to predict acute drug-induced neutropenia: an international blind trial to validate a prediction model for the maximum tolerated dose (MTD) of myelosuppressive xenobiotics.

In a previous study of prevalidation, a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed, to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up, by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper, we describe an international blind trial designed to apply this model to the clinical neutropenia, by testing 20 drugs, including 14 antineoplastics (Cytosar-U, 5-Fluorouracil, Myleran, Thioguanine, Fludarabine, Bleomycin, Methotrexate, Gemcitabine, Carmustine, Etoposide, Teniposide, Cytoxan, Taxol, Adriamycin); two antivirals (Retrovir, Zovirax,); three drugs for other therapeutic indications (Cyclosporin, Thorazine, Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin, Bleomycin, Etoposide, Fludarabine, 5-Fluorouracil, Myleran, Taxol, Teniposide, Thioguanine, and Thorazine), IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine, Cyclosporin, Cytosar-U, Cytoxan, Gemcitabine, Indocin, Lindane, Methotrexate, Retrovir, and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had "actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD, instead of the 4-fold range required by the model, whereas the prediction with Cytosar-U was approximately 10-fold in error. A possible explanation for the failure in the prediction of these three drugs, which are pyrimidine analogs, is discussed. We concluded that our model correctly predicted the human MTD for 20 drugs out of 23, since the other three drugs (Topotecan, PZA, and Flavopiridol) were tested in the prevalidation study. The high percentage of predicitivity (87%), as well as the reproducibility of the SOP testing, confirm that the model can be considered scientifically validated in this study, suggesting promising applications to other areas of research in developing validated hematotoxicological in vitro methods.

A phase I clinical trial of spicamycin derivative KRN5500 (NSC 650426) using a phase I accelerated titration "2B" design.

The spicamycin derivative KRN5500 was considered as a potential anti-cancer agent based on in vitro and preclinical studies. A Phase I study involving 24 cancer patients in whom tumors were refractory to all other conventional therapies was conducted to determine the dose limiting toxicity, maximum tolerated dose, effectiveness, and pharmacokinetic parameters of this drug administered by 1-h IV infusion daily for five consecutive days every 3 weeks. Using an accelerated dose titration strategy, 8.4 mg/m2/d x 5 days was the maximum administered dose. Severe gastrointestinal and hepatic toxicities were observed at doses at or above 4.3 mg/m2/d x 5. The recommended Phase II dose i s 4.3mg/m2/d x 5. The distribution of KRN5500 followed a two-compartment model, and clearance did not decrease significantly over the dose range 0.8-8.4 mg/m2/d x 5. No significant correlation was observed between plasma levels and toxicity. No tumor responses were observed among the 14 patients evaluable for response.

The investigational new drug XK469 induces G(2)-M cell cycle arrest by p53-dependent and -independent pathways.

PURPOSE: XK469 (2-[4-(7-chloro-2-quinoxalinyloxy) phenoxy]propionic acid), a synthetic quinoxaline phenoxypropionic acid derivative, has broad activity against murine tumors and is entering Phase I clinical development as a topoisomerase IIbeta inhibitor. This study investigated the underlying molecular mechanism of XK469's effects on the cell cycle. EXPERIMENTAL DESIGN: Growth inhibition, cell cycle arrest, induction of p53 and p21 mRNA and protein, and cdc2 phosphorylation and kinase activity were studied in treated cells from the H460 lung cancer line and p21 and p53 knockout cells of the HCT 116 colon cancer line. RESULTS: XK469 arrested H460 cells at G(2)-M, which was associated with cdc2 phosphorylation and decreased cdc2 kinase activity. Moreover, XK469 stabilized p53 and subsequently increased p21(WAF1/CIP1). Furthermore, HCT116 p21(-/-) cells were less sensitive than wild-type cells to XK469-induced growth inhibition, but p53(+/+) and p53(-/-) cells were equally sensitive despite the absence of p21 induction in the p53(-/-) cells. CONCLUSIONS: When considered with published data, our study suggests a complex mechanism of XK469-mediated anticancer activity involving multiple pathways, including p53-dependent and -independent G(2)-M arrest via inactivation of cdc2-cyclin B1 kinase activity.

Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay.

This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential.

High-performance liquid chromatographic method for the estimation of the novel investigational anti-cancer agent SR271425 and its metabolites in mouse plasma.

A simple and reliable HPLC method was developed for the estimation of a new anti-cancer agent that belongs to the thioxanthone class, SR271425 in mouse plasma. SR271425, it's metabolites and internal standard (SR233377) were separated from plasma by liquid-liquid extraction using dichloromethane after quenching the plasma proteins with acetonitrile. Chromatography was performed on a reversed-phase C18 column using methanol-10 mM phosphate buffer, pH 3.5 (45:55) as mobile phase at a flow-rate of 0.8 ml/min for first 10 min and 1.4 ml/min for the next 15 min with UV-Vis detection at 264 nm and SR233377 as internal standard. The retention times of SR271425 and internal standard were 18.6 and 14.8 min, respectively. The limit of detection was 40 ng/ml and the limit of quantification was 78 ng/ml. This method was also able to detect the three metabolites of SR271425. The intra- and inter-day relative standard deviations were less than 13% at all concentrations. This analytical method was precise and reproducible for pharmacokinetics and metabolism studies of the drug in mice. SR271425 is proceeding to phase I clinical trials in 2001.

Latent hematopoietic stem cell toxicity associated with protracted drug administration.

OBJECTIVE: The protracted administration of near-conventional daily doses of chemotherapeutic agents is a strategy to increase dose intensity and, potentially, efficacy as well. However, protracted therapy carries the risk of damage to stem cells in proliferative tissues that are not targeted by intermittent schedules. Therefore, we have investigated the effects produced by the protracted administration of two anticancer drugs on hematopoietic stem cell function. MATERIALS AND METHODS: We used the competitive repopulating assay to assess stem cell damage caused by protracted daily drug treatment of mice. RESULTS: Treatment with acetyldinaline for 10 consecutive days mediated a modest effect on the short-term repopulating cells (STRCs) but spared the long-term repopulating cells (LTRCs). Gemcitabine for 10 days led to a modest decline in both the STRCs and LTRCs. Extending treatment with gemcitabine for 28 days resulted in more severe repopulating cell (RC) damage, which was much worse than in acetyldinaline-treated mice. As expected, melphalan for 10 or 28 days mediated a marked reduction in all of the RCs of treated mice. The analysis of the RCs from mice that were allowed a 1-year recovery period after completing the 28-day treatment with either acetyldinaline or gemcitabine showed normal levels of neutrophils and bone marrow (BM) progenitors. However, a reduction in the RCs was observed in both groups, with larger reductions in gemcitabine-treated mice. CONCLUSIONS: Our data show that protracted treatment with gemcitabine, but not acetyldinaline, of mice caused severe permanent damage to the stem cell components. Therefore, although 28-day therapy with acetyldinaline or gemcitabine appeared to be well tolerated at the level of peripheral blood and bone marrow progenitors, gemcitabine produces permanent stem cell damage when used in long-term administration regimens that should perhaps only be explored clinically with stem cell support available.

In vitro human tissue models in risk assessment: report of a consensus-building workshop.

Advances in the technology of human cell and tissue culture and the increasing availability of human tissue for laboratory studies have led to the increased use of in vitro human tissue models in toxicology and pharmacodynamics studies and in quantitative modeling of metabolism, pharmacokinetic behavior, and transport. In recognition of the potential importance of such models in toxicological risk assessment, the Society of Toxicology sponsored a workshop to evaluate the current status of human cell and tissue models and to develop consensus recommendations on the use of such models to improve the scientific basis of risk assessment. This report summarizes the evaluation by invited experts and workshop attendees of the current status of such models for prediction of human metabolism and identification of drug-drug interactions, prediction of human toxicities, and quantitative modeling of pharmacokinetic and pharmaco-toxicodynamic behavior. Consensus recommendations for the application and improvement of current models are presented.

Cytotoxic chemotherapy regimens that increase dose per cycle (dose intensity) by extending daily dosing from 5 consecutive days to 28 consecutive days and beyond.

Dose intensity, defined as dose administered per unit time, has emerged as a potentially important measurement of anticancer drug exposure and determinant of efficacy. There are several strategies for increasing dose intensity, one being a protracted daily dosing strategy without major dose reduction for toxicity. This strategy involves continued therapy during periods of recovery from reversible toxicity, and it inherently challenges our understanding that renewing tissues cannot repopulate (recover) in the continued presence of cytotoxic drug. We have tested this idea directly in a murine preclinical trial. Specifically, we have tested whether acutely myelotoxic doses of gemcitabine (i.p. injection, 6.0 mg/m2/day), acetyldinaline [CI-994; GOE 5549; PD 123 654; 4-acetylamino-N-(2'-aminophenyl)-benzamide, 150 mg/m2/day p.o.], and/or melphalan (i.p. injection, 7.2 mg/m2/day) can be tolerated for 28 consecutive days and whether suppressed bone marrow function recovers despite this protracted daily therapy. The three drugs all caused acute neutropenia and suppression of medullary hematopoiesis. Damage to progenitor populations exposed to acetyldinaline and gemcitabine was not as severe as that caused by melphalan, in which case absolute neutrophil count, mature progenitors (colony-forming unit granulocyte/macrophage), and immature progenitors (colony-forming unit-S) progressively declined to severely depressed levels. Marrow recovery was observed during continued daily treatment with acetyldinaline and gemcitabine but not melphalan, and marrow function completely recovered after finishing the 28-day course. Pharmacology studies proved that protracted therapy causes little, if any, change in cellular drug tolerance or systemic exposure.

Taxanes: an overview of the pharmacokinetics and pharmacodynamics.

Paclitaxel and docetaxel have emerged in the last two decades as effective antitumor agents in a variety of malignancies. Paclitaxel is a semisynthetic taxane isolated from bark of the Pacific yew tree. Docetaxel is a semisynthetic taxane derived from the needles of the European yew (Taxus baccata). These compounds bind to tubulin, leading to microtubule stabilization, mitotic arrest and, subsequently, cell death. Plasma clearance of paclitaxel exhibits nonlinear kinetics, which results in a disproportionate change in plasma concentration and area under the curve (AUC) with dose alterations. In contrast, docetaxel has a linear disposition over the dose ranges used clinically, so its concentration changes linearly with changes in the dosage. Premedicating with corticosteroids and histamine H1 and H2 receptor antagonists is advocated prior to paclitaxel administration; prior to docetaxel administration, premedication with corticosteroids is suggested. The taxanes are metabolized in the liver by the cytochrome P-450 enzymes and are eliminated in the bile. The known metabolites are either inactive or less potent than the parent compounds. The toxic effects associated with paclitaxel therapy are mainly neutropenia, peripheral neuropathy, and, rarely, cardiotoxicity. Docetaxel toxicity produces mainly myelosuppression and a cumulative dose fluid retention syndrome. Paclitaxel demonstrates sequence-dependent interactions with cisplatin, cyclophosphamide, and doxorubicin. Docetaxel has shown increased myelosuppression with preceding ifosfamide in a preliminary study. The future holds increasing indications for taxanes in newer combination regimens; consideration of their pharmacologic characteristics is an important aspect of designing and applying new taxane-based treatment regimens.

Predicting hematological toxicity (myelosuppression) of cytotoxic drug therapy from in vitro tests.

Several clinical oncology units are studying the roles of in vitro hematotoxicology in phase I evaluations. At the same time, the European Center for the Validation of Alternative Methods (ECVAM) is supporting a validation study of the CFU-GM assay. It is important that these activities be coordinated so that high performance, optimized technical protocols are used for prospective and retrospective clinical evaluations. The EROTC, the NCI and ECVAM could provide support for these coordinated efforts. There is an opportunity for medical oncologists involved in early clinical trials to participate in the evaluation of in vitro tests and their clinical application . Fundamental to acceptance of these assays by oncologists and regulatory scientists, they must predict clinical outcome for myelosuppressive agents and then improve phase I design and performance. These achievements would justify more aggressive dose escalation schemes using guidance from in vitro studies without compromising patient safety. Success in predicting neutropenia might also stimulate the research required to understand how to predict other hematologic toxicities, such as a thrombocytopenia. The complexity of a validation study in hematotoxicology is that it seeks to predict the level of exposure that causes neutropenia, in contrast to other validation studies that have sought to classify a xenobiotic as toxic or not. It may be that the clinical relevance of a new assay is not just a yes-no answer. This important distinction came from the realization that the xenobiotic tolerance in other organ systems of the body must be the same or greater than marrow in order for myelosuppression to be a clinical consequence of exposure. Pharmacological principles of system exposure and toxicity that are integrated into the prediction model provide the links to clinical oncology. It is also important to anticipate future applications of in vitro hematotoxicology. If the maximum tolerated level of drug exposure for human hematopoietic cells can be predicted, then in vitro hematotoxicology could play an important role in new drug discovery. One concept involves screening for compounds that show efficacy at the IC level that predicts maximum tolerated exposure levels in the human. 'Therapeutic index based' drug discovery has been applied to the tallimustine family with some success.

Alternative testing systems for evaluating noncarcinogenic, hematologic toxicity.

Hematopoietic tissues are the targets of numerous xenobiotics. Clinical hematotoxicity is either a decrease or an increase in peripheral blood cell counts in one or more cell lineages--a cytopenia or a cytosis, respectively--that carries a risk of an adverse clinical event. The purpose of in vitro hematotoxicology is the prediction of these adverse hematologic effects from the effects of the toxicants on human hematopoietic targets under controlled experimental conditions in the laboratory. Building on its important foundations in experimental hematology and the wealth of hematotoxicology data found in experimental oncology, this field of alternative toxicology has developed rapidly during the past decade. Although the colony-forming unit-granulocyte/monocyte neutrophil progenitor is most frequently evaluated, other defined progenitors and stem cells as well as cell types found in the marrow stroma can be evaluated in vitro. End points have been proposed for predicting toxicant exposure levels at the maximum tolerated dose and the no observable adverse effect level for the neutrophil lineage, and several clinical prediction models for neutropenia have developed to the point that they are ready for prospective evaluation and validation in both preclinical species and humans. Known predictive end points are the key to successful comparisons across species or across chemical structures when in vitro dose-response curves are nonparallel. Analytical chemistry support is critical for accurate interpretation of in vitro data and for relating the in vitro pharmacodynamics to the in vivo pharmacokinetics. In contrast to acute neutropenia, anemia and acute thrombocytopenia, as well as adverse effects from chronic toxicant exposure, are much more difficult to predict from in vitro data. Pharmacologic principles critical for clinical predictions from in vitro data very likely will apply to toxicities to other proliferative tissues, such as mucositis.

13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies for organ toxicity.

In the past decade in vitro tests have been developed that represent a range of anatomic structure from perfused whole organs to subcellular fractions. To assess the use of in vitro tests for toxicity testing, we describe and evaluate the current status of organotypic cultures for the major target organs of toxic agents. This includes liver, kidney, neural tissue, the hematopoietic system, the immune system, reproductive organs, and the endocrine system. The second part of this report reviews the application of in vitro culture systems to organ specific toxicity and evaluates the application of these systems both in industry for safety assessment and in government for regulatory purposes. Members of the working group (WG) felt that access to high-quality human material is essential for better use of in vitro organ and tissue cultures in the risk assessment process. Therefore, research should focus on improving culture techniques that will allow better preservation of human material. The WG felt that it is also important to develop and make available relevant reference compounds for toxicity assessment in each organ system, to organize and make available via the Internet complete in vivo toxicity data, including human data, containing dose, end points, and toxicokinetics. The WG also recommended that research should be supported to identify and to validate biological end points for target organ toxicity to be used in alternative toxicity testing strategies.

Preclinical antitumor activity of XK469 (NSC 656889).

XK469 (NSC 656889) is a water-soluble member of the novel quinoxaline family of antitumor agents. In vitro, XK469 demonstrated selective cytotoxicity for several murine solid tumors including colorectal and mammary adenocarcinoma cell lines, when compared to both leukemia and normal epithelial cells. In vivo, XK469 was active against 7/7 murine tumors tested, including pancreatic ductal carcinomas #02 and #03, colon adenocarcinomas #38 and #51/A, mammary adenocarcinoma #16/C and the Adriamycin resistant mammary adenocarcinomas #16/C/ADR and #17/ADR. XK469 was efficacious both intravenously and orally. Regardless of dosing schedule, conventional mice tolerated higher total doses than SCID or nu/nu mice did. Despite these reduced doses, XK469 was active against xenografts of 4/6 human tumor lines including mammary adenocarcinoma MX-1, the small cell lung cancer DMS 273, the prostate model LNCaP and the CNS tumor SF295. The lower doses in the xenograft studies were below curative levels. The dose-limiting toxicity appeared to be myelosuppression with rapid host recovery (5-8 days), and in vitro assays of XK469 toxicity to murine bone marrow neutrophil progenitors CFU-GM (colony forming unit-granulocyte/macrophage) demonstrated concentration-dependent toxicity from 0.5-30 microg/mL. The difference in drug tolerance between BDF1 and SCID mice was detected in vitro as a 3-fold difference in the IC90 for CFU-GM, despite similar IC50 values. Comparative in vitro hematotoxicology studies revealed that human bone marrow CFU-GM tolerated XK469 as well as their SCID counterparts (IC90 values 5.7 vs. 7.4 microg/mL). Based on comparison with previously tested anti-cancer agents, these data suggest that humans will be able to tolerate XK469 doses that are efficacious against human tumor xenografts.

Topoisomerase I inhibitors and drug resistance.

DNA topoisomerase I is a nuclear enzyme which catalyzes the conversion of the DNA topology by introducing single-strand breaks into the DNA molecule. This enzyme represents a novel and distinct molecule target for cancer therapy by antitopoisomerase drugs belonging to the campthotecin series of antineoplastics. As many tumors can acquire resistance to drug treatment and become refractary to the chemotherapy it is very important to investigate the mechanisms involved in such a drug resistance for circumventing the phenomenon. This article describes the role of topoisomerase I in cell functions and the methods used to assess its in vitro catalytic activity. It reviews the mechanisms of cytotoxicity of the most specific antitopoisomerase I drugs by considering also the phenomenon of drug resistance. Some factors useful to drive the future perspectives in the development of new topoisomerase I inhibitors are also evidenced and discussed.

Differential toxicity of camptothecin, topotecan and 9-aminocamptothecin to human, canine, and murine myeloid progenitors (CFU-GM) in vitro.

PURPOSE: 20(S)-Camptothecin (CAM), topotecan (TPT, active ingredient in Hycamtin) and 9-amino-20(S)-camptothecin (9AC) are topoisomerase I inhibitors that cause similar dose-limiting toxicities to rapidly renewing tissues, such as hematopoietic tissues, in humans, mice, and dogs. However, dose-limiting toxicity occurs at tenfold lower doses in humans than in mice. The purpose of the current study was to determine whether hematopoietic progenitors of the myeloid lineage from humans, mice, and dogs exhibit the differential sensitivity to these compounds that is evident in vivo. METHODS: Drug-induced inhibition of in vitro colony formation by a myeloid progenitor in human, murine, and canine marrow colony-forming unit-granulocyte/macrophage (CFU-GM) provided the basis for interspecies comparisons at concentrations which inhibited colony formation by 50% (IC50) and 90% (IC90). RESULTS: Murine IC90 values were 2.6-, 2.3-, 10-, 21-, 5.9-, and 11-fold higher than human values for CAM lactone (NSC-94600) and sodium salt (NSC-100880), TPT (NSC-609699), and racemic (NSC-629971), semisynthetic and synthetic preparations (NSC-603071) of 9AC, respectively. In contrast, canine IC90 values were the same as, or lower than, the human IC90 values for all six compounds. CONCLUSIONS: The greater susceptibility of humans and dogs to the myelotoxicity of camptothecins, compared to mice, was evident in vitro at the cellular level. Differential sensitivity between murine and human myeloid progenitors explains why the curative doses of TPT and 9AC in mice with human tumor xenografts are not achievable in patients. Realizing the curative potential of these compounds in humans will require the development of therapies to increase drug tolerance of human CFU-GM at least to a level equal to that of murine CFU-GM. Because these interspecies differences are complicated by species-specific effects of plasma proteins on drug stability, not all in vitro assay conditions will yield results which can contribute to the development of such therapies.

Stage-dependent genetically-based deformities of the regenerating newt limb from 4-nitroquinoline-N-oxide mutagenesis: potential embryonic regulation of cancer.

The effect of the mutagenic carcinogen 4-nitroquinoline-N-oxide (4NQO) on limb regeneration was studied in adult Triturus cristatus newts. Delayed limb regeneration was observed when a 10- to 15-micrograms crystal of 4NQO was implanted at the late dedifferentiation or late bud stage. Additionally, 4NQO administration at these stages caused developmental deformities in skeletal elements of the subsequent regenerates. In contrast, 4NQO implanted at the wound healing stage did not affect skeletal morphogenesis. These critical stages of limb regeneration affected by carcinogen exposure (but not other manipulations) represent mechanisms distinct from disruption of basement membrane deposition. Secondary regeneration, initiated by reamputation of the 4NQO-treated abnormal regenerates 4-5 mm proximal to the site of carcinogen implantation, produced normal regenerates, setting a limit on the diffusion distance of 4NQO in the limb. Distal reamputation through regenerates at the level of the abnormality resulted in regeneration of the original bone deformity, suggesting that the teratogenic effect of 4NQO is mediated via heritable mutational events in blastema cells. These results extend a previously published conclusion that 4-NQO is mutagenic in non-regenerative tissue to include regenerative tissue as well. However, in spite of mutagenic activity, squamous carcinomas were not induced by 4NQO exposure at any stage of regeneration. Therefore, the resistance of regenerative tissue to 4'-NQO carcinogenesis is not due to resistance to mutations, but rather to other mechanisms perhaps similar to those which regulate malignant cells in murine embryos.

Automated imaging and quantitation of tumor cells and CFU-GM colonies in microcapillary cultures: toward therapeutic index-based drug screening.

Human colony forming units (CFUs) from both malignant and hematopoietic tissues can be assayed in vitro in microcapillary cultures, an alternative cloning system to the Petri dish methodology. For technical reasons, microcapillary culture may be ideally suited for new drug screening by therapeutic index. To achieve the high output required by screening programs, automated quantitation of CFUs is required. Toward this end, this paper reports the development of a prototype CapScan, an image analysis system that uses a novel axial laser illumination system to detect tumor cell colonies and, with technical modifications, CFU-granulocyte-macrophage (CFU-GM) colonies in microcapillary cultures. As currently configured, the CapScan can quantify colonies grown in a rack of 18 microcapillary cultures in 30 minutes or less. The sensitivity and detection specificity of tumor cell colonies is >90% with a coefficient of variance of 5-40%, dependent upon colony number. Over a range of colony numbers, CapScan and manual colony counts showed a linear correlation > -0.9, and yielded identical results in assays of doxorubicin inhibition of clonogenic P388 cells. As an additional advantage, the growth kinetics of individual colonies can also be monitored with the CapScan, making distinctions between cytotoxic and cytostatic drugs possible; colonies of freshly isolated human tumor cells can also be quantified. Thus, a microcapillary-based human tumor cloning assay that tests for resistance and/or sensitivity to chemotherapeutic agents may be useful in drug development programs and may also facilitate the development of chemotherapy for individual patient tumors, especially when tumor availability is limited.

Myelotoxic effects of the bifunctional alkylating agent bizelesin on human, canine and murine myeloid progenitor cells.

Bizelesin is a potent synthetic derivative of the anticancer agent CC-1065 that preferentially alkylates and binds the minor grove of DNA. Preclinical animal studies have found bizelesin to be more toxic to beagle dogs than to rodents and that myelosuppression was the dose-limiting toxicity. This toxicity was dose- and time-dependent in all species. Due to the significant difference in the in vivo myelotoxicity between species, it was important to determine which one most closely resembles humans on a pharmacodynamic basis. Therefore, hematopoietic clonal assays were utilized to evaluate the effects of bizelesin on granulocyte-macrophage (CFU-gm) colony formation. Marrow cells were exposed in vitro to bizelesin (0.001-1000 nM) for 1 or 8 h and then assayed for colony formation. There was a 3-log difference in drug concentration at which 100% colony inhibition occurred (1 or 8 h) for murine CFU-gm versus human or canine CFU-gm. The IC70 value after an 8-h bizelesin exposure for human CFU-gm (0.006 +/- 0.002 nM) was 2220-times lower than for murine CFU-gm (13.32 +/- 8.31 nM). At any given concentration, an 8 h drug exposure resulted in greater colony inhibition than a 1 h exposure for all species (P < 0.05). Increasing exposure time from 1 to 8 h increased toxicity to human and canine CFU-gm much more than to murine CFU-gm. The clinically formulated drug solution was a more potent inhibitor of human colony formation than drug dissolved in DMSO. The IC70 value after a 1-h exposure was 1.7 times lower for human CFU-gm with formulated bizelesin (0.106 +/- 0.105 nM) than bulk drug in DMSO (0.184 +/- 0.044 nM). The results of these in vitro clonal assays were qualitatively consistent with those seen in whole animal studies, suggesting that bizelesin will be a potent myelosuppressive agent in the clinic. Since the dose-limiting toxicity in preclinical models is myelosuppression and the in vitro sensitivity of human and canine CFU-gm is similar, the canine maximum tolerated dose (MTD) is better than the murine MTD to determine a safe starting dose for phase I clinical trials.

In vivo-in vitro correlation of myelotoxicity of 9-methoxypyrazoloacridine (NSC-366140, PD115934) to myeloid and erythroid hematopoietic progenitors from human, murine, and canine marrow.

BACKGROUND: 9-Methoxypyrazoloacridine (PZA) is an anticancer agent that shows selectivity of action for carcinomas over leukemias. It also has nearly equal potency against cycling and quiescent or hypoxic and normoxic target cells. Phase I trials of PZA in humans are nearing completion. PURPOSE: This study was conducted to determine (a) if PZA is directly inhibitory to hematopoietic cells and, if it is, to characterize the inhibition pharmacodynamically, (b) whether species-specific differences in direct toxicity could explain differences in myelosuppression in mice, dogs, and humans, and (c) whether in vitro data correlate with in vivo myelosuppression data. METHODS: In vitro clonogenic assays of hematopoietic progenitors of myeloid and erythroid lineages from human, canine, and murine femoral marrow were used to measure the direct toxicity of PZA. Results from these assays were compared on an area-under-the-curve (AUC) basis to clinical myelosuppression data. RESULTS: On the basis of maximum tolerated concentrations, canine hematopoietic progenitors are most susceptible to PZA, followed by human and then murine progenitors. We found no difference in susceptibility to PZA toxicity between the human progenitors of myeloid and erythroid lineages. Both concentration and duration of exposure contribute to the in vitro toxicity of PZA. In contrast to antimetabolites, the in vitro toxicity of PZA could be minimized at a given AUC by lowering drug concentration and prolonging the period of exposure. On an AUC basis, the in vitro data are consistent with limited in vivo myelosuppression data from preclinical models and correlate with neutropenia data from a phase I trial. CONCLUSIONS: PZA directly inhibits hematopoietic progenitors, an action that is responsible for the myelosuppression observed in humans. Human marrow appears able to compensate for the loss of up to 35% of its myeloid progenitors, in that peripheral neutrophil counts remain unchanged at that level of loss. Although in vivo studies show that prolonged infusion reduces myelosuppression at a given total dose in both rodent and canine models, pharmacokinetic differences make it unlikely that this approach will benefit human patients. IMPLICATIONS: The in vitro data quantitatively predict the AUCs at maximum tolerated dose in preclinical models and human patients. Thus, in vitro clonogenic assays of myelotoxic agents can provide data that make both preclinical toxicology testing and clinical trial planning and interpretation more efficient and accurate.

Megakaryocyte colony-stimulating factor (Meg-CSF) is a unique cytokine specific for the megakaryocyte lineage.

The regulation of megakaryocytopoiesis and platelet production has not yet been clearly elucidated. Several cytokines have been shown to be capable of producing megakaryocyte colonies from bone marrow [i.e. Interleukin (IL)-3, granulocyte-macrophage (GM)-colony-stimulating factor (CSF), erythropoietin (Epo)]. In addition, other activities have been reported to stimulate megakaryocyte precursors, yet a megakaryocyte-CSF (Meg-CSF) has not been purified to homogeneity and IL-3, GM-CSF and/or Epo often contaminate purification attempts which could account for the activities. A Meg-CSF has been isolated from the urine of patients with aplastic anaemia and purified by sequential ultrafiltration, cation exchange, G-50 chromatography, preparative PAGE, chromatofocusing and cation exchange HPLC. The activity of this material is 2-4 x 10(4) CFU-Meg/mg as measured in a murine marrow, serum-containing assay. This activity also stimulates CFU-Meg in the absence of adherent accessory cells and in serum-free cultures, indicative of the direct stimulation on CFU-Meg. Immunoassays, colony forming assays, and proliferation assays demonstrate that purified Meg-CSF has no GM-CSF, IL-3, M-CSF, G-CSF or IL-1 alpha, -3, -6, -9 and -11. In confirmation of these results, neutralizing antibody to IL-6 also did not abrogate Meg-CSF activity. Therefore the previously-reported megakaryocyte colony-stimulating activity in purified aplastic anaemia patient urine is due to a unique cytokine: Meg-CSF.