Characterization of a three-drug nonlinear mixture response model.

Our group recently developed a response-surface modeling paradigm (White et al: Curr Drug Metab 2, 399-409, 2003) and tested its application to both mixtures of anticancer agents and antifungals. This new model is a Hill-type equation, with the slope and potency parameters being functions of the normalized drug ratios, using polynomial expressions. Response surface methods allow one to model and interpret all of the information present in the full concentration-effect data set, to visualize local regions of synergy, additivity and antagonism, and even to quantify the degree of synergy or antagonism, both globally, and across local regions of the response surface. In the present article, we study the effect of changes in the different parameters of the polynomial expressions for two-drug and three-drug mixtures, on the geometrical shapes of several 2-dimensional representations of the 3-dimensional concentration-effect surface. A secondary goal of this report is to compare the mathematical characteristics of the rival White and (Minto et al: Anesthesiol 92, 1603-1616, 2000) modeling paradigms.

D-optimal designs for parameter estimation for indirect pharmacodynamic response models.

This report generates efficient experimental designs (dose, sampling times) for parameter estimation for four basic physiologic indirect pharmacodynamic response (IDR) models. The principles underlying IDR models and their response patterns have been well described. Each IDR model explicitly contains four parameters, k (in) (production), k (out) (loss), I (max)/S (max) (capacity) and IC (50)/SC (50) (sensitivity). The pharmacokinetics of an IV dose of drug described by a monoexponential function of time with two parameters, V and k (el), is assumed. The random errors in the response variable are assumed to be additive, independent, and normal with zero mean and variance proportional to some power of the mean response. Optimal design theory was used extensively to assess the role of both dose and sampling times. Our designs were generated in Mathematica (ADAPT 5 typically produces identical results). G-optimality was used to verify that the generated designs were indeed D-optimal. Such designs are efficient and robust when good prior knowledge of the estimated parameters is available. The efficiency of unconstrained D-optimal designs (4 dose, sampling time pairs) does not improve much when the drug doses are allowed to differ, compared with constrained single dose designs (4 sampling times) with one maximal feasible dose. Also, explored were efficiencies of alternative study designs and results from parameter misspecification. This analysis substantiates the importance of larger doses yielding greater certainty in parameter estimation in pharmacodynamics.

Extrapolation concepts for dealing with multiple contamination in environmental risk assessment.

Mixture exposure against several stressors is what organisms in the environment typically experience, and combined effects resulting from this are well documented. Risk management often still focuses on the assessment of individual priority compounds. Because of the large number of possible mixtures and their variability in time, experimental investigation of every conceivable mixture for their adverse effects is not a viable option. Instead, during the past decade, modeling approaches have been discussed in ecotoxicology that allow the prediction of expected combination effects based on the knowledge of the biological activity of the individual components. This contribution summarizes mixture exposure situations where consensus has been achieved about extrapolation techniques. In particular, for simultaneous exposure and for sequential exposure with no intervening recovery, currently available evidence demonstrates reasonable predictability of combined effects from the information of the individual mixture components. By contrast, when organisms are exposed to pulsed exposure with recovery periods or when nonchemical stressors interfere, there is still an open research field as how to account for these types of interaction.

Simultaneous modeling of concentration-effect and time-course patterns in gene expression data from microarrays.

BACKGROUND: Time-course and concentration-effect experiments with multiple time-points and drug concentrations provide far more valuable information than experiments with just two design-points (treated vs. control), as commonly performed in most microarray studies. Analysis of the data from such complex experiments, however, remains a challenge. MATERIALS AND METHODS: Here we present a semi-automated method for fitting time profiles and concentration-effect patterns, simultaneously, to gene expression data. The submodels for time-course included exponential increase and decrease models with parameters, such as initial expression level, maximum effect, and rate-constant (or half-time). The submodel for concentration-effect was a 4-parameter Hill model. RESULTS: The method was applied to an Affymetrix HG-U95Av2 dataset consisting of 51 arrays. The specific study focused on the effects of two platinum drugs, cisplatin and oxaliplatin, on A2780 human ovarian carcinoma cells. Replicates were available at most time points and concentrations. Eighteen genes were selected, and after selection, time-course and concentration-effect were modeled simultaneously. CONCLUSION: Comparisons of model parameters helped to distinguish genes with different expression patterns between the two drug treatments. This overall paradigm can help in understanding the molecular mechanisms of the agents, and the timing of their actions.

Modeling the combination of amphotericin B, micafungin, and nikkomycin Z against Aspergillus fumigatus in vitro using a novel response surface paradigm.

Response surface methods for the study of multiple-agent interaction allow one to model all of the information present in full concentration-effect data sets and to visualize and quantify local regions of synergy, additivity, and antagonism. In randomized wells of 96-well plates, Aspergillus fumigatus was exposed to various combinations of amphotericin B, micafungin, and nikkomycin Z. The experimental design was comprised of 91 different fixed-ratio mixtures, all performed in quintuplicate. After 24 h of drug exposure, drug effect on fungal viability was assessed using the tetrazolium salt 2,3-bis {2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide} (XTT) assay. First, we modeled each fixed-ratio combination alone using the four-parameter Hill concentration-effect model. Then, we modeled each parameter, including the 50% inhibitory concentration (IC(50)) effect, versus the proportion of each agent using constrained polynomials. Finally, we modeled the three-agent response surface overall. The overall four-dimensional response surface was complex, but it can be explained in detail both analytically and graphically. The grand model that fit the best included complex polynomial equations for the slope parameter m and the combination index (equivalent to the IC(50) for a fixed-ratio concentration, but with concentrations normalized by the respective IC(50)s of the drugs alone). There was a large region of synergy, mostly at the nikkomycin Z/micafungin edge of the ternary plots for equal normalized proportions of each drug and extending into the center of the plots. Applying this response surface method to a huge data set for a three-antifungal-agent combination is novel. This new paradigm has the potential to significantly advance the field of combination antifungal pharmacology.

Platinum drug effects on the expression of genes in the polyamine pathway: time-course and concentration-effect analysis based on Affymetrix gene expression profiling of A2780 ovarian carcinoma cells.

PURPOSE: As a follow-up to our previous findings that platinum drugs induce a key enzyme in polyamine catabolism, gene expression profiling and mathematical modeling were used to define the effects of cisplatin and oxaliplatin on the expression of polyamine metabolic pathway genes in A2780 human ovarian carcinoma cells. METHODS: Time-course and concentration-effect experiments were each carried out with cisplatin or oxaliplatin in two separate experiments and cells subjected to gene expression profiling using Affymetrix array technology. Time-course data were modeled using exponential increase and decrease models. Concentration-effect data were modeled using a four parameter Hill model. RESULTS: Gene expression profiling of human ovarian carcinoma A2780 cells after exposure to either cisplatin or oxaliplatin indicates that the expression of several genes involved in polyamine pathway is affected by the platinum drugs. Mathematical/Statistical modeling of the data from time-course and concentration-effect experiments of gene expression from nine polyamine pathway genes represented on the HGU95Av2 chip, indicates that three biosynthetic pathway genes (SAMDC, ODC1 and SRM) are down-regulated and one catabolic pathway gene (SSAT) is up-regulated. Expression changes were similar for different probesets for a given gene on the array. Studies on the induction of SSAT by platinum drugs suggested by the Affymetrix data have been previously validated from this laboratory (Hector et al. in Mol Cancer Ther 3:813-822, 2004). Here, the effects of oxaliplatin exposure on SAMDC and ODC observed by Affymetix are validated with real time QRT-PCR. CONCLUSION: The data indicate a concerted effect of platinum drugs on the polyamine metabolic pathway with down-regulation in the expression of several enzyme genes involved in biosynthesis and many-fold up-regulation in expression of SSAT, an acetylating enzyme gene that is critically involved in polyamine catabolism and export.

On the mode of action of N-phenyl-2-naphthylamine in plants.

N-phenyl-2-naphthylamine, a sediment contaminant previously identified as a major toxicant of site-specific importance was investigated for its mode of toxic action. From short-term bioassays with daphnids, fish eggs, bacteria, and algae it appears that this compound has specific phytotoxic properties at concentrations below 100 microg/L, which cannot be explained assuming an unspecific narcosis type of action in plants. Also, hydroxy-, nitro-, and methylderivatives show clear excess toxicity as compared to baseline toxic effects. Of several plant-specific growth and development processes investigated, only photosynthesis could be demonstrated to be affected at short exposure times and low concentrations. Disturbance of primary photosynthetic reactions such as oxygen evolution and fluorescence quenching, however, becomes only apparent after 2-3 h of exposure, which is in sharp contrast to known specific inhibitors targeting processes such as electron transport or ATP production. This, and concentration-time-effect modeling lead to the suggestion that N-phenyl-2-naphthylamine acts intracellular as a reactive compound in cell membranes producing irreversible, and thus cumulative, damage over time in algae. The effects may become first apparent in membrane-rich compartments such as the algal chloroplast.

Clinical pharmacokinetics of the PDT photosensitizers porfimer sodium (Photofrin), 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (Photochlor) and 5-ALA-induced protoporphyrin IX.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) uses a photosensitizer activated by light, in an oxygen-rich environment, to destroy malignant tumors. Clinical trials of PDT at Roswell Park Cancer Institute (RPCI) use the photosensitizers Photofrin, Photochlor, and 5-ALA-induced protoporphyrin IX (PpIX). In some studies the concentrations of photosensitizer in blood, and occasionally in tumor tissue, were obtained. Pharmacokinetic (PK) data from these individual studies were pooled and analyzed. This is the first published review to compare head-to-head the PK of Photofrin and Photochlor. STUDY DESIGN/MATERIALS AND METHODS: Blood and tissue specimens were obtained from patients undergoing PDT at RPCI. Concentrations of Photofrin, Photochlor, and PpIX were measured using fluorescence analysis. A non-linear mixed effects modeling approach was used to analyze the PK data for Photochlor (up to 4 days post-infusion; two-compartment model) and a simpler multipatient-data-pooling approach was used to model PK data for both Photofrin and Photochlor (at least 150 days post-infusion; three-compartment models). Physiological parameters were standardized to correspond to a standard (70 kg; 1.818 m2 surface area) man to facilitate comparisons between Photofrin and Photochlor. RESULTS: Serum concentration-time profiles obtained for Photofrin and Photochlor showed long circulating half-lives, where both sensitizers could be found more than 3 months after intravenous infusion; however, estimated plasma clearances (standard man) were markedly smaller for Photofrin (25.8 ml/hour) than for Photochlor (84.2 ml/hour). Volumes of distribution of the central compartment (standard man) for both Photofrin and Photochlor were about the size (3.14 L, 4.29 L, respectively) of plasma volume, implying that both photosensitizers are almost 100% bound to serum components. Circulating levels of PpIX were generally quite low, falling below the level of instrument sensitivity within a few days after topical application of 5-ALA. CONCLUSION: We have modeled the PK of Photochlor and Photofrin. PK parameter estimates may, in part, explain the relatively long skin photosensitivity attributed to Photofrin but not Photochlor. Due to the potential impact and limited experimental PK data in the PDT field further clinical studies of photosensitizer kinetics in tumor and normal tissues are warranted.

Efficacy of lidocaine or bupivacaine combined with ephedrine in rat sciatic nerve block.

BACKGROUND AND OBJECTIVES: The adrenergic drug ephedrine inhibits Na(+) current in cultured cells expressing Na(+) channels and provides dose-dependent reversible rat sciatic nerve blockade. The dosage required for peripheral nerve blockade in humans would cause unacceptable cardiovascular side effects. We therefore hypothesized that either lidocaine or bupivacaine would show synergy with ephedrine in a rat sciatic nerve block model, allowing a dose reduction and limiting side effects while improving efficacy. METHODS: Sciatic nerves of anesthetized rats were exposed by lateral incision of the thighs; 0.2 mL of different concentrations of the following drugs was injected sub-fascially: bupivacaine, lidocaine, and ephedrine alone and bupivacaine or lidocaine combined with ephedrine (n=8 per group). After animals recovered from anesthesia, a blinded investigator evaluated motor blockade (push against a balance) and sensory/nociceptive blockade (to pinch of the fifth toe) at predefined time intervals. RESULTS: Ephedrine combined with bupivacaine interacted additively for both motor and sensory blockade, and ephedrine combined with lidocaine interacted antagonistically (sub-additive) for sensory blockade and additively for motor blockade. CONCLUSIONS: A combination of ephedrine with either lidocaine or bupivacaine is unlikely to allow a significant dose reduction because of a lack of synergy. Furthermore, the cardiovascular side effects will limit the maximal tolerable dosage of ephedrine.

Effect of amphotericin B and micafungin combination on survival, histopathology, and fungal burden in experimental aspergillosis in the p47phox-/- mouse model of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by recurrent life-threatening bacterial and fungal infections. We characterized the effects of single and combination antifungal therapy on survival, histopathology, and laboratory markers of fungal burden in experimental aspergillosis in the p47phox-/- knockout mouse model of CGD. CGD mice were highly susceptible to intratracheal Aspergillus fumigatus challenge, whereas wild-type mice were resistant. CGD mice were challenged intratracheally with a lethal inoculum (1.25 x 10(4) CFU/mouse) of A. fumigatus and received one of the following regimens daily from day 0 to 4 after challenge (n = 19 to 20 per treatment group): (i) vehicle, (ii) amphotericin B (intraperitoneal; 1 mg/kg of body weight), (iii) micafungin (intravenous; 10 mg/kg), or (iv) amphotericin B plus micafungin. The rank order of therapeutic efficacy based on prolonged survival, from highest to lowest, was as follows: amphotericin B plus micafungin, amphotericin B alone, micafungin alone, and the vehicle. Lung histology showed pyogranulomatous lesions and invasive hyphae, but without hyphal angioinvasion or coagulative necrosis. Treatment with micafungin alone or combined with amphotericin B produced swelling of invasive hyphae that was not present in mice treated with the vehicle or amphotericin B alone. Assessment of lung fungal burden by quantitative PCR showed no significant difference between treatment groups. Serum galactomannan levels were at background despite documentation of invasive aspergillosis by histology. Our findings showed the superior efficacy of the amphotericin B and micafungin combination compared to either agent alone after A. fumigatus challenge and also demonstrated unique features of CGD mice as a model for experimental aspergillosis.

Mild skin photosensitivity in cancer patients following injection of Photochlor (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a; HPPH) for photodynamic therapy.

PURPOSE: To measure skin photosensitivity in cancer patients infused with the new second-generation photodynamic sensitizer Photochlor (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a). A major disadvantage of using the clinically approved photosensitizer Photofrin is potentially prolonged and sometimes severe cutaneous phototoxicity. PATIENTS AND METHODS: Forty-eight patients enrolled in Phases 1 and 2 clinical trials underwent two or more exposures to four graded doses (44.4, 66.6, 88.8 or 133.2 J/cm2) of artificial solar-spectrum light (SSL) before and after administration of Photochlor at a dose of 2.5, 3, 4, 5 or 6 mg/m2 . RESULTS: The most severe skin response, experienced by only six of the subjects, was limited to erythema without edema and could only be elicited by exposure to the highest light dose. Conversely, eight subjects had no discernible reaction to SSL at any light dose. For nearly all the patients, the peak skin response was obtained when the interval between sensitizer injection and exposure to SSL was 1 day and, generally, their sensitivity to SSL decreased with increasing sensitizer-light interval. For example, a 2-day sensitizer-SSL interval resulted in less severe reactions than those obtained with the 1-day interval in 79% of the subjects, while 90% of the subjects exposed to SSL 3 days after Photochlor infusion had responses that were less severe than those obtained with either the 1- or 2-day sensitizer-SSL interval. CONCLUSIONS: Photochlor, at clinically effective antitumor doses, causes only mild skin photosensitivity that declines rapidly over a few days.

Sequential administration of irinotecan and cytarabine in the treatment of relapsed and refractory acute myeloid leukemia.

PURPOSE: Based on reported synergy of the topoisomerase-I (topo-I) inhibitor irinotecan with antimetabolites, irinotecan and cytarabine (Ara-C) were administered sequentially to patients with acute myeloid leukemia (AML) refractory to or relapsed following high-dose Ara-C and anthracycline therapy. Pharmacokinetic and pharmacodynamic studies were performed with the first irinotecan dose. EXPERIMENTAL DESIGN: In vitro synergy of irinotecan followed by Ara-C was confirmed in a human AML cell line as a basis for the clinical trial. Irinotecan was administered daily for 5 days, with Ara-C 1 g/m2 12 h after each irinotecan dose. Irinotecan was initiated at 5 mg/m2, and the dose was escalated by 5 mg/m2 increments in cohorts of three patients and in individual patients. Pre-treatment samples were studied for topo-I activity and serial samples after the first irinotecan dose were analyzed for pharmacokinetics and for pharmacodynamic effects, including DNA damage and DNA synthesis rate. RESULTS: The irinotecan dose reached 15 mg/m2 in three-patient cohorts without reaching the maximum tolerated dose, and reached 30 mg/m2 in individual patients. The AUC and Cmax of both irinotecan and its active metabolite SN38 increased linearly in proportion to dose, and the mean half-lives of irinotecan conversion to SN38 and SN38 elimination were 6.2 h (CV 171%) and 7.2 h (CV 48%). Irinotecan rapidly induced DNA damage, and DNA synthesis inhibition varied among patients and treatment cycles. All courses resulted in rapid cytoreduction, and two patients achieved complete remission. Topo-I activity did not predict response. CONCLUSION: Irinotecan can be safely administered with Ara-C. This combination is active in refractory AML and warrants further study.

Antiangiogenic chimeric anti-endoglin (CD105) antibody: pharmacokinetics and immunogenicity in nonhuman primates and effects of doxorubicin.

We generated a human/mouse chimeric antibody c-SN6j of human IgG1 isotype from a murine anti-human endoglin (EDG) monoclonal antibody (mAb) SN6j that suppressed angiogenesis, tumor growth and metastasis in mice. We determined pharmacokinetics (PKs) and immunogenicity of c-SN6j in monkeys after multiple i.v. injections. A dose-escalation study was performed by administration of c-SN6j into six monkeys at the dose of 1 mg, 3 mg and 10 mg per kg body weight. In addition, both c-SN6j (3 mg/kg) and doxorubicin (0.275 mg/kg) were injected into two monkeys. c-SN6j and doxorubicin were injected twice a week for 3 weeks. We developed a unique and sensitive ELISA by sequentially targeting the common and idiotypic epitopes of c-SN6j-Fv to quantify plasma c-SN6j. Application of the ELISA showed that increasing the c-SN6j dose resulted in a proportional increase in the circulating c-SN6j after the first injection. In addition, the estimated area under the curve (AUC) for the first injection of c-SN6j is proportional to dose. We carried out detailed analyses of PKs of c-SN6j during and after the repeated injections. Our model of PKs fitted the empirical data well. Addition of doxorubicin modulated the PK parameters. We developed two ELISAs to separately determine the immune responses to the murine part and the human part of c-SN6j in monkeys. Interestingly, the murine part induced a weaker immune response than the human part. Doxorubicin potentiated the immune responses. Increasing the dose of c-SN6j increased plasma levels of c-SN6j but did not increase the immune responses to c-SN6j.

Gene expression profiling of a clonal isolate of oxaliplatin-resistant ovarian carcinoma cell line A2780/C10.

The efficacy of platinum drugs in the treatment of cancer is often restricted by the acquisition of tumor cell resistance subsequent to treatment. To better understand mechanisms involved in this phenomenon, a clonal subline (A2780/C10B) isolated from an oxaliplatin-resistant human ovarian carcinoma cell line (A2780/C10) was developed, as reported previously. This cell line is 18-fold resistant to oxaliplatin and shows a 3-fold cross resistance to cisplatin. Here, we report on the gene expression analysis using Affymetrix HG-U95Av2 oligonucleotide arrays of cells in log phase growth from both the parental cell line and drug-resistant variant. Probe level analysis was perfomed using the model based expression index (dChip) and robust multichip average (RMA) methods. Genes that were differentially expressed between the two groups were identified using the significance analysis of microarrays (SAM) method with a minimum false discovery rate <1%. We identified 43 genes that were overexpressed, and 39 underexpressed in the drug-resistant cell line. Collagen VI (COL6A3) was overexpressed 62-fold and the most highly up-regulated gene. This finding is consistent with other published data based on serial analysis of gene expression (SAGE) profiling of cisplatin-resistant and sensitive ovarian carcinoma cells. Among the significant functional groups of overexpressed genes in our study were extracellular matrix genes (9 of 43) and those involved in signal transduction (7 of 43). Extracellular matrix genes included two matrix metalloproteinases (MMP3 and MMP12). Integrin alpha 1 (ITGA1) and WNT5A were also overexpressed. Genes that encode for extracellular matrix proteins were also among those found down-regulated in the resistant cell line. Several genes involved in the regulation of cell cycle and growth were found to be underexpressed, including the suppressor of cytokine signaling 2 (SOCS2), necdin (NDN), and glypicans (GPC3 and GPC4). The mRNA levels of six differentially expressed genes (COL6A3, MMP12, MMP3, WNT5A, NID, and HMGB2) were validated using real-time quantitative RT-PCR. The identification of these genes should aid in a better understanding of the pathways resulting in platinum drug resistance.

Cyclosporin A is a broad-spectrum multidrug resistance modulator.

PURPOSE: Overexpression of the multidrug resistance proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) is associated with treatment failure in acute myeloid leukemia (AML) and other malignancies. The Pgp modulator cyclosporin A has shown clinical efficacy in AML, whereas its analogue PSC-833 has not. Cyclosporin A is known to also modulate MRP-1, and we hypothesized that broad-spectrum multidrug resistance modulation might contribute to its clinical efficacy. EXPERIMENTAL DESIGN: We studied the effects of cyclosporin A and PSC-833 on in vitro drug retention and cytotoxicity in resistant cell lines overexpressing Pgp, MRP-1, and BCRP and on nuclear-cytoplasmic drug distribution and cytotoxicity in cells overexpressing LRP. Cellular drug content was assessed by flow cytometry and nuclear-cytoplasmic drug distribution by confocal microscopy. RESULTS: Cyclosporin A enhanced retention of the substrate drug mitoxantrone in cells overexpressing Pgp (HL60/VCR), MRP-1 (HL60/ADR), and BCRP (8226/MR20, HEK-293 482R) and increased cytotoxicity 6-, 4-, 4-, and 3-fold, respectively. Moreover, cyclosporin A enhanced nuclear distribution of doxorubicin in 8226/MR20 cells, which also express LRP, and increased doxorubicin cytotoxicity 12-fold without an effect on cellular doxorubicin content, consistent with expression of wild-type BCRP, which does not efflux doxorubicin. Cyclosporin A also enhanced nuclear doxorubicin distribution in a second cell line with LRP overexpression, HT1080/DR4. PSC-833 enhanced mitoxantrone retention and cytotoxicity in cells overexpressing Pgp, but had no effect in cells overexpressing MRP-1, BCRP, or LRP. CONCLUSIONS: Cyclosporin A modulates Pgp, MRP-1, BCRP, and LRP, and this broad-spectrum activity may contribute to its clinical efficacy.

Breast cancer resistance protein (BCRP/MXR/ABCG2) in adult acute lymphoblastic leukaemia: frequent expression and possible correlation with shorter disease-free survival.

Drugs used in treatment of adult acute lymphoblastic leukaemia (ALL) are substrates for breast cancer resistance protein (BCRP, MXR, ABCG2), which may thus play a role in resistance in this disease. Pretreatment blasts from 30 adult ALL patients were studied for BCRP mRNA by quantitative reverse transcription polymerase chain reaction analysis, BCRP protein by immunophenotyping with three antibodies and BCRP function by fumitremorgin C modulation of intracellular mitoxantrone retention, measured by flow cytometry. BCRP mRNA in all cases encoded wild type protein (BCRP(R482)), which mediates mitoxantrone and methotrexate resistance, but only low-level anthracycline resistance. The BXP-21, BXP-34 and anti-ABCG2 antibodies stained blasts in 13, 11 and 14 cases (43%, 37% and 47%); BXP-21 correlated well with BXP-34 and anti-ABCG2, but BXP-34 and anti-ABCG2 did not correlate, and antibody staining did not correlate with mRNA levels. BCRP function was seen in 21 cases (70%), but correlated poorly with antibody staining. An exploratory statistical analysis indicated that BXP-21 staining was predictive of shorter disease-free survival (DFS) (P = 0.0374) in this small patient population. Poor correlations between mRNA, protein and function indicate the complex biology of BCRP in adult ALL, and the possible correlation of BCRP expression with DFS should be studied in larger series.

Expression of the neural cell adhesion molecule CD56 is not associated with P-glycoprotein overexpression in core-binding factor acute myeloid leukemia.

Acute myeloid leukemia (AML) with rearrangement of the core-binding factor (CBF) alpha or beta subunit gene has a favorable prognosis, but CD56 expression in CBFalpha-AML is associated with short disease-free survival. A proposed mechanism is overexpression of the multidrug resistance (MDR) protein P-glycoprotein (Pgp). CD56 expression, Pgp expression and function, and expression of the additional MDR proteins multidrug resistance protein-1 (MRP-1), lung resistance protein (LRP) and breast cancer resistance protein (BCRP) were studied in pretreatment blasts from 25 CBF-AML patients. CD56 expression was frequent in CBFalpha but rare in CBFbeta, and Pgp expression and function were frequent in both subtypes. CD56 expression did not correlate with Pgp expression or function, nor with expression of the other MDR proteins. Treatment failure associated with CD56 expression in CBFalpha-AML is not likely attributable to Pgp.

Prostate derived Ets transcription factor shows better tumor-association than other cancer-associated molecules.

We previously reported that prostate derived Ets transcription factor (PDEF) is a breast tumor-associated molecule. To obtain further insights into PDEF expression in other human tumor types, a cDNA library database from human adult normal and tumor tissues was compiled and searched for PDEF distribution. The results showed that PDEF is present at relative higher frequencies in the cDNA libraries from brain, breast, lung and ovarian tumors in comparison to those from the corresponding normal tissues. RT/PCR analysis of PDEF expression in ovarian tumors confirmed that PDEF is expressed in 36 out of 51 (71%) ovarian tumors. Further comparison of the distribution of PDEF with other widely recognized cancer-associated molecules showed that PDEF has more restricted distributions than Her-2/neu, Bcl-2, survivin or telomerase in cDNA libraries from normal human tissues and more increased distribution than Her-2/neu, CA-125, Bcl-2, survivin and telomerase in cDNA libraries from brain (except survivin), breast, lung and ovarian tumors. These data together show a better tumor-association for PDEF and suggest that PDEF is a more suitable target for developing specific cancer therapies.

Photodynamic therapy: a means to enhanced drug delivery to tumors.

Using the photosensitizer 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a, we have determined that photodynamic therapy (PDT) can be used to facilitate the delivery of macromolecular agents. PDT regimens that use low fluences and fluence rates were the most successful. This effect was demonstrated for fluorescent microspheres with diameters ranging from 0.1 to 2 microm. Such treatment given immediately before administration of Doxil, a liposomally encapsulated formulation of doxorubicin with an average diameter of 0.1 microm, significantly enhanced its accumulation in transplanted murine Colo 26 tumors. The combination of PDT and Doxil led to a highly significant potentiation in tumor control without concomitant enhancement of systemic or local toxicity. Interestingly, concentration-effect modeling suggested that the enhanced cure rate was greater than what was predicted based on the increase in intratumor Doxil concentration. In summary, we have developed a novel PDT treatment that enhances the delivery and efficacy of macromolecule-based cancer therapies such as Doxil.