Enhancing drug discovery through in silico screening: strategies to increase true positives retrieval rates.

Computational chemistry software for lead discovery has become well established in pharmaceutical industry and has found its way to the desktop computers of medicinal chemists for different purposes, providing insight on the mode of action and binding properties, and creating new ideas for lead structure refinement. In this review we investigate the performance and reliability of recent state-of-the-art data modeling techniques, as well as ligand-based and structure-based modeling approaches for 3D virtual screening. We discuss and summarize recently published success stories and lately developed techniques. Parallel screening is one of these emerging approaches allowing for efficient activity in silico profiling of several compounds against different targets or anti-targets simultaneously. This is of special interest to medicinal chemists, as the approach allows revealing unknown binding modes ('target-fishing') as well as integrated ADME profiling or--more generally--the prediction of off-target effects.

Human adherent cell contact-mediated modulation of normal myeloid colony formation.

The effects of coincubation of normal nonadherent bone marrow cells on adherent monolayers created from human peripheral blood mononuclear cells or marrow cells were investigated. Nonadherent marrow cells were coincubated for 4 hours with peripheral blood adherent cells at ratios of adherent cells to marrow cells of 2:1 to 5:1. This coincubation suppressed subsequent neutrophilic agar colony growth but not eosinophilic growth. Further studies suggested that this suppression was a cell-cell-mediated process and not secondary to soluble factors. However, coincubation on marrow adherent cells caused increased neutrophilic colony recovery. The possible in vivo relevance is discussed.

Effect of diethylaminoethyl-dextran on colony formation of human tumor cells in semisolid suspension cultures.

The effect of diethylaminoethyl-dextran on colony formation of established and primary human ovarian and breast tumor cells in semisolid suspension cultures was investigated. At the concentration of 250 to 300 micrograms/ml used in the human tumor stem cell assay, diethylaminoethyl-dextran inhibited in vitro growth of 3 of 4 human breast tumor cell lines and of 10 of 19 primary tumors. It did not affect growth in 7 primary tumors and enhanced colony formation in 2 tumors. We suggest that diethylaminoethyl-dextran may interact in the human tumor stem cell assay with other sites additional to sulfuric acid residues of the agar and that these effects may cancel or outweigh its purported beneficial effects.

Normalization of in vitro sensitivity testing of human tumor clonogenic cells.

The use of normal bone marrow (granulocyte-macrophage colony-forming units) as a point of reference to normalize the in vitro activities of anticancer agents has been investigated. The cytotoxic effects of four substituted anthraquinone derivatives, and of vinblastine on myeloid progenitors of different donors were reproducible up to a cell kill of approximately 60%. Equitoxic in vitro concentrations for normal bone marrows did not correlate with in vivo pharmacokinetic concentrations of these drugs. Breast tumor progenitor cells of 46 specimens were more sensitive than were bone marrow progenitors to the anthraquinone derivatives in 26 to 39% of instances, ratios which are similar to the clinically observed response rates of patients with breast carcinoma to these agents. Tumors were either sensitive or resistant to all four drugs in 68% (10 tumors were more sensitive, and 21 tumors were less sensitive than normal bone marrow); but in 32% of instances there were differences in tumor sensitivity for the four drugs, and the assay could select one to three drugs for which the tumor sensitivity was greater than that of bone marrow. Correlations of in vitro sensitivity and of clinical response to single agent treatments were determined in 21 patients, and the concordance was 71%. The value of the assay in predicting clinical response ranked best for sensitivity determinations within the normalized dose ranges, when testing within three different dose ranges was compared in a group of six patients. The concordance was higher in the small (1 or 2 metastatic sites) than in the large (greater than or equal to 3 metastatic sites) tumors (85 versus 50%), indicating a confounding influence of tumor load on the ability of the assay to predict efficacy of treatment. A rule of thumb is proposed for altering the in vitro sensitivity test results for large tumors that improves the overall concordance to 90%.

In vitro activity of bleomycin, tallysomycin S10b, and liblomycin against fresh human tumor cells.

The objective of this study was to compare the relative in vitro cytotoxicity of bleomycin to that of two newer-generation analogues, tallysomycin S10b and liblomycin. The latter compound is of particular interest as it has recently been shown in preclinical studies to be free of a potential to cause pulmonary injury and yet to possess only a minor potential to produce myelotoxicity. Using the adhesive tumor cell culture system, we evaluated the activity of these three drugs against a panel of 13 human tumors of various types. The range of concentrations chosen was determined and normalized using a nonleukemic permanent mouse hematopoietic progenitor cell line. Those drug concentrations achieving 90% inhibition of growth (IC90) against the murine cell line were: 6.11 microM bleomycin; 7.53 microM tallysomycin S10b; and 0.6 microM liblomycin. When tested against fresh human tumors at equally myelotoxic IC90 concentrations, bleomycin and tallysomycin S10b (nonmyelotoxic compounds) both achieved 90% growth inhibition of all tumors, while liblomycin (a myelotoxic compound) produced an IC90 inhibition in 69% of all tumors. A comparison of drug IC90 values against individual fresh tumors indicated a correlation between bleomycin and its structurally related analogue tallysomycin S10b. No such correlation, however, was seen with liblomycin in comparison to either bleomycin or tallysomycin S10b. The relative activity of liblomycin versus that of bleomycin and tallysomycin S10b varied with individual tumors tested. The response rate of liblomycin, a myelotoxic compound within this normalized range, appears promising. These data represent the first comparison of liblomycin to bleomycin against a spectrum of fresh human tumors using a stem cell assay technique.

Correlation of DNA distribution abnormalities with cytogenetic findings in human adult leukemia and lymphoma.

Pulse cytophotometric analysis of bone marrow cells from 175 patients with leukemia or lymphoma showed abnormalities of cellular DNA distribution in 29 patients for an overall incidence of 16.6 percent. Comparative standard cytogenetic examination indicated that high-degree chromosomal aberrations (less than or equal to 44, greater than or equal to 53 chromosomes) can generally be detected on DNA histograms, whereas patients with diploid, pseudodiploid, 45-hypodiploid, and 47-hyperdiploid abnormalities usually escape recognition by this technique. There were 11 patients with normal diploid or near-diploid karyotypes exhibiting marked DNA deviations; this discrepancy may reflect lack of proliferation of some leukemic clones which is a prerequisite for cytogenetic identification.

Possible mechanisms of action of lithium on augmentation of in vitro spontaneous myeloid colony formation.

To understand the possible mechanisms of lithium carbonate-induced neutrophilia, the in vitro effect on human myeloid progenitor cells was examined. A significant increase in spontaneous colony formation (15 of 24 experiments) was observed with the addition of lithium. Increased colony formation seldom occurred when human placental conditioned media as a source of colony-stimulating activity (CSA) was simultaneously added to the cultures. Further data suggest that lithium requires an adherent marrow cell population for this action and that increases in CSA-containing cultures may be due to suboptimal CSA concentrations. Lithium was shown to release CSA from marrow cells and adherent cell population prepared from human bone marrow. Lithium possibly increases spontaneous human myeloid colony development indirectly through CSA release by adherent cells.

Constitutive and induced expression of growth factors in normal and chronic phase chronic myelogenous leukemia Ph1 bone marrow stroma.

Study of growth factor RNA levels in the stromal cells derived from the adherent layer of long-term bone marrow culture demonstrated constitutive expression of transforming growth factor beta 1 (TGF-beta 1) and macrophage colony-stimulating factor. These cells did not express granulocyte colony-stimulating factor, granulocyte-monocyte colony-stimulating factor, interleukin (IL) 1 alpha, IL-1 beta, IL-3, and IL-6. However, granulocyte colony-stimulating factor expression could be induced by recombinant human IL-1 beta; while IL-6 could be induced by both IL-1 beta and tumor necrosis factor-alpha. No differences could be detected between adherent layers established from normal and benign phase Ph1 chronic myelogenous leukemia bone marrow. The uninduced expression of TGF-beta 1, a potent hematopoietic cell growth inhibitor, suggests that stromal cells play an inherent role in regulating the proliferation of adjacent bone marrow hematopoietic progenitor cells. However, a defect in stromal TGF-beta 1 production cannot account for the profoundly expanded myeloid compartment in chronic phase chronic myelogenous leukemia. In contrast to the constitutive expression of TGF-beta 1 and macrophage colony-stimulating factor, hematopoietic growth factors are only expressed following a proper stimulation.

Antitumor activity against human tumor samples of cis-diamminedichloroplatinum(II) and analogues at equivalent in vitro myelotoxic concentrations.

We compared the antitumor activity of cis-diamminedichloroplatinum(II) (cisplatin; CDDP) with three CDDP analogues: cis-diammine-1,1-cyclobutanedicarboxylateplatinum(II) (CBDCA), N-methyliminodiacetato-1,2-diamino(cyclohexane)platinum(II) (MIDP), and N-(2-hydroxyethyl)-iminodiacetato-1,2-diamino(cyclohexane)platinum (II) (HIDP). Fresh human tumor samples in the adhesive tumor culture system were utilized for this comparison. The equitoxic concentrations of all four drugs were derived based on their inhibitory activity against human bone marrow samples. For these normalized concentrations, CDDP proved to have a higher cytotoxic activity than its analogues. CBDCA's in vitro activity had a significant correlation with CDDP activity (r = 0.67) in vitro. However, the structurally similar substances MIDP and HIDP demonstrated a much greater degree of association (r = 0.90). Our data suggest that CBDCA, HIDP, and MIDP have overall less activity than CDDP when tested at equitoxic in vitro concentrations. Close association between CDDP and CBDCA also reflects known clinical experience with these two drugs, suggesting the method of comparison used here is probably appropriate. These conclusions, however, must be validated by clinical trials.

Drug and radiation sensitivity measurements of successful primary monolayer culturing of human tumor cells using cell-adhesive matrix and supplemented medium.

The limitations of the agar suspension culture method for primary culturing of human tumor cells prompted development of a monolayer system optimized for cell adhesion and growth. This method grew 83% of fresh human tumor cell biopsy specimens, cultured and not contaminated, from a heterogeneous group of 396 tumors including lung cancer (93 of 114, 82%); melanoma (54 of 72, 75%); sarcoma (46 of 59, 78%); breast cancer (35 of 39, 90%); ovarian cancer (16 of 21, 76%); and a miscellaneous group consisting of gastrointestinal, genitourinary, mesothelioma, and unknown primaries (78 of 91, 86%). Cell growth was characterized morphologically with Papanicolaoustained coverslip cultures and cytogenetically with Giemsastained metaphase spreads. Morphological features such as nuclear pleomorphism, chromatin condensation, basophilic cytoplasm, and melanin pigmentation were routinely seen. Aneuploid metaphases were seen in 90% of evaluable cultures, with 15 of 28 showing 70% or more aneuploid metaphases. Colony-forming efficiency ranged between 0.01 and 1% of viable tumor cells, with a median efficiency of 0.2%. This culture system uses a low inoculum of 25,000 viable cells per well which permitted chemosensitivity testing of nine drugs at four doses in duplicate from 2.2 X 10(6) viable tumor cells and radiation sensitivity testing at five doses in quadruplicate from 0.6 X 10(6) cells. Cultures were analyzed for survival by computerized image analysis of crystal violet-stained cells. Drug sensitivity studies showed variability in sensitivity and in survival curve shape with exponential cell killing for cisplatin, Adriamycin, and etoposide, and shouldered survival curves for 5-fluorouracil frequently seen. Radiation sensitivity studies also showed variability in both sensitivity and survival curve shape. Many cultures showed exponential cell killing, although others had shouldered survival curves. This method for growing cells from primary human biopsy specimens is more efficient than the agar culture method, enables easier and better biological analysis of the actual cells grown, and permits improved characterization of drug and radiation survival curves.

Biological effect of epidermal growth factor on the in vitro growth of human tumors.

The effect of epidermal growth factor (EGF) on the in vitro growth of 186 malignant human tumor specimens (45 melanomas, 32 sarcomas, and 56 lung, 16 gynecological, 14 breast, 12 genitourinary, and 11 gastrointestinal carcinomas) was evaluated in the cellular adhesive matrix human tumor culture system supplemented with transferrin, insulin, hydrocortisone, and estradiol. EGF increased tumor growth by at least 50% in 81% of the 186 tumors and by over 100% in 54%. The enhanced growth induced by EGF was related to an accelerated cellular division independent of tumor type and not to an increase in the actual number of clonogenic units. The drug concentrations of cell cycle-independent Adriamycin and cisplatin needed to achieve a 90% tumor cell kill were not altered by the responsiveness of the tumor to EGF.

Involvement of chromosome 7 in primary lung tumor and nonmalignant normal lung tissue.

By using the newly developed adhesive tumor cell culture system, we analyzed the chromosomal constitutions of primary lung tumor and nonmalignant normal lung tissue from 10 previously untreated patients with non-small cell lung cancer. Chromosomal analyses were successfully carried out in banded chromosome preparations from 10 tumor and 8 normal lung tissue samples. All analyzed tumor and normal lung tissue samples had a predominantly normal diploid chromosome number. However, there was at least one structural or numerical alteration in every tumor and lung tissue sample analyzed. Chromosomes 1, 3, 4, 6, 7, 8, 9, 12, 15, and 20 were more often involved in rearrangement. The most consistent finding was trisomy 7; 4 patients had trisomy 7 in both tumor and normal lung tissue, and another 2 had this anomaly in tumor tissue only. Of the 4 patients without trisomy 7, 2 had a homogeneously staining region in the short arm of chromosome 7 in tumor tissue. Phytohemagglutinin-stimulated peripheral blood lymphocytes from 7 patients, including 5 patients with trisomy 7 in tumor tissue, did not show trisomy 7. These cytogenetic data suggest that chromosome 7 may be associated with lung cancer development and that trisomy 7 may be the hallmark of premalignant changes, at least in a subgroup of patients with non-small cell lung cancer.

The true predictive value of the human tumor stem cell assay: does a workable assay select for treatment responders?

In practice, the human tumor clonogenic assay is workable for less than half of the patient population to which it is applied, since the remainder of the specimens fail to produce sufficient numbers of colonies. Thereby a bias may be introduced which could result in a false predictive value positive of the test. It is therefore necessary to compare the responses to treatment of patients whose tumors could be assayed in vitro to those whose tumors failed to grow adequately, to assure that the prevalence of treatment responders has not changed within the group of patients for which the assay worked. From an analysis of the treatment response of 70 patients with stage III and IV ovarian carcinomas and 70 patients with stage IV breast cancer, no selection bias did occur and no preferential in vitro growth of tumor samples from patients with treatment response was found.

A phase II study of mitoxantrone, etoposide, and thiotepa with autologous marrow support for patients with relapsed breast cancer.

To further improve the effect of high-dose chemotherapy in the treatment of locally advanced and metastatic breast cancer, we sought to develop a second active high-dose noncross-resistant regimen to use in tandem with our customary high-dose regimen of cyclophosphamide, etoposide, and cisplatin (CVP). We performed a phase II trial of high-dose mitoxantrone 30 mg/m2, etoposide 200 mg/m2 every 12 hours x 6, and thiotepa 250 mg/m2 x 3 days (MVT) in 31 patients with heavily pretreated metastatic breast cancer and one with locally advanced chemotherapy-refractory breast cancer. These patients were ineligible for high-dose CVP chemotherapy because of the amount of prior treatment and poor-response status. Of the 32 patients, 14 responded to cycle 1, did not experience any grade 4 toxicity, and received a second cycle of MVT. Overall, seven of 31 patients achieved a complete response (CR; 23%). Four of the 14, who were partial responders to the first cycle, achieved a CR after the second cycle. The overall response rate was 19 of 31 (61%) with an overall median freedom from progression of 4 to 5 months and an overall median survival of 9 months. Toxicity consisted primarily of mucositis (grade 3 or 4 in 69%). The results indicate that high-dose MVT produces significant activity, even in heavily pretreated patients. Administration of a second cycle of high-dose therapy with MVT increased the CR rate, and the morbidity and mortality from the second cycle were not greater than that for the first cycle. Because of the high incidence of grade 3 or 4 mucositis with this regimen, we are currently completing a follow-up study of high-dose mitoxantrone and thiotepa alone.

Comparison between clinical response and in vitro drug sensitivity of primary human tumors in the adhesive tumor cell culture system.

The newly described adhesive tumor cell culture system (ATCCS) offers a distinct advantage over other assays in that it has a high plating efficiency requiring low cell inoculum, it affords workable assays in approximately 70% of specimens from the heterogenous tumor types, and it has the ability to assay up to nine drugs at four different concentrations. Clinical correlations based on the ATCCS were obtained in 65 patients undergoing 71 clinical trials. Patients with melanoma, lung cancer, and sarcoma dominated the group. The most active in vitro drug was correlated per clinical trial. Thirteen of 17 (76%) sensitive in vitro predictions and 51 of 54 (94%) resistant in vitro predictions were accurate. The assay in this study had a sensitivity of 81% and specificity of 93%. These preliminary results are encouraging and warrant prospective trials to establish the true value of this assay to patients.

Treatment of estrogen receptor-negative or hormonally refractory breast cancer with double high-dose chemotherapy intensification and bone marrow support.

We have used high-dose therapy followed by randomization to receive or not receive autologous bone marrow infusion in 58 stage IV breast cancer patients (all were estrogen receptor-negative [ER-] or primary hormonal refractory). Patients received a median of four courses of induction chemotherapy and if stable or responding received two courses of intensive therapy with cyclophosphamide 4.5 to 5.25 g/m2, etoposide 750 to 1,200 mg/m2, and cisplatin 120 to 180 mg/m2 (CVP). The complete remission (CR) rate after completion of the induction and intensive phases was 55%. Median progression-free interval from induction is 57 weeks with a projected 2-year progression-free survival of approximately 25%. Six of seven patients followed for greater than 2 years are still progression-free. Three favorable features predicted for improved progression-free survival are the following: (1) absent liver involvement, (2) absent soft tissue involvement, and (3) less than or equal to two disease sites (P values of .001, .013, and .048, respectively). Hormonal and menopausal status did not predict for progression-free survival. Major toxicities were infectious secondary to neutropenia, with a 93% incidence of fever and a 38% incidence of sepsis. The treatment-related mortality rate was 9%, with three infectious, one coronary, and one late hemorrhage-related death of unknown cause. Longer follow-up is still needed to truly evaluate the possibility of long-term disease-free survival, but further studies of this approach to high-dose intensification in poor prognostic groups of stage IV breast cancer appear warranted.

Piperazinedione, total body irradiation, and autologous bone marrow transplantation in chronic myelogenous leukemia.

Eleven patients with Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) in blast crisis (ten patients) or accelerated disease (one patient) were treated with piperazinedione (PIP) and fractionated total body irradiation (TBI) followed by autologous bone marrow transplantation (ABMT). Three patients were transplanted with marrow from which the Ph1 clone had been eradicated by prior intensive chemotherapy. All patients responded with disappearance of blasts in bone marrow and peripheral blood. Six patients achieved a second chronic phase lasting 3 to 14 months (median, 6 months). Two patients had incomplete recovery, and three patients failed to engraft and died from infection. Transplantation with Ph1-negative bone marrow did not improve response duration or survival. Recurrence of blast crisis and incomplete engraftment continue to be the two major problems in this patient group, and more active regimens need to be investigated.

High-dose intensification therapy with autologous bone marrow support for limited small-cell bronchogenic carcinoma.

The efficacy and toxicity of high-dose chemotherapy with autologous bone marrow transplantation (ABMT) was studied in 32 patients with untreated limited small-cell bronchogenic carcinoma (SCBC). Ten patients received three courses of induction therapy consisting of vincristine (VCR) (1.5 mg X 2), ifosfamide (5 g/m2), and Adriamycin (Ad; Adria Laboratories, Columbus, Ohio) (60 mg/m2). Patients then received two courses of intensification therapy with cyclophosphamide (CYT) (4.5 g/m2), 4' demethyl-epipodophyllotoxin-d-D-ethylidene glucoside (VP-16-213) (600 mg/m2) and VCR (2 mg) with ABMT. Another 22 patients received induction therapy with VCR, CYT (600 mg/m2), Ad (50 mg/m2), and VP-16-213 (180 mg/m2). All 22 patients also received intensification therapy of the same dose of CYT (4.5 g/m2) and VP-16-213 (600 mg/m2). Nine patients also received high-dose methotrexate (MTX), four patients received Ad (40 to 60 mg/m2), and two patients received both Ad and MTX. After intensification, patients received elective prophylactic brain irradiation (3,000 rad) and chest irradiation (5,000 rad). After induction therapy, there were 13 (41%) complete remissions (CR) and 17 (53%) partial remissions (PR). After intensification therapy, there were 22 CRs (69%) and 10 PRs (31%). Median survival for all patients was 14 months (range, 5 to 59+). Of the 13 patients who received intensification therapy in CR, five remain disease free (DF), four for 4 years or longer. Of the nine patients to achieve CR with intensification, only one is DF. No patient died during intensification. In conclusion, intensification with high-dose chemotherapy can increase the CR rate, and this approach is most likely to show long-term benefits in patients with minimal disease (CR) at the beginning of intensification therapy.

A comparison of marrow transplantation with chemotherapy for adults with acute leukemia of poor prognosis in first complete remission.

From July 1980 to November 1985, 109 patients with acute myelogenous and lymphoblastic leukemia who had reached a complete remission (CR) following induction treatment were assigned to a study comparing marrow transplantation with chemotherapy as a postremission treatment. Sixty-nine patients did not have a human leukocyte antigen (HLA)-identical donor, and therefore served as chemotherapy controls; 40 patients had HLA-identical donors, and therefore were assigned to the transplant arm. Of these, 23 were transplanted in first remission and 17 were not. Ten of these 17 were subsequently transplanted in relapse. Initially, only patients with poor prognosis determined by a predictive model were entered into the study. Subsequently, patients with moderately poor prognosis were admitted. Comparing the chemotherapy group with the patients transplanted in first CR, significant control of leukemia relapse in transplanted patients was seen in the subgroup with acute myelogenous leukemia (AML) (P less than .1), and acute lymphoblastic leukemia (ALL) (P less than .01), in the poor (P = .01) and intermediate subgroup (P = .01), and in the good-prognostic groups (P = .05). The survival was affected significantly in only the poor and intermediate subgroups. The use of predictive models might help to select patients for whom bone marrow transplantation is appropriate in first remission and those for whom bone marrow transplantation can be left as the initial treatment of relapse. Predictive models could further be helpful in comparing studies performed at different transplant centers.

Recombinant human granulocyte colony-stimulating factor hastens granulocyte recovery after high-dose chemotherapy and autologous bone marrow transplantation in Hodgkin's disease.

To determine whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) can accelerate granulocyte recovery after high-dose combination chemotherapy with autologous bone marrow transplantation (ABMT) in patients with Hodgkin's disease, we performed a nonrandomized phase II study using historical controls as a comparison. Eighteen relapsed/refractory Hodgkin's disease patients who received cyclophosphamide at 1.5 g/m2/day (days -6 to -3), carmustine (BCNU) at 300 mg/m2 (day -6), and etoposide (VP-16) at 125 mg/m2 every 12 hours (days -6 to -4), followed by ABMT (day 0) were treated with rhG-CSF at 60 micrograms/kg/day for a maximum of 28 days beginning on day 1. rhG-CSF dosage was gradually diminished and stopped once an adequate granulocyte count was maintained. rhG-CSF significantly accelerated absolute granulocyte count (AGC) compared with historical controls recovery to the 100/microL level (median, 9 days v 13 days; P = .103 x 10(-4), 500/microL level (median, 13 days v 22 days; P = 0.189 x 10(-2), and 1000/microL level (median, 16 days v 30 days levels; P = .125 x 10(-5). Platelet recovery to 50,000/microL was not significantly altered (P = .370). rhG-CSF was well tolerated, bone pain and myalgia being the only side effects noted. rhG-CSF hastens granulocyte recovery after high-dose chemotherapy with ABMT in patients with relapsed/refractory Hodgkin's disease without significant toxicity.