Doxorubicin and the alkylating anthracycline 3'-deamino-3'-(3-cyano-4-morpholinyl) doxorubicin: comparative in vitro potency against leukemia and bone marrow cells.

The new anthracycline analogue 3'-deamino-3'-(3-cyano-4-morpholinyl) doxorubicin (MRA-CN) is an intensely potent compound that has been shown to be 100-1,000 times more potent than doxorubicin (DOX) in vivo and in vitro. In addition, MRA-CN has been non-cross-resistant with DOX in DOX-selected models of multidrug resistance. We now report the effect of MRA-CN (and DOX) on leukemia cell lines established from patients with common, T-cell, and B-cell acute lymphoblastic leukemia, as well as with monoblastic leukemia. The effect of MRA-CN on the leukemia cells was compared to its toxicity on normal myeloid progenitors (therapeutic ratio) and to the effect of DOX on the leukemia and normal cells. MRA-CN was found to be 100 times more potent than DOX against normal myeloid progenitors--colony-forming units, granulocyte-macrophage (CFU-GM)--and 40-240 times more potent than DOX against leukemia cell lines. In addition, the therapeutic ratio was uniformly greater than 1, indicating that each leukemia cell line tested was more sensitive than CFU-GM to MRA-CN in vitro. There was a lack of correlation between MRA-CN and DOX at a drug concentration at which the colony formation is inhibited by 50% in the leukemia cell lines (correlation coefficient = 0.38), which supported the previous reports of non-cross-resistance between these two agents. The favorable therapeutic ratio, the non-cross-resistance with DOX, and the previously described lack of cardiac toxicity all make MRA-CN an attractive candidate for clinical trials in patients with acute leukemia.

Adaptive Design for a Confirmatory Basket Trial in Multiple Tumor Types Based on a Putative Predictive Biomarker.

Increasingly, tumors are defined on a molecular basis rather than only on histology, and targeted agents, which address these molecular subtypes, are being approved. This profusion of molecular subtypes creates "rare" diseases as subsets of common cancers, leading to difficulties in enrolling sufficiently large cohorts for confirmatory trials. However, if the molecular subtype is shared across various histologies, these may be pooled into a basket trial. To date, basket trials have been primarily for exploratory early development. In this perspective, we consider qualitative designs for confirmatory basket trials. These confirmatory basket designs will provide patients in niche indications with enhanced access to novel therapies, facilitate development and full approval for niche indications, allow accelerated approval for indications within a basket based on a surrogate endpoint, reduce development cost by combining trials, and enhance the ability of regulatory authorities to evaluate risk and benefit in niche indications.

PIPELINEs: Creating Comparable Clinical Knowledge Efficiently by Linking Trial Platforms.

Adaptive, seamless, multisponsor, multitherapy clinical trial designs executed as large scale platforms, could create superior evidence more efficiently than single-sponsor, single-drug trials. These trial PIPELINEs also could diminish barriers to trial participation, increase the representation of real-world populations, and create systematic evidence development for learning throughout a therapeutic life cycle, to continually refine its use. Comparable evidence could arise from multiarm design, shared comparator arms, and standardized endpoints-aiding sponsors in demonstrating the distinct value of their innovative medicines; facilitating providers and patients in selecting the most appropriate treatments; assisting regulators in efficacy and safety determinations; helping payers make coverage and reimbursement decisions; and spurring scientists with translational insights. Reduced trial times and costs could enable more indications, reduced development cycle times, and improved system financial sustainability. Challenges to overcome range from statistical to operational to collaborative governance and data exchange.

Oral topotecan as single-agent second-line chemotherapy in patients with advanced ovarian cancer.

PURPOSE: To evaluate oral topotecan as single-agent, second-line therapy in patients with ovarian cancer previously treated with a platinum-based regimen. PATIENTS AND METHODS: Patients (N = 116) received oral topotecan 2.3 mg/m2 daily for 5 days every 21 days. Eligibility criteria included histologic diagnosis of International Federation of Gynecology and Obstetrics stage III or IV epithelial ovarian cancer, bidimensionally measurable disease, prior platinum-containing chemotherapy, age > or = 18 years, performance status < or = 2, and life expectancy > or = 12 weeks. RESULTS: Overall response rate was 21.6% (25 of 116 patients). Median duration of response was 25.0 weeks; median time to response was 8.4 weeks. Median time to progression was 14.1 weeks; median survival was 62.2 weeks. Grade 4 neutropenia was experienced by 50.4% of patients in 13.4% of courses administered. Grade 4 thrombocytopenia was experienced by 22.1% of patients in 5.1% of courses. Grade 3 or 4 anemia was experienced by 29.2% of patients in 8.5% of courses. Most frequent nonhematologic toxicities were predominantly (> 90%) grade 1 or 2 and included nausea, alopecia, diarrhea, and vomiting. CONCLUSION: Second-line oral topotecan administered at 2.3 mg/m2 for 5 days every 21 days demonstrated activity in patients with progressive or recurrent ovarian cancer after first-line platinum-based chemotherapy. This activity was comparable to that seen in previous studies with intravenous topotecan. Grade 4 neutropenia was less frequent with oral topotecan than previously reported for intravenous topotecan. Oral topotecan is an active, tolerable, and convenient formulation of an established agent for the second-line treatment of advanced epithelial ovarian cancer and may also facilitate exploring prolonged treatment schedules.

A randomised trial of oral versus intravenous topotecan in patients with relapsed epithelial ovarian cancer.

A multicentre, randomised study was carried out in Europe, South Africa and North America to compare the activity and tolerability of oral versus intravenous (i.v.) topotecan in patients with relapsed epithelial ovarian cancer. Patients who had failed first-line therapy after one platinum-based regimen, which could have included a taxane, were randomised to treatment with either oral (p.o.) topotecan, 2.3 mg/m(2)/day or i.v. topotecan 1.5 mg/m(2)/day for 5 days every 21 days. Patients were stratified by prior paclitaxel exposure, interval from previous platinum therapy and tumour diameter. 266 patients were randomised. Response rates were 13% orally (p.o.) and 20% (i.v.) with a complete response in 2 and 4 patients, respectively. The difference in the response rates was not statistically significant. Median survival was 51 weeks (p.o.) and 58 weeks (i.v.) with a risk ratio of death (p.o. to i.v. treatment) of 1.361 (95% confidence interval (CI): 1.001, 1.850). Median time to progression was 13 weeks (p.o.) and 17 weeks (i.v.). The principal toxicity was myelosuppression although grade 3/4 neutropenia occurred less frequently in those receiving oral topotecan. Toxicity was non-cumulative and infectious complications were relatively infrequent. Non-haematological toxicity was generally mild or moderate. The incidence of grade 3/4 gastrointestinal events was slightly higher for oral than i.v. topotecan. Oral topotecan shows activity in second-line ovarian cancer and neutropenia may be less frequent than with the i.v. formulation. A small, but statistically significant, difference in survival favoured the i.v. formulation, but the clinical significance of this needs to be interpreted in the context of second-line palliative treatment. Oral topotecan is convenient and well tolerated and further studies to clarify its role are ongoing.

Pretreatment with ranitidine does not reduce the bioavailability of orally administered topotecan.

PURPOSE: The purpose of this randomized, two-period crossover study was to determine the pharmacokinetics of orally administered topotecan in the presence and absence of oral ranitidine. METHODS: Patients with solid malignant tumors refractory to standard treatment were given topotecan orally on a daily times five schedule repeated every 21 days. Topotecan was given initially at 2.3 mg/m2 per day; dose adjustments were permitted after the first dose of course 2 if necessary. Blood samples for pharmacokinetic assessments were drawn at protocol-specified times for up to 10 h following oral administration of topotecan on day 1 of courses 1 and 2. Patients were randomly assigned to receive a total of nine doses of ranitidine: 150 mg twice daily for 4 days before day 1 of one of the first two courses and 150 mg given 2 h before the first topotecan dose. Plasma samples were assayed for concentrations of active topotecan lactone (TPT-L) and total topotecan (TPT-T, lactone plus open-ring carboxylate form) using high-performance liquid chromatography with fluorescence detection. After completion of courses 1 and 2, patients could continue on therapy for days 1-5 of every 21 days if not withdrawn due to unacceptable toxicity, disease progression, protocol violation, or by request. Patients continued on treatment for a maximum of six courses. RESULTS: No pharmacokinetic parameter for either TPT-L or TPT-T differed significantly during administration of topotecan with ranitidine compared with topotecan alone (n = 13). Geometric mean ratios (95% confidence intervals, CIs) of areas under the curve in the presence and absence of ranitidine were 0.94 (0.80, 1.10) for TPT-L and 0.97 (0.80, 1.16) for TPT-T. Corresponding ratios (CIs) of peak plasma concentrations in the presence and absence of ranitidine were 1.06 (0.78, 1.44) for TPT-L and 1.07 (0.84, 1.38) for TPT-T. The median difference in time to peak plasma concentration was 0.0 h for TPT-L and -0.5 h for TPT-T (i.e. slightly faster in the presence of ranitidine). CONCLUSIONS: Administration of ranitidine prior to oral topotecan resulted in a similar extent of absorption. A slightly faster rate of absorption of topotecan was also observed, which is unlikely to be of clinical significance. Dosage adjustments of orally administered topotecan should not be necessary in patients who are pretreated with ranitidine, an H2 antagonist, or another agent that comparably raises gastric pH.

Multi-stage proofreading in DNA replication.

The mechanisms by which DNA polymerases achieve their remarkable fidelity, including base selection and proofreading, are briefly reviewed. Nine proofreading models from the current literature are evaluated in the light of steady-state and transient kinetic studies of E. coli DNA polymerase I, the best-studied DNA polymerase. One model is demonstrated to predict quantitatively the response of DNA polymerase I to three mutagenic probes of proofreading: exogenous pyrophosphate, deoxynucleoside monophosphates, and the next correct deoxynucleoside triphosphate substrate, as well as the response to combinations of these probes. The theoretical analysis allows elimination of many possible proofreading mechanisms based on the kinetic data. A structural hypothesis links the kinetic analysis with crystallographic, NMR and genetic studies. It would appear that DNA polymerase I proofreads each potential error twice, at the same time undergoing two conformational changes within a catalytic cycle. Multi-stage proofreading is more efficient, and may be utilized in other biological systems as well. In fact, recent evidence suggests that fidelity of transfer RNA charging may be ensured by a similar mechanism.

On the fidelity of DNA synthesis. Pyrophosphate-induced misincorporation allows detection of two proofreading mechanisms.

The effect of pyrophosphate on the fidelity of in vitro DNA synthesis has been examined. Pyrophosphate enhances misincorporation by Escherichia coli DNA polymerase I in copying phi X174 DNA. The increased misincorporation is directly proportional to the extent of inhibition of the rate of polymerization. In contrast, pyrophosphate is not detectably mutagenic with avian myeloblastosis virus DNA polymerase or DNA polymerases alpha and beta from animal cells, which lack associated proofreading activities. This suggests that increased misincorporation by pyrophosphate is not due to an increase in misinsertions by DNA polymerase, but rather due to inhibition of proofreading by pyrophosphate. However, the pyrophosphate-induced infidelity has a different specificity from, and is not competitive with, two experimental markers of 3'----5' exonuclease proofreading; i.e. the effects of the next nucleotide or the addition of deoxynucleoside monophosphates. These distinctive features suggest a second mode of proofreading susceptible to inhibition by pyrophosphate. This concept is discussed in relation to models for proofreading described in the literature.

On the fidelity of DNA replication: manganese mutagenesis in vitro.

Manganese is mutagenic in vivo and in vitro in studies with a variety of enzymes and templates. Using Escherichia coli DNA polymerase I with poly[d(A-T)] and phi X174 DNA templates, we analyzed the mechanism of manganese mutagenesis by determining the dependence of error rate on free Mn2+ concentration and comparing this to measured dissociation constants of Mn2+ from enzyme, template, and deoxynucleoside triphosphate substrates. This comparison suggests several conclusions: (1) At very low Mn2+ concentrations, the enzyme is activated at high fidelity. Thus, it is unlikely that activation with manganese per se significantly alters the conformation of the enzyme so as to affect nucleotide selection. (2) At low free Mn2+ concentrations (less than 100 microM), manganese causes errors in incorporation via its interaction with the DNA template. The concentration dependence of mutagenesis is determined by the strength of binding Mn2+ to the particular DNA template used. The data do not allow one to rule out the possibility that Mn2+-deoxynucleoside triphosphate interactions contribute to mutagenesis in selected situations. This range of free Mn2+ concentrations is the one of greatest relevance for in vivo mutagenesis. (3) At higher concentrations (between 500 microM and 1.5 mM), further mutagenesis by Mn2+ occurs. This mutagenesis probably is due either to binding of manganese to single-stranded regions within the DNA or to weak accessory sites on the enzyme.

Solution structure of horse heart ferricytochrome c and detection of redox-related structural changes by high-resolution 1H NMR.

A model for the solution structure of horse heart ferricytochrome c has been determined by nuclear magnetic resonance spectroscopy combined with hybrid distance geometry-simulated annealing calculations. Forty-four highly refined structures were obtained using a total of 1671 distance constraints based on the observed magnitude of nuclear Overhauser effects and 58 torsion angle restrains based on the magnitude of determined J-coupling constants. The model incorporates six long-lived water molecules detected by pseudo-two-dimensional NOESY-TOCSY spectra. The all-residue root mean square deviation about the average structure is 0.33 +/- 0.04 A for the backbone N, C alpha, and C' atoms and 0.83 +/- 0.05 A for all heavy atoms. The overall topology of the model for solution structure is very similar to that seen in previously reported models for crystal structures of homologous c-type cytochromes though there are a number of significant differences in detailed aspects of the structure. Two of the three main helices display localized irregularities in helical hydrogen bonding resulting in bifurcation of main chain hydrogen bond acceptor carbonyls. The N- and C-terminal helices are tightly packed and display several interhelical interactions not seen in reported crystal models. To provide an independent measure of the accuracy of the model for the oxidized protein, the expected pseudocontact shifts induced by the spin 1/2 iron were compared to the observed redox-dependent chemical shift changes. These comparisons confirm the general accuracy of the model for the oxidized protein and its observed differences with the structure of the reduced protein. The structures of the reduced and oxidized states of the protein provide a template to explain a range of physical and biological data spanning the redox properties, folding, molecular recognition, and stability of the cytochrome c molecule. For example, a redox-dependent reorganization of surface residues at the heme edge can be directly related to the redox behavior of the protein and thereby provides a previously undocumented linkage between structural change potentially associated with molecular recognition of redox partners and the fundamental parameters governing electron transfer.

Statistical strategy for stereospecific hydrogen NMR assignments: validation procedures for the floating prochirality method.

We examine the statistical and other considerations which determine the validity and reproducibility of stereospecific hydrogen NMR assignments obtained by the floating prochirality method. In this method, the assignment of a prochiral configuration of hydrogens at selected centers is allowed to 'float' during the structure refinement, and the distribution of prochiral orientations in highly refined structures is subjected to statistical analysis. The underlying statistical basis for this approach is examined and potential limitations of current approaches are identified. As an example, approximately 1300 distance constraints obtained from NOESY spectra of oxidized horse cytochrome c have been used to examine several computational strategies. Repeated calculations were done by several different methods on both the whole molecule (104 residues plus heme) and on a 23-residue fragment containing two helices, a turn, and flanking residues. The results show that, even with NOE constraints alone, one third of the centers may be reproducibly assigned, provided appropriate precautions are taken. These precautions include adjustments for multiple statistical comparisons and characterization of statistical interactions between prochiral centers. The analysis demonstrates that inadequately constrained systems, such as fragments from a larger molecule, may produce misleading results, raising concerns about methods which rely solely on intraresidue and sequential interresidue constraints. A mathematical model describing interactions among prochiral centers is described and validated, and protocols for assignment and statistical validation are presented.

On the fidelity of DNA replication. Nucleoside monophosphate generation during polymerization.

During catalysis by homogeneous procaryotic DNA polymerases, nucleoside monophosphates are generated by a 3' leads to 5'-exonucleolytic activity. Using Escherichia coli DNA polymerase I and poly[d(A-T)] as a template, the contribution of this activity to the fidelity of DNA synthesis has been evaluated by three different criteria. 1) The ratio between the rates of monophosphate generation and incorporation of the noncomplementary nucleotide with Mg2+ as an activating cation was 0.6 +/- 0.6, which is insufficient to account for the high fidelity of polymerization. 2) Inhibition of polymerization by pyrophosphate fails to diminish fidelity, although some kinetic models suggest that optimal error correction via monophosphate release requires the polymerization reaction to be strongly driven by pyrophosphate release. 3) The addition of deoxynucleoside monophosphates in concentrations as great as 10 mM to the reaction mixture does not alter the fidelity of DNA synthesis. These observations argue against the kinetic proofreading mode to account for the fidelity of E. coli DNA polymerase I when copying poly[d(A-T)] in a Mg2+-activated reaction. Furthermore, they suggest that the polymerase may enhance specificity at the base-selection step. However, the 3' leads to 5' exonuclease plays a larger role when the polymerase is activated with Mn2+ and may also be important in copying natural DNA where lower error rates are observed in vitro.

On the fidelity of DNA replication. Effect of the next nucleotide on proofreading.

The contribution of proofreading to the fidelity by which Escherichia coli DNA polymerase I copies natural DNA has been analyzed by two independent criteria. With phi X174 am 3 DNA as a template, there is approximately a 25-fold increase in noncomplementary base substitutions at position 587 when the concentration of the next correct nucleotide, dATP, is increased. Sequence analysis indicates that the mistakes represent misincorporation of C in place of T at position 587. This mutagenic response is presumed to result from a decrease in the probability of excision by the 3' leads to 5' exonuclease of Pol I and is considered within the context of current theories on proofreading. No enhanced mutagenicity is observed with avian myeloblastosis virus DNA polymerase, which lacks a 3' leads to 5' exonuclease. Using a second approach, an enhancement in mutagenesis as large as 30-fold is observed to result from the addition of deoxynucleoside monophosphates to the Pol I reaction. This mutagenicity occurs with any of the four deoxynucleoside monophosphates and is independent of a significant inhibition of DNA synthesis, thus supporting proofreading models in which sites of excision and incorporation are independent. The results of both approaches suggest that the exonucleolytic activity of Pol I can increase fidelity by approximately 30-fold on natural DNA, a value much higher than previous estimates with polynucleotide templates. The effect of the next correct nucleotide in decreasing accuracy provides an in vitro probe for screening eukaryotic cells for putative proofreading functions.

Interaction of the RNA-binding domain of the hnRNP C proteins with RNA.

The hnRNP C proteins are among the most abundant and avid pre-mRNA-binding proteins and they contain a consensus sequence RNA-binding domain (RBD) that is found in a large number of RNA-binding proteins. The interaction of the RBD of the hnRNP C proteins with an RNA oligonucleotide [r(U)8] was monitored by nuclear magnetic resonance (NMR). 15N and 13C/15N-labelled hnRNP C protein RBD was mixed with r(U)8 and one- and two-dimensional (1D and 2D) NMR spectra were recorded in a titration experiment. NMR studies of the uncomplexed 93 amino acid hnRNP C RBD (Wittekind et al., 1992) have shown that it has a compact folded structure (beta alpha beta beta alpha beta), which is typical for the RBD of this family of proteins and which is comprised of a four-stranded antiparallel beta-sheet, two alpha-helices and relatively unstructured amino- and carboxy-terminal regions. Sequential assignments of the polypeptide main-chain atoms of the hnRNP C RBD-r(U)8 complex revealed that these typical structural features are maintained in the complex, but significant perturbations of the chemical shifts of amide group atoms occur in a large number of residues. Most of these residues are in the beta-sheet region and especially in the terminal regions of the RBD. In contrast; chemical shifts of the residues of the well conserved alpha-helices, with the exception of Lys30, are not significantly perturbed. These observations localize the candidate residues of the RBD that are involved in the interaction with the RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Continuous octreotide infusion for the treatment of secretory diarrhea caused by acute intestinal graft-versus-host disease in a child.

This report describes the use of octreotide, a synthetic somatostatin analogue, for severe diarrhea caused by acute intestinal graft-versus-host disease (GVHD) after bone marrow transplantation. A 22-month-old boy suffered grade 4 intestinal GVHD, with profuse diarrhea, intestinal inflammation, and grossly bloody stools after matched, unrelated donor transplant for biphenotypic leukemia. He required intensive blood product support. In addition to aggressive anti-GVHD therapy, octreotide acetate was initiated at 30 microg (2 microg/kg) intravenously 3 times per day and escalated to continuous infusion at 15 microg/hr (1 microg/kg per hour). The diarrhea did not improve with anti-GVHD treatment. However, moderate dose octreotide therapy resulted in prompt control of the bloody diarrhea, which rebounded on cessation of octreotide therapy. Rebound diarrhea responded promptly when the dose of octreotide was escalated. Octreotide was associated with an exacerbation of preexisting hypertension, but it appeared to be effective for control of severe, bloody diarrhea caused by acute GVHD in a child, with manageable side effects. Further studies of this application in infants and children are warranted.

Topotecan given as a 21-day infusion in the treatment of advanced ovarian cancer.

A phase II programme was carried out in both Europe and North America to evaluate the activity of topotecan administered as a 21-day continuous intravenous infusion to patients with recurrent ovarian cancer. The European results are reported here. Patients who had failed first line therapy with a platinum-based regimen received topotecan 0.4 mg/m(2)/day, as a 21-day infusion every 28 days. Patients were only permitted one prior regimen. 35 patients were enrolled and evaluable for response. 3 patients (8.6%) had a partial response to treatment (95% CI 1.8%, 23.1%) with a median time to response of 8.1 weeks and a median duration of response of 17.6 weeks. Response was also evaluated by CA125 and was also found to be 8%. For all 35 patients, median time to progression was 16.1 weeks and median survival was 43.6 weeks. The principal toxicity was myelosuppression although grade 4 neutropenia occurred in only 8.8% of patients (2.1% of courses) and infectious complications were relatively infrequent. Non-haematological toxicity was generally mild and mainly consisted of gastrointestinal events, alopecia and fatigue. A prolonged infusion of topotecan was well tolerated with a low incidence of severe neutropenia. Responses were seen in both North American and European patients. Response rates varied between the 2 studies possibly due to differences in patient demographics.