Dose Escalation Experiments Research Articles

Design and Development of Small Molecules for Specific Targeted Therapy of Diffuse Large B-Cell Lymphoma.

BCL6 is the most commonly involved oncogene in diffuse large B-cell lymphoma (DLBCL). BCL6 mediates lymphomagenesis through direct transcriptional repression of target genes including p53 and ATR. BCL6 represses these genes by recruiting three corepressors: SMRT, N-CoR and BCoR. This interaction is mediated by an 18 amino acid corepressor sequence with a unique “lateral groove” motif located in the BCL6 BTB domain. We previously showed that a peptide mimic of the corepressor BCL6-binding sequence could inhibit BCL6 and kill DLBCL cells in vitro and in vivo, without toxicity to normal tissues. Although, transcription factors such as BCL6 that function through protein-protein interactions were traditionally regarded as “undruggable”, we hypothesized that a detailed structural and biochemical analysis might facilitate the design of small molecule inhibitors. Based on our crystal structure of the BCL6-corepressor complex, we used a computational strategy to screen one million small molecules for their ability to potentially dock to a specific lateral groove region. The chemistry of protein contacts in this region led us to predict that small molecules that could dock to this site would have the best chance of destabilizing the BCL6-corepressor complex. Among the top-scoring 100 molecules from this screen, we identified 10 compounds that could specifically inhibit the repressor activity of the BCL6 BTB domain in reporter assays and that displayed direct binding to purified BCL6 BTB domains. Using these leads as molecular scaffolds we generated small libraries of molecules derived from each parental compound. The most active of these families was called “the 57 series”. Series 57 compounds could all specifically block BCL6 repression in reporter assays, and disrupt corepressor/BCL6 complexes at low micromolar concentrations as shown in fluorescence polarization assays. X-ray crystallography of the most active member of the 57 family (called “57-6”) showed that the small molecule docked as predicted in the critical region of the lateral groove. Moreover, 57-6 induced an allosteric conformational change in the entire lateral groove that explains how these small molecules so effectively disrupt the BCL6/corepressor complex. 57-6 was also biologically active, since it could induce expression of BCL6 target genes including p53 and ATR in BCL6-positive DLBCL cells as shown by QPCR. 57-6 had no effect on negative control genes nor in BCL6-negative DLBCL cells. The mechanism of action was confirmed in ChIP assays showed that 57-6 abrogated BCL6 mediated corepressor recruitment to BCL6 target genes but had no effect on negative control genes. Most importantly, 57-6 specifically killed BCL6-positive DLBCL cells but had no effect on BCL6-negative DLBCL cells. A dose escalation experiment in mice revealed no toxic effects. In xenotransplantation experiments, 57-6 potently inhibited the growth of already established human DLBCL tumors in mice, again without toxicity to other organs. In summary, we used a rational approach to design specific and potent small molecule inhibitors of BCL6, which could serve as targeted agents for DLBCL in clinical trials. Our data show that transcription factors are druggable targets that can be harnessed to potentially improve cancer therapy.

DEC-205 antigen targeting at a dendritic cell-enriched site and CD40L-directed dendritic cell activation enhances CD4+ T cell priming. (47.12)

Abstract To enhance DNA vaccine efficacy, we tested the hypothesis that inoculation of calves with a single dose of a DNA vaccine capable of DC recruitment, DC antigen-targeting and activation will prime significant CD4+ T cell responses. To test this hypothesis, we developed a DNA construct, pCC98MSP1, in which a DEC-205-specific single chain antibody was used for DC-targeting of an Anaplasma marginale MSP1a DR-restricted CD4+ T cell antigen. A DNA construct, pCD40L-ED, encoding CD40 ligand was used for DC activation and DNA-encoded Flt3L/GM-CSF were used for DC recruitment. Recombinant CC98MSP1 binds to DEC-205 in vitro and to skin DCs in situ. In a dose-escalation experiment, DR-matched calves were immunized with a single dose of pCC98MSP1 (0.25–3mg) mixed with pCD40L-ED, pFlt-3L and pGM-CSF (0.5mg each). Immunization with 0.25mg pCC98MSP1 primed CD4+ T cell responses detectable in fresh PBMC 1wk post-immunization (PI). A dose dependent CD4+ T cell response was detected and surprisingly, at 2wks PI, the response induced by 0.25mg was not statistically different from the responses induced by 0.5–3mg pCC98MSP1. The responses were maintained for >9wks PI and underwent rapid recall 4 days post-boost with 0.25mg pCC98MSP1. This data supports the hypothesis that immunization with a single dose of a DNA vaccine capable of DC recruitment, DC-antigen targeting and activation enhances vaccine efficacy. Funding: USDA-CSREES 2005-01693

Inhibition of Experimental Gingivitis in Beagle Dogs With Topical Mercaptoalkylguanidines

Nitric oxide is a free radical produced in host tissues by constitutive and inducible forms of the enzyme nitric oxide synthase. Nitric oxide plays physiological roles, but it is also involved in the pathophysiology of several inflammatory conditions, including arthritis, ulcerative colitis, and circulatory shock. Local increases in inducible nitric oxide synthase (iNOS) and reactive nitrogen products have also been demonstrated in humans and animals with periodontal disease. This masked, randomized, placebo-controlled preclinical investigation examined the effect of two mercaptoalkylguanidines, mercaptoethylguanidine (MEG) and guanidinoethyldisulfide (GED), which are iNOS inhibitors and reactive nitrogen scavenging compounds, on the development of experimental gingivitis in beagle dogs. Fifteen female, 1-year-old beagles first completed a 2-week dose-escalation experiment during which a maximum tolerated dose was determined for MEG and GED gels. Thereafter, all animals were brought to optimal gingival health by mechanical scaling, followed by rigorous daily toothbrushing over a 4-week washout period. Experimental gingivitis was then induced, with cessation of plaque control and institution of a soft diet over 8 weeks. Beagles randomly received 0.3% MEG, 0.3% GED, or placebo (vehicle) gels, topically applied twice daily to premolar teeth. Gingival inflammation, bleeding tendency, and supragingival plaque were clinically measured at baseline and at 2, 3, 4, 6, and 8 weeks. Comparisons among groups and between group pairs (active versus placebo) were made using Kruskal-Wallis tests. From baseline to day 7, all groups expressed similar indices. Thereafter, significant and time-dependent increases in the plaque index (PI), gingival index (GI), and percentage of bleeding on probing (%BOP) were observed in placebo-treated beagles. Mean GI scores for beagles treated with GED or MEG gels remained at or below baseline levels for the entire treatment period. At weeks 2, 3, 4, and 8, GI scores were significantly lower for MEG and GED groups compared to the placebo group (P<0.05). In addition, MEG and GED gels significantly reduced gingival bleeding responses by 8 weeks (P<0.05). Although placebo-treated beagles demonstrated %BOP scores of 43% at week 8, GED- and MEG-treated beagles exhibited %BOP scores of 21% and 26%, respectively. Since no statistical difference among PI scores was noted for any of the time points, neither mercaptoalkylguanidine appeared to affect supragingival plaque levels. The data from this preclinical study indicate that mercaptoalkylguanidines, topically administered, may significantly reduce experimental gingivitis in the beagle dog.

Suberoylanilide hydroxamic acid (SAHA) has potent anti‐glioma properties in vitro, ex vivo and in vivo

Current treatment modalities for malignant gliomas do not allow long-term survival. Here, we identify suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylases (HDAC), as an effective experimental anti-glioma agent. Administration of SAHA to various glioma cell lines obtained from human, rat and mouse inhibited tumour cell growth in a range of 1-10 microm. This anti-glioma property is associated with up-regulation of the cell cycle control protein p21/WAF, as well as the induction of apoptosis. A novel tumour invasion model using slice cultures of rat brain corroborated the anti-glioma properties of SAHA in the organotypic brain environment. In this model, glioma invasion compromised adjacent brain parenchyma, and this tumour-associated cytotoxicity could be inhibited by SAHA. In addition, a 10-fold dose escalation experiment did not challenge the viability of cultured brain slices. In vivo, a single intratumoural injection of SAHA 7 days after orthotopic implantation of glioma cells in syngeneic rats doubled their survival time. These observations identify chromatin-modifying enzymes as possible and promising targets for the pharmacotherapy of malignant gliomas.

Evaluation of quinolone derivatives for antitrypanosomal activity.

About 160 fluoroquinolones and derivatives were tested for antitrypanosomal activity in a drug sensitivity assay followed by fluorometric evaluation. The most active quinolone compounds had IC50 values in the range from 100 to 900 ng/ml, while several derivatives were not active at a concentration of 100 microg/ml. In a structure activity relationship study, modification of the quinolones at position R1, R2, R3 and R8 did not influence trypanocidal activity. An exchange of the fluor at position 6 may contribute to an increase in activity but does not entirely control it. Pyrrolidine substituents at position R7 generally were more active than other substituents at this position. Tetracyclic quinolone derivatives were amongst the most active compounds with IC50 values in the range of 0.3-8.8 microg/ml. The in vitro cytotoxicity on HT-29 cells was determined for active compounds with IC50 values below 1 microg/ml. In addition, six drugs with an IC50 below 1 microg/ml and a selectivity index of more than 10 were chosen for in vivo experiments. Dose escalation experiments with a maximum dose of 100 mg/kg/bid were performed in a mouse model without central nervous system involvement. For unknown reasons the in vitro effect of the drugs could not be confirmed in vivo, but the class of compound remains of interest for their mode of action, the low toxicity, pharmacological properties and the availability of a large number of synthesized compounds.

In vivo targeting of leukemic cells using diphtheria toxin fused to murine GM-CSF.

We have previously demonstrated that diphtheria toxin (DT) fused to human GM-CSF effectively eliminates human long-term leukemia initiating cells in SCID mice. However, because huGM-CSF does not react with the murine GM-CSF receptor possible side-effects to nonleukemic tissues could not be analyzed in the AML/SCID model. To overcome this problem, we used murine GM-CSF fused to DT and studied the therapeutic index in the rat leukemia model BNML/LT12. In DT-mGM-CSF dose escalation experiments, severe dose-dependent toxicity to organs such as liver, kidney and lung was observed. Therefore, the antileukemic effects were evaluated with the lower doses. Daily intraperitoneal bolus injections of 75 microg/kg/day for 7 days induced a 3 log leukemic cell kill. The dose of 75 microg/kg/day had no effect on the hemopoietic progenitor cell subsets. These in vivo studies show that the DT-GM-CSF fusion protein can be used for specifically targeting leukemic cells and thus has potential as a therapeutic agent in the treatment of AML.

Increased injection number enhances adenoviral genetic radiotherapy.

Intratumoral injection of an adenoviral vector containing radiation-inducible DNA sequences of the early growth response gene (Egr-1) promoter ligated to a cDNA encoding tumor necrosis factor-alpha (TNF-alpha; Ad.Egr-TNF) increases the radiation killing of a human radioresistant xenograft (SQ-20B). Viral dose-escalation experiments demonstrated that SQ-20B growth inhibition correlated with viral titer. Injection of 5 x 10(8) PFU Ad.Egr-TNF produced regression to a mean volume of 22 +/- 13% of the original tumor volume, 1 x 10(8) PFU to a mean of 62 +/- 24%, and 5 x 10(7) PFU to a mean of 67 +/- 27%. No regression was observed when tumors were injected with 1 x 10(7) PFU Ad.Egr-TNF or with the null viral vector (Ad.null). When two injections of vector (2 x 10(8) PFU Ad.Egr-TNF) were combined with 50 Gy, a significant increase in tumor regression was observed compared with injection of buffer, Ad.Egr-TNF, or 50 Gy. The interactive killing between TNF and radiation was enhanced significantly (P = 0.05) when the number of injections was increased from two to five while maintaining a constant viral titer (2 x 10(8) PFU Ad.Egr-TNF) and a constant radiation dose (50 Gy). Significant TNF-alpha levels were present in irradiated vs. unirradiated tumors following injection with Ad.Egr-TNF. Taken together, these data suggest that the volumetric reduction produced by the combined effects of Ad.Egr-TNF and radiation is enhanced with increasing vector concentration and the number of vector injections.

Radiotoxicity of systemically administered [ 211At]astatide in B6C3F 1 and BALB/ c ( nu/nu) mice: A long-term survival study with histologic analysis

Purpose : The present study undertook to establish the dose (LD) of systemically administered (via tail vein) sodium [ 211At]astatide that would kill 10% (LD 10) of exposed animals in two mouse models and to evaluate the resulting histologic lesions. Methods and Materials : Three dose escalation experiments were carried out using groups of 10 3- to 4-week-old, 20 ± 2 g B6C3F 1 mice, and one dose escalation experiment was carried out with groups of 10 4- to 6-week-old, 22 ± 2 g BALB/c (nu/nu) mice. All animals were weighed daily and checked twice daily for general health; autopsies were performed within 12 h of death. Results : The LD 10 (95% confidence interval) level of free [ 211At]astatide at 360 days was 15.1 μCi (5.2–19.1 μCi) in B6C3F 1 mice and was associated with a 37.8% weight difference from saline controls ( p < 0.001). In the BALB/c (nu/nu) mice, the LD 10 at 360 days was 7.7 μCi (0–14.2 μCi), while a dose of 10 μCi (0.42 μCi g −1) was associated with a 9.44% weight difference vs. saline controls ( p < 0.05). Exclusive of the well-known effects on thyroid, [ 211At]astatide activity levels were associated with severe bone marrow depression, testicular atrophy, focal alopecia, and nuclear atypia of the epidermoid mucosa of the fore-stomach in the B6C3F 1 mice; at activity levels approximating LD 10 at 360 days, mild changes in the heart, liver, stomach, and spleen were observed. For BALB/c (nu/nu) mice, administration of 10 μCi was associated at autopsy with mild histologic lesions in the heart, stomach, liver, and spleen. Conclusions : These studies provide a basis for the design of further investigations of [ 211At]-labeled compounds as therapeutic agents.