David E. Thurston

Synthesis of DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepines (PBDs).

Pyrrolobenzodiazepines (PBD) are an important class of sequence-selective DNA-interactive agents that bind covalently to guanine bases within the minor groove of DNA. The PBD monomers are remarkable in possessing a 3-dimensional shape that allows them to fit perfectly within the minor groove of DNA, partly due to the longitudinal twist created by the chiral center at their C11a-position. A PBD produced synthetically, semisynthetically, or isolated from natural sources can exist as a mixture of two or even three forms or can exist predominantly as just one. Various biochemical and structural studies on PBD-DNA adducts have suggested that the molecules locate themselves with their N10-position pointing toward the floor of the minor groove. A further important feature of the PBDs is that they interact selectively with specific DNA sequences. A significant effort has also been made to increase the base-pair span and sequence-selectivity of PBD molecules so that they might be used as gene-targeting agents in biological experiments and possibly as therapeutic agents.

Chemical approaches to the discovery and development of cancer therapies.

The chemical sciences are essential for the process of anticancer-drug discovery, and a range of chemical research techniques is needed to develop clinically effective drugs. Improved understanding of the cellular, molecular and genetic basis of cancer has increased the number of drug targets available. What chemical approaches are used to develop agents that target specific features of cancer cells and make these therapeutics more effective? We outline the roles that chemical synthesis and understanding of drug uptake have had in drug discovery over the past 100 years, as well as the chemical insights derived from knowledge of the three-dimensional structure of targets.

A Potent Anti-CD70 Antibody-Drug Conjugate Combining a Dimeric Pyrrolobenzodiazepine Drug with Site-Specific Conjugation Technology

A highly cytotoxic DNA cross-linking pyrrolobenzodiazepine (PBD) dimer with a valine-alanine dipeptide linker was conjugated to the anti-CD70 h1F6 mAb either through endogenous interchain cysteines or, site-specifically, through engineered cysteines at position 239 of the heavy chains. The h1F6239C-PBD conjugation strategy proved to be superior to interchain cysteine conjugation, affording an antibody-drug conjugate (ADC) with high uniformity in drug-loading and low levels of aggregation. In vitro cytotoxicity experiments demonstrated that the h1F6239C-PBD was potent and immunologically specific on CD70-positive renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines. The conjugate was resistant to drug loss in plasma and in circulation, and had a pharmacokinetic profile closely matching that of the parental h1F6239C antibody capped with N-ethylmaleimide (NEM). Evaluation in CD70-positive RCC and NHL mouse xenograft models showed pronounced antitumor activities at single or weekly doses as low as 0.1 mg/kg of ADC. The ADC was tolerated at 2.5 mg/kg. These results demonstrate that PBDs can be effectively used for antibody-targeted therapy.

Targeting protein-protein interactions for therapeutic intervention: a challenge for the future

BACKGROUND Over the last two decades, an increasing research effort in academia and industry has focused on the modulation (both inhibition and stabilization) of protein-protein interactions (PPIs) in order to develop novel therapeutic approaches and target-selective agents in drug discovery. DISCUSSION The diversity and complexity of highly dynamic systems such as PPIs present many challenges for the identification of drug-like molecules with the ability to modulate the PPI with the necessary selectivity and potency. In this review, a number of these strategies will be presented along with a critical overview of the challenges and potential solutions relating to the exploitation of PPIs as molecular targets. CONCLUSIONS Both traditional drug discovery approaches and some more recently developed innovative strategies have already provided valuable tools for the discovery of PPI modulators, and a number of successful examples have highlighted the potential of targeting PPIs for therapeutic intervention, especially in the oncology area.

SJG-136 (NSC 694501), a Novel Rationally Designed DNA Minor Groove Interstrand Cross-Linking Agent with Potent and Broad Spectrum Antitumor Activity Part 1: Cellular Pharmacology, In vitro and Initial In vivo Antitumor Activity

SJG-136 (NSC 694501) is a rationally designed pyrrolobenzodiazepine dimer that binds in the minor groove of DNA. It spans 6 bp with a preference for binding to purine-GATC-pyrimidine sequences. The agent has potent activity in the National Cancer Institute (NCI) anticancer drug screen with 50% net growth inhibition conferred by 0.14 to 320 nmol/L (7.4 nmol/L mean). Sensitive cell lines exhibit total growth inhibition and 50% lethality after treatment with as little as 0.83 and 7.1 nmol/L SJG-136, respectively. COMPARE and molecular target analysis of SJG-136 data versus that of >60,000 compounds tested in the NCI 60 cell line screen shows that, although the agent has similarity to other DNA binding agents, the pattern of activity for SJG-136 does not fit within the clusters of any known agents, suggesting that SJG-136 possesses a distinct mechanism of action. Testing in the NCI standard hollow fiber assay produced prominent growth inhibition in 20 of 24 i.p. and 7 of 24 s.c. test combinations with 5 of 12 cell lines exhibiting cell kill. In addition, SJG-136 produced antitumor activity in mice bearing CH1 and CH1cisR xenografts, a cisplatin-resistant human ovarian tumor model, and also in mice bearing LS174T xenografts, a human colon tumor model. SJG-136 produces DNA interstrand cross-links between two N-2 guanine positions on opposite strands and separated by 2 bp. In human tumor cell lines, the cross-links form rapidly and persist compared with those produced by conventional cross-linking agents such as nitrogen mustards. In mice bearing the LS174T human colon xenograft, DNA interstrand cross-links can be detected in tumor cells using a modification of the single cell gel electrophoresis (comet) assay after administration of a therapeutic dose. Cross-links in the tumor increase with dose and are clearly detectable at 1 hour after i.v. administration. The level of cross-linking persists over a 24-hour period in this tumor in contrast to cross-links produced by conventional cross-linking agents observed over the same time period.

Chemistry and Pharmacology of Anticancer Drugs

INTRODUCTION TO CANCER Terminology Metastases Diagnosis and Screening Formation of Cancer Cells (Tumorigenesis) Mechanisms of Genomic Damage Treatments Discovery of Anticancer Drugs and Preclinical Evaluation Accessibility of Drugs to Tumor Cells Achieving Selective Toxicity Limiting the Toxicity of Chemotherapeutic Agents Overview of Mechanisms of Action of Chemotherapeutic Agents Drug Resistance Combination Chemotherapy Use of Adjuvants Infertility Following Cancer Treatments ANTIMETABOLITES DHFR Inhibitors (Antifolates) Purine Antimetabolites Pyrimidine Antimetabolites Thymidylate Synthase Inhibition Adenosine Deaminase Inhibition Ribonucleotide Reductase Inhibition DNA-INTERACTIVE AGENTS Alkylating Agents Cross-Linking Agents Intercalating Agents Topoisomerase Inhibitors DNA-Cleaving Agents ANTITUBULIN AGENTS Vinca Alkaloids The Taxanes MOLECULARLY TARGETED AGENTS Kinase Inhibitors Inhibition of Ras Pathway Signaling Cell Cycle Inhibitors Proteasome Inhibitors mTOR Inhibitors HORMONAL THERAPIES Breast Cancer Prostatic Cancer Neuroendocrine Tumors: Somatostatin Analogs Estrogen Therapy Progestogen Therapy TUMOR-TARGETING STRATEGIES Antibody-Based Approaches Vascular-Targeting Strategies X-DEPT (Biphasic) Strategies Enzymatic Targeting Photoactivated Drugs (Photodynamic Therapy) Boron Neutron Capture Therapy Novel Drug Delivery Approaches BIOLOGICAL AGENTS Biological Response Modifiers Immunotherapy Enzyme-Based Therapies Vaccines THE FUTURE Novel Biological Targets and Therapeutic Strategies New Research Tools and Methodologies Chemopreventive Agents PERSONALIZED TREATMENTS Screening for Risk of Disease Development Prognosis and Staging Screening for Risk of Recurrence of Disease Selecting Best Treatments for Patients Predicting Side Effects of Chemotherapeutic Agents Pharmacogenomics in Clinical Trials ADJUNCT THERAPIES Anti-emetic Agents Steroidal Agents Adjuvent Enzymes Other Therapies Index *Each chapter includes an Introduction and a Further Reading section.

The XPF-ERCC1 endonuclease and homologous recombination contribute to the repair of minor groove DNA interstrand crosslinks in mammalian cells produced by the pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136

SJG-136, a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer, is a highly efficient interstrand crosslinking agent that reacts with guanine bases in a 5′-GATC-3′ sequence in the DNA minor groove. SJG-136 crosslinks form rapidly and persist compared to those produced by conventional crosslinking agents such as nitrogen mustard, melphalan or cisplatin which bind in the DNA major groove. A panel of Chinese hamster ovary (CHO) cells with defined defects in specific DNA repair pathways were exposed to the bi-functional agents SJG-136 and melphalan, and to their mono-functional analogues mmy-SJG and mono-functional melphalan. SJG-136 was >100 times more cytotoxic than melphalan, and the bi-functional agents were much more cytotoxic than their respective mono-functional analogues. Cellular sensitivity of both SJG-136 and melphalan was dependent on the XPF-ERCC1 heterodimer, and homologous recombination repair factors XRCC2 and XRCC3. The relative level of sensitivity of these repair mutant cell lines to SJG-136 was, however, significantly less than with major groove crosslinking agents. In contrast to melphalan, there was no clear correlation between sensitivity to SJG-136 and crosslink unhooking capacity measured using a modified comet assay. Furthermore, repair of SJG-136 crosslinks did not involve the formation of DNA double-strand breaks. SJG-136 cytotoxicity is likely to result from the poor recognition of DNA damage by repair proteins resulting in the slow repair of both mono-adducts and more importantly crosslinks in the minor groove.

SJG-136 (NSC 694501), A Novel Rationally Designed DNA Minor Groove Interstrand Cross-Linking Agent with Potent and Broad Spectrum Antitumor Activity Part 2: Efficacy Evaluations

Pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136 (NSC 694501) selectively cross-links guanine residues located on opposite strands of DNA, and exhibits potent in vitro cytotoxicity. In addition, SJG-136 is highly active in vivo in hollow fiber assays. In the current investigation, SJG-136 was evaluated for in vivo efficacy in 10 tumor models selected on the basis of sensitivity of cells grown in the hollow fiber and in vitro time course assays: LOX IMVI and UACC-62 (melanomas); OVCAR-3 and OVCAR-5 (ovarian carcinomas); MDA-MB-435 (breast carcinoma); SF-295 and C-6 (gliomas); LS-174T (colon carcinoma); HL-60 TB (promyelocytic leukemia); and NCI-H522 (lung carcinoma). SJG-136 was active against small (150 mg) and large (250–400 mg) xenografts with tumor mass reductions in all 10 models. In addition, significant growth delays occurred in nine models, cell kill in six models ranged between 1.9 and 7.2 logs, and there were 1 to 4/6 tumor-free responses in six models. SJG-136 is active following i.v. bolus injections, as well as by 5-day continuous infusions. Of all of the schedules tested, bolus administrations for 5 consecutive days (qd×5) conferred the greatest efficacy. SJG-136 is active over a wide dosage range in athymic mouse xenografts: on a qd×5 schedule, the maximum-tolerated dose was ∼120 μg/kg/dose (total dose: 0.6 mg/kg = 1.8 mg/m2) and the minimum effective dose in the most sensitive model (SF-295) was ∼16 μg/kg/dose (total dose: 0.08 mg/kg = 0.24 mg/m2). Results of this study extend the initial in vivo observations reported in the reference above and confirm the importance of expediting more detailed preclinical evaluations on this novel agent in support of phase I clinical trials in the United Kingdom and the United States, which are planned to commence shortly.

The Pyrrolobenzodiazepine Dimer SJG-136 Forms Sequence-Dependent Intrastrand DNA Cross-Links and Monoalkylated Adducts in Addition to Interstrand Cross-Links

SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinical trials. Using a HPLC/MS-based methodology developed to evaluate the binding of DNA-interactive agents to oligonucleotides of varying length and sequence, we have demonstrated that, in addition to the previously known interstrand cross-link at Pu-GATC-Py sequences, 1 can form a longer interstrand cross-link at Pu-GAATC-Py sequences, an intrastrand cross-link at both shorter Pu-GATG-Py and longer Pu-GAATG-Py sequences, and, in addition, monoalkylated adducts at suitable PBD binding sites where neither intra- or interstrand cross-links are feasible because of the unavailability of two appropriately positioned guanines. Crucially, we have demonstrated a preference for the extended intrastrand cross-link with Pu-GAATG-Py, which forms more rapidly than the other cross-links (rank order: Pu-GAATG-Py > Pu-GATC-Py >> Pu-GATG-Py and Pu-GAATC-Py). However, thermal denaturation studies suggest that the originally reported Pu-GATC-Py interstrand cross-link is more stable, consistent with the covalent joining of both strands of the duplex and a lower overall distortion of the helix according to modeling studies. These observations impact on the proposed mechanism of action of SJG-136 (1) both in vitro and in vivo, the repair of its adducts and mechanism of resistance in cells, and potentially on the type of pharmacodynamic assay used in clinical trials.

SG2285, a Novel C2-Aryl-Substituted Pyrrolobenzodiazepine Dimer Prodrug That Cross-links DNA and Exerts Highly Potent Antitumor Activity

The pyrrolobenzodiazepines (PBD) are naturally occurring antitumor antibiotics, and a PBD dimer (SJG-136, SG2000) is in phase II trials. Many potent PBDs contain a C2-endo-exo unsaturated motif associated with the pyrrolo C-ring. The novel compound SG2202 is a PBD dimer containing this motif. SG2285 is a water-soluble prodrug of SG2202 in which two bisulfite groups inactivate the PBD N10-C11 imines. Once the bisulfites are eliminated, the imine moieties can bind covalently in the DNA minor groove, forming an interstrand cross-link. The mean in vitro cytotoxic potency of SG2285 against human tumor cell lines is GI(50) 20 pmol/L. SG2285 is highly efficient at producing DNA interstrand cross-links in cells, but they form more slowly than those produced by SG2202. Cellular sensitivity to SG2285 was primarily dependent on ERCC1 and homologous recombination repair. In primary B-cell chronic lymphocytic leukemia samples, the mean LD(50) was significantly lower than in normal age-matched B and T lymphocytes. Antitumor activity was shown in several human tumor xenograft models, including ovarian, non-small cell lung, prostate, pancreatic, and melanoma, with cures obtained in the latter model with a single dose. Further, in an advanced-stage colon model, SG2285 administered either as a single dose, or in two repeat dose schedules, was superior to irinotecan. Our findings define SG2285 as a highly active cytotoxic compound with antitumor properties desirable for further development.