The paradox-breaking panRAF plus SRC family kinase inhibitor, CCT3833, is effective in mutant KRAS-driven cancers.

BACKGROUND: KRAS is mutated in ∼90% of pancreatic ductal adenocarcinomas, ∼35% of colorectal cancers and ∼20% of non-small-cell lung cancers. There has been recent progress in targeting G12CKRAS specifically, but therapeutic options for other mutant forms of KRAS are limited, largely because the complexity of downstream signaling and feedback mechanisms mean that targeting individual pathway components is ineffective. DESIGN: The protein kinases RAF and SRC are validated therapeutic targets in KRAS-mutant pancreatic ductal adenocarcinomas, colorectal cancers and non-small-cell lung cancers and we show that both must be inhibited to block growth of these cancers. We describe CCT3833, a new drug that inhibits both RAF and SRC, which may be effective in KRAS-mutant cancers. RESULTS: We show that CCT3833 inhibits RAF and SRC in KRAS-mutant tumors in vitro and in vivo, and that it inhibits tumor growth at well-tolerated doses in mice. CCT3833 has been evaluated in a phase I clinical trial (NCT02437227) and we report here that it significantly prolongs progression-free survival of a patient with a G12VKRAS spindle cell sarcoma who did not respond to a multikinase inhibitor and therefore had limited treatment options. CONCLUSIONS: New drug CCT3833 elicits significant preclinical therapeutic efficacy in KRAS-mutant colorectal, lung and pancreatic tumor xenografts, demonstrating a treatment option for several areas of unmet clinical need. Based on these preclinical data and the phase I clinical unconfirmed response in a patient with KRAS-mutant spindle cell sarcoma, CCT3833 requires further evaluation in patients with other KRAS-mutant cancers.

Development of an outpatient finger-prick glomerular filtration rate procedure suitable for epidemiological studies.

Development of an outpatient finger-prick glomerular filtration rate (GFR) procedure suitable for epidemiological studies. In clinical practice, reference GFR procedures are rarely used; in large-scale research studies, a great deal of effort and experience is required to obtain them, which is a considerable disincentive to using GFR as an end point. The major problem for both clinical staff and the subject is the length of time that the procedure takes, requiring continuous attendance in the outpatient clinic or its vicinity. Using iohexol as a marker, we therefore propose an alternative approach, which addresses this fundamental deterrent to a more widespread use of GFR measurement. Eighty-two GFR measurements were performed in a mixture of healthy subjects and patients with differing degrees of renal impairment with a wide range of GFRs. Serum was obtained from blood samples to enable a reference GFR to be calculated. Blood spots were collected on filter paper at the same intervals (120, 180, and 240 min), allowed to dry, and then sent through the post. Serum and blood spots were analyzed simultaneously for each individual by automated reverse-phase high-pressure liquid chromatography. Standard linear regression analyses confirmed a good agreement (r2 = 0.953) between the iohexol serum GFR and iohexol blood spots GFR. Bland-Altman analysis confirmed that there was no concentration bias. Paired comparisons (Wilcoxon's paired signed rank test) showed no significant difference between the two measurements. Capillary sampling is simple, effective, and significantly reduces the time and costs of performing plasma clearance GFR measurements. This approach will make the GFR measurement more accessible for clinical practice and large-scale epidemiological studies may become feasible.

Significant differences in biological parameters between prodrugs cleavable by carboxypeptidase G2 that generate 3,5-difluoro-phenol and -aniline nitrogen mustards in gene-directed enzyme prodrug therapy systems.

Nine new nitrogen mustard compounds derived from 2,6-difluoro-4-hydroxy- (3a-e) and 2,6-difluoro-4-amino- (4a-d) aniline were synthesized as potential prodrugs. They were designed to be activated to their corresponding 3,5-difluorophenol and -aniline (4)-nitrogen mustards by the enzyme carboxypeptidase G2 (CPG2) in gene-directed enzyme prodrug therapy (GDEPT) models. The compounds were tested for cytotoxicity in the MDA MB-361 breast adenocarcinoma. The cell line was engineered to express stably either CPG2 tethered to the cell surface stCPG2-(Q)3 or beta-galactosidase (beta-Gal) as control. The cytotoxicity differentials were calculated between CPG 2-expressing and -nonexpressing cells and yielded different results for the two series of prodrugs despite their structural similarities. While the phenol compounds are ineffective as prodrugs, their aniline counterparts exhibit outstanding activity in the tumor cell lines expressing CPG2. [3,5-Difluoro-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl-l-glutamic acid gave a differential of >227 in MDA MB361 cells as compared with 19 exhibited by 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-l-glutamic acid, 1a, which has been in clinical trials.

Thymitaq (Zarix).

Zarix is developing Thymitaq (nolatrexed dihydrochloride) for the potential treatment of cancer. It is conducting a phase III trial in the US, Canada, South Africa and certain European countries [382746], [397955], [4061471, after review and comment by the FDA. One clinical trial may be sufficient for registration [405928]. In April 2000, Thymitaq was designated a Fast Track product for the treatment of unresectable hepatocellular carcinouna (HCC) by the FDA [364887], based on the survival analysis (intent-to-treat) of the Agouron phase II data and medical necessity [405928]. The company was expected to file an NDA in 2002 [367559]. However, as of April 2001, the company was expecting to file an NDA in the third quarter of 2003 [405928]. In January 1999, Zarix licensed worldwide rights to Thymitaq from Agouron [311213]. Agouron discontinued development of Thymitaq, based on an interim analysis of phase II/III trials which showed that although the compound was effective as a single agent in head and neck and liver tumors, it was not sufficiently better than existing therapeutics to justify further development [211844], [270994]. However, in its efforts to complete the monitoring of these studies, Zarix discovered that a great majority of patients obtained stabilization of disease with Thymitaq treatment [405928]. Zarix will focus development efforts on the treatment of HCC. Zarix is developing Thymitaq in an iv formulation for the treatment of HCC and, in May 2000, planned to initiate phase III trials in the fourth quarter of 2000 [364887]. Patient enrollment in a pivotal, phase III trial in patients with unresectable HCC comparing Thymitaq to doxorubicin was initiated in September 2000. Unresectable HCC is the first indication being pursued for FDA approval and the trial design was deemed acceptable in August 2000. The study is a multicenter, multinational trial that will utilize approximately 50 sites. It is anticipated that patient accrual will be completed within 24 months [378514], 1382746], [384018]. The company plans to file for regulatory approval of the product in North America, Europe and Japan, and will pursue development of the drug in a variety of oncology indications [311213]. The multicenter study is to be conducted as a global program with sites in the US, Canada, Europe and South Africa. In January 2001, the Canadian Therapeutic Products Directorate indicated that the design of the phase IX trial was acceptable. Zarix expected patient enrollment in Canadian clinical sites to begin in Spring 2001 [397955], [405928]. By April 2001, patient enrollment was underway in the US, Italy, Canada, Romania, South Africa and the UK. At this time, Zarix also expected to initiate trials in the Czech Republic and Germany in the near future and Poland in the summer of 2001 [406147]. By May 1997, three clinical trials had been conducted in a total of 149 patients with HCC [405928]. In two US phase II trials, patients with unresectable HCC treated with Thymitaq had a median survival (intent-to-treat) of over 23 weeks with published survivals (intent-to-treat) of less than 15 weeks. Thymitaq has no end-organ toxicity. Side effects are typical of other antimetabolites but are of shorter duration [364887], [405928].

Definition of chromosome aberrations in testicular germ cell tumor cell lines by 24-color karyotyping and complementary molecular cytogenetic analyses.

Many of the reported karyotypes for adult testicular germ cell tumors (GCTs) are complex and incomplete, although the presence of an isochromosome 12p, i(12p), and gain of 12p material have consistently been found. Here, an accurate definition of the chromosome aberrations associated with four cell lines derived from GCTs (GCT27, H12.1, Tera1, and Tera2) has been produced using 24-color karyotyping by mulifluor in situ hybridization, comparative genomic hybridization analysis, and further fluorescence in situ hybridization analysis to confirm some chromosomal assignments and refine involvement of specific regions of 12p. There was karyotypic heterogeneity. Isochromosomes in addition to i(12p) were found, as were other rearrangements with breakpoints at or near centromeric regions. The most frequent non-centromeric breakpoints were at 1p31 approximately p32, 1p21 approximately p22, 11q13, and Xq22, although consistent partner chromosomes were not involved. One cell line (Tera1) showed a subtle dosage increase in the copy number of a 12p probe known to be within the smallest overlapping region of amplification that has been defined in a number of testicular GCTs with amplicons at 12p11 approximately p12. The chromosome rearrangements and associated imbalances may be significant in GCT progression and the characterized cell lines can be used to investigate these further.

NCI-EORTC-AACR--11th Symposium on New Drugs in Cancer Therapy: Gene therapy and new drugs. 7-10 November 2000, Amsterdam, The Netherlands.

A number of advances in cancer therapy during the last two years were reported, including new strategies, new targets and new compounds. New strategies were developed in gene therapy, drug delivery, drug targeting and prodrug design. New targets within the broad family of kinases, histone deacetylases, proteasomes and hypoxic factors were defined. Improved compounds including tubulin interacting agents, bioreductive agents, angiogenesis inhibitors, DNA interactive agents, anthracyclines, taxanes, cyclin- and tyrosine kinase inhibitors have recently reached the stage of clinical trials. Special attention has been given to chemoprevention and also to structure-activity relationships as a tool for speeding the development of better drugs. The aim of the meeting was to present and discuss the recent results in this area with respect to the R and D of new generations of anticancer compounds. This review focuses on a number of the most recent advances in the area of strategies and anticancer drug and prodrug design presented and discussed in plenary and poster sessions.

ZD-9331 AstraZeneca.

AstraZeneca (formerly Zeneca) is developing ZD-9331, a non-polyglutamatable thymidylate synthase inhibitor, as a potential treatment for solid tumors and other neoplasia, including colorectal tumors [216476,179954,179955]. ZD-9331 is being developed as both an oral and an i.v. formulation, both of which are in phase II trials as of December 1999 [349551,352095]. As of June 1998, ZD-9331 was in phase II trials for advanced colorectal and other solid tumors [315489], with drug filings not expected until 2002 [349551]. A clinical study presented at the 36th Annual Meeting of the American Society of Clinical Oncology (ASCO) demonstrated that treatment with ZD-9331 resulted in a period of intracellular 2'-deoxyuridine (dUrd) elevation, a surrogate marker of thymidylate synthase inhibition, with observed myelosuppression being no greater than that seen with raltitrexed and less than with bolus 5-FU [369475]. Results from a 56-patient phase I study were presented at the 1999 ASCO meeting. Dose escalation followed a two-stage procedure. As in previous studies myelosuppression was the dose-limiting toxicity, occurring at 4.8 and 7.5 mg/m2/day, with one patient at each of these two doses experiencing a DLT. The MTD was not achieved until 12 to 16 mg/m2/day, based on which a fixed dose of 25 mg/day was being evaluated [326935]. A number of other studies are ongoing, comparing once to twice daily dosages as well as the pharmacokinetics of the compound. Encouraging phase I data have been seen in melanoma, ovarian, colon and breast cancer; myelosuppression is the dose limiting toxicity in the majority of these studies [326938,326943,326945,327399]. A phase I dose-escalation trial was conducted to evaluate the feasibility of a once 3-weekly 30-min i.v. infusion of ZD-9331, with doses ranging from 4.8 to 370 mg/m2. The regimen was overall well tolerated up to 370 mg/m2, with grade IV myelosuppression and grade IV diarrhea being observed in a small number of patients [288959,377842]. In June 2000, Deutsche Bank predicted sales of $12 million in 2002 [374500]. In January 1999, ABN Amro predicted sales of US $8 million in 2002 rising to $66 million in 2005 [316250, 328676]. In March 1999, Lehman Brothers predicted a 30% probability that the drug would reach the worldwide markets, and be launched in 2002 [336599].

Technology evaluation: gemtuzumab ozogamicin, Celltech Group.

Gemtuzumab ozogamicin (CMA-676) is a cytotoxic drug (calichaemicin) linked to a human monoclonal antibody that targets CD33, and which has been jointly developed and launched by Celltech Group and the Wyeth-Ayerst Research division of American Home Products as a treatment for acute myeloid leukemia (AML). Wyeth-Ayerst submitted an NDA to the US FDA in October 1999 for approval to treat AML patients who have relapsed after initial chemotherapy [346494]. In January 2000, the FDA assigned CMA-676 Priority Review status [351745], and in March 2000, the US FDA's Oncologic Drugs Advisory Committee voted that there was sufficient evidence of improved safety and acceptable efficacy to support accelerated approval for the treatment of AML in patients who have relapsed following initial chemotherapy [360113], [361883]. In May 2000, the FDA gave final approval for CMA-676 for the treatment of patients of 60 years and over in first relapse with CD33+ AML who are not considered candidates for cytotoxic chemotherapy [367212], [367215]. CMA-676 was launched (as Mylotarg) in the US on 1 June 2000 by AHP. An EMAA is in preparation and is expected to be submitted by the end of 2000 [382163]. In September 2000, Merill Lynch predicted sales of 2.9 million Pounds in 2002, rising to 17.9 million Pounds in 2004 [383742], while in January 2000, Lehman Brothers predicted peak sales in excess of US$200 million [360128]. In December 1999, Lehman Brothers predicted peak sales of US$150 million, with Celltech receiving royalties from AHP. Possible first-line therapy could follow depending on further studies, creating a potential market of US$300 million [352078].

Self-immolative anthracycline prodrugs for suicide gene therapy.

Four novel potential prodrugs derived from daunorubicin (8, 10) and doxorubicin (12, 14) were designed and synthesized. They are self-immolative prodrugs for suicide gene therapy activation by the enzyme carboxypeptidase G2 (CPG2) subsequently releasing the corresponding anthracyclines, by a 1,6-elimination mechanism. A mammary carcinoma cell line (MDA MB 361) was engineered to express CPG2 intracellularly (CPG2) or extracellularly, tethered to the outer cell membrane (stCPG2(Q)3). The prodrugs derived from doxorubicin showed prodrug/drug cytotoxicity differentials of 21-fold (compound 12) and 23-fold (compound 14). Prodrug 12 underwent an 11-fold activation when assayed in the cell line expressing externally surface-tethered CPG2.

Prodrugs for antibody- and gene-directed enzyme prodrug therapies (ADEPT and GDEPT).

Antibody- and gene-directed enzyme prodrug therapy are two-step targeting strategies designed to improve the selectivity of antitumour agents. The approaches are based on the activation of specially designed prodrugs by antibody-enzyme conjugates targeted to tumour-associated antigens (ADEPT) or by enzymes expressed by exogenous genes in tumour cells (GDEPT). Herein the design, synthesis, physico-chemical and biological properties, kinetics and clinical trials of the prodrugs and the enzymes carboxypeptidase G2 and nitroreductase are reviewed for ADEPT and GDEPT.

Recent developments in gene-directed enzyme prodrug therapy (GDEPT) for cancer.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising two-step treatment for solid malignant tumors. In the first step, the gene for a foreign enzyme is administered and directed to the tumor, where it may be expressed using specific transcriptional elements. In the second step, prodrugs are administered and activated by the foreign enzyme expressed at the tumor. This review focuses on the progress from the end of 1997 to date. Important issues, such as viral and non-viral vectors, new enzyme/prodrug systems, new strategies, advances in the understanding of the bystander effects, the comparison of different systems used in GDEPT and clinical trials are outlined.

Self-immolative nitrogen mustard prodrugs for suicide gene therapy.

Four new potential self-immolative prodrugs derived from phenol and aniline nitrogen mustards, four model compounds derived from their corresponding fluoroethyl analogues and two new self-immolative linkers were designed and synthesized for use in the suicide gene therapy termed GDEPT (gene-directed enzyme prodrug therapy). The self-immolative prodrugs were designed to be activated by the enzyme carboxypeptidase G2 (CPG2) releasing an active drug by a 1, 6-elimination mechanism via an unstable intermediate. Thus, N-[(4-¿[4-(bis¿2-chloroethyl¿amino)phenoxycarbonyloxy]methyl¿pheny l)c arbamoyl]-L-glutamic acid (23), N-[(4-¿[4-(bis¿2-chloroethyl¿amino)phenoxycarbonyloxy]methyl¿pheno xy) carbonyl]-L-glutamic acid (30), N-[(4-¿[N-(4-¿bis[2-chloroethyl]amino¿phenyl)carbamoyloxy]methyl¿+ ++phen oxy)carbonyl]-L-glutamic acid (37), and N-[(4-¿[N-(4-¿bis[2-chloroethyl]amino¿phenyl)carbamoyloxy]methyl¿+ ++phen yl)carbamoyl]-L-glutamic acid (40) were synthesized. They are bifunctional alkylating agents in which the activating effects of the phenolic hydroxyl or amino functions are masked through an oxycarbonyl or a carbamoyl bond to a benzylic spacer which is itself linked to a glutamic acid by an oxycarbonyl or a carbamoyl bond. The corresponding fluoroethyl compounds 25, 32, 42, and 44 were also synthesized. The rationale was to obtain model compounds with greatly reduced alkylating abilities that would be much less reactive with nucleophiles compared to the corresponding chloroethyl derivatives. This enabled studies of these model compounds as substrates for CPG2, without incurring the rapid and complicated decomposition pathways of the chloroethyl derivatives. The prodrugs were designed to be activated to their corresponding phenol and aniline nitrogen mustard drugs by CPG2 for use in GDEPT. The synthesis of the analogous novel parent drugs (21b, 51) is also described. A colorectal cell line was engineered to express CPG2 tethered to the outer cell surface. The phenylenediamine compounds were found to behave as prodrugs, yielding IC50 prodrug/IC50 drug ratios between 20- and 33-fold (for 37 and 40) and differentials of 12-14-fold between CPG2-expressing and control LacZ-expressing clones. The drugs released are up to 70-fold more potent than 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoic acid that results from the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA) which has been used previously for GDEPT. These data demonstrate the viability of this strategy and indicate that self-immolative prodrugs can be synthesized to release potent mustard drugs selectively by cells expressing CPG2 tethered to the cell surface in GDEPT.

Recent advances in anticancer drugs.

During the last few years, research in anticancer drug development has revealed a number of new approaches. These include new types of prodrugs, small peptides and synthetic molecules affecting signal transduction pathways and cell cycle, topoisomerase inhibitors, antisense compounds, anti-angiogenic and antimetastatic agents, gene therapy and vaccines. The aim of the meeting was to present and discuss the recent results in these areas, with respect to the R and D of new anticancer compounds. This review focuses on the most recent advances in anticancer drugs presented and discussed in the plenary sessions.

Antibody-directed enzyme prodrug therapy (ADEPT): a review.

Antibody-directed enzyme prodrug therapy (ADEPT) is a therapeutic strategy which aims to improve the selectivity of anticancer drugs. ADEPT is a two-step antibody targeting system that has benefits over a one-step chemo-, toxin- or radioimmunoconjugate. The basic principles of ADEPT are discussed alongside the requirements of the components: antibodies, enzymes and prodrugs. The design and syntheses of prodrugs are detailed particularly prodrug/drug systems of potential clinical use, the rationale behind their design and the in vitro and in vivo results obtained. The main features of ADEPT, such as targeting of cancer cells by the antibody-enzyme conjugates, enzymic activation of the prodrugs, selection of the prodrug/drug and enzyme/prodrug systems are reviewed.

Gene-directed enzyme prodrug therapy: a review of enzyme/prodrug combinations.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising, new, two-step treatment for cancer chemotherapy. In the first step, the gene for a foreign enzyme is administered and is directed to the tumour, where it is expressed by the use of specific promoters. In the second step, injected prodrugs are activated by the foreign enzyme. The design and synthesis of prodrugs able to undergo enzymatic activation in such systems is an essential component. This review focuses on the requirements which must be fulfilled by the components of GDEPT systems in order for this therapy to be considered a realistic possibility. A special emphasis is placed on the description of the prodrugs used in GDEPT protocols and the requirement for a bystander effect is also discussed.

ZD2767, an improved system for antibody-directed enzyme prodrug therapy that results in tumor regressions in colorectal tumor xenografts.

ZD2767 represents an improved version of antibody-directed enzyme prodrug therapy. It consists of a conjugate of the F(ab')2 A5B7 antibody fragment and carboxypeptidase G2 (CPG2) and a prodrug, 4-[N,N-bis(2-iodoethyl)amino]phenoxycarbonyl L-glutamic acid. The IC50 of the prodrug against LoVo colorectal tumor cells was 47 microM, and cleavage by CPG2 released the potent bis-iodo phenol mustard drug (IC50 = 0.34 microM). The drug killed both proliferating and quiescent LoVo cells. Administration of the ZD2767 conjugate to nude mice bearing LoVo colorectal xenografts resulted in approximately 1% of injected ZD2767 conjugate localizing/g of tumor after 72 h, and blood and normal tissue levels of the conjugate were 10-50-fold lower. A single round of therapy involving the administration of the prodrug 72 h after the conjugate to athymic mice bearing established LoVo xenografts resulted in approximately 50% of the tumors undergoing complete regressions, tumor growth delays greater than 30 days, and little toxicity (as judged by body-weight loss). Similar studies using a control antibody-CPG2 conjugate that does not bind to LoVo tumor cells resulted in a growth delay of less than 5 days, confirming the tumor specificity of this approach. These studies demonstrate the potential of ZD2767 for the treatment of colorectal cancer.

Optimization of alkylating agent prodrugs derived from phenol and aniline mustards: a new clinical candidate prodrug (ZD2767) for antibody-directed enzyme prodrug therapy (ADEPT).

Sixteen novel potential prodrugs derived from phenol or aniline mustards and their 16 corresponding drugs with ring substitution and/or different alkylating functionalities were designed. The [[[4-]bis(2-bromoethyl)-(1a), [[[4-[bis(2-iodoethyl)-(1b), and [[[4-[(2-chloroethyl)-[2-(mesyloxy)ethyl]amino]phenyl]oxy] carbonyl]-L-glutamic acids (1c), their [[[2- and 3-substituted-4-[bis(2-chloroethyl)amino]phenyl]oxy]carbonyl]-L- glutamic acids (1e-1), and the [[3-substituted-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl]-L- glutamic acids (1o-r) were synthesized. They are bifunctional alkylating agents in which the activating effect of the phenolic hydroxyl or amino function is masked through an oxycarbonyl or a carbamoyl bond to a glutamic acid. These prodrugs were designed to be activated to their corresponding phenol and aniline nitrogen mustard drugs at a tumor site by prior administration of a monoclonal antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2) in antibody-directed enzyme prodrug therapy (ADEPT). The synthesis of the analogous novel parent drugs (2a-r) is also described. The viability of a colorectal cell line (LoVo) was monitored with the potential prodrugs and the parent drugs. The differential in the cytotoxicity between the potential prodrugs and their corresponding active drugs ranged between 12 and > 195 fold. Compounds 1b-d,f,o exhibited substantial prodrug activity, since a cytotoxicity differential of > 100 was achieved compared to 2b-d,f,o respectively. The ability of the potential prodrugs to act as substrates for CPG2 was determined (kinetic parameters KM and kcat), and the chemical stability was measured for all the compounds. The unsubstituted phenols with different alkylating functionalities (1a-c) proved to have the highest ratio of the substrates kcat:KM. From these studies [[[4-[bis(2-iodoethyl)amino]phenyl]oxy]carbonyl]-L-glutamic acid (1b) emerges as a new ADEPT clinical trial candidate due to its physicochemical and biological characteristics.