Individuals receiving a standardised daily dose of 2000 mg of the n-3 highly unsaturated fatty acid (HUFA) eicosapentaenoic acid (EPA) for 12 months in the seAFOod polyp prevention trial demonstrated wide inter-individual variability in red blood cell (RBC) EPA levels (expressed as

pre|CISION® peptide drug conjugates comprise a peptide moiety bound to a payload by a linker that is specifically cleaved by FAP, a protease selectively overexpressed in the tumor microenvironment (TME) of many solid tumors. The pre|CISION® peptide moiety prevents cellular entry of the conjugate unless cleaved by FAP, enabling targeted delivery of released payload directly to tumors. Two payloads are described using this platform: AVA6000 (doxorubicin [dox]; pre-clinical and clinical) and AVA6103 (exatecan, pre-clinical). Methods: Pharmacokinetic (PK; plasma and intra-tumoral) parameters of the conjugate and released payload were investigated pre-clinically (for both AVA6000 and AVA6103) in tumor-bearing mice. Clinical validation was sought for AVA6000 given intravenously q2w or q3w in a first-in-human, 3+3 dose escalation Phase 1 trial exploring the safety, PK and preliminary efficacy. Preclinical data with both AVA6000 and AVA6103 demonstrate substantial tumor-specific payload release, with high tumor-to-plasma ratios ranging from 10:1 to > 200:1 indicating selective release and uptake within the tumor. Preliminary analysis of PK data with AVA6103 in murine models demonstrated a differentiated PK profile versus AVA6000 with sustained payload release in the tumor (tumor:plasma of 10:1 longer than 24 hours) with no systemic exatecan exposure beyond 4 hours. In the Phase 1 trial of AVA6000, 63 patients received escalating doses of AVA6000 from 80mg/m2 (equivalent to 54 mg/m2 dox) to 385 mg/m2 (260 mg/m2 dox) with no MTD identified in the trial. Importantly, of the 63 patients dosed, 53 patients received molar equivalent doses of dox higher than the conventional 75 mg/m2 dose. PK proof of principle was demonstrated in patients treated with AVA6000, with elevated released tumor dox concentrations (median tumor:plasma 100:1 at 24 hours post first dose) and lower plasma Cmax of released dox (77.9-92.5% reduction). The AUC of released dox was lower (4.8-77%) across dose levels when compared with published data for conventional dox (75 mg/m2). In addition, released dox had a 40% lower volume of distribution and a >40% increase in plasma elimination half-life compared with conventional dox (75 mg/m2, historical data). Clinical toxicities were reduced relative to historical data and preliminary signals of efficacy were seen, including a disease control rate of 90% in patients with salivary gland cancers (n=10) treated at >250 mg/m2 doses. In the Phase 1 trial, AVA6000 exhibits promising tumor-targeting characteristics with encouraging clinical activity and reduced systemic toxicities validating the tumor-specific FAP cleavage mode of action. These AVA6000 data along with the preclinical PK data with AVA6103 have informed the predictive modeling to support the clinical development of AVA6103 by optimizing the delivery of the exatecan payload. Gezim Lahu, Udai Banerji, William C. Tap, Natalie Cook, Jeff T. Evans, Ruth Plummer, Alan Anthoney, Christopher Plummer, Rhona Dawson, Paul Loadman, Huw Jones, Nelson Kinnersley, Ruairidh Edwards, Curtis Rink, Ellen Watts, Gilad Gordon, Lee D. Cranmer, Chris Twelves. Comparative pharmacokinetics and tumor activation of fibroblast activation protein (FAP)-enabled pre|CISION® peptide drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT151.

Supplementary Table 1 shows the list of SNPs that were analysed by the Fluidigm assay.

Supplementary Figure 3 shows the distribution of individual total (combined adenomatous and serrated polyp) colorectal polyp counts according to seAFOod trial treatment allocation by factorial margins and TP53 rs104522 genotype.

Abstract Background: AVA6000 is a peptide drug conjugate, consisting of a peptide moiety that is specifically cleaved by FAP in the TME that is bound to doxorubicin. FAP is selectively overexpressed in many solid tumors. The peptide moiety linker (pre|CISIONTM) prevents cellular entry of doxorubicin unless cleaved by FAP, thus enabling targeted delivery of doxorubicin to tumors. Design: The safety, PK and preliminary efficacy of AVA6000 was studied in a first-in-man, multicenter dose escalation phase I trial. AVA6000 was administered intravenously q3w using a standard 3+3 design in patients with locally advanced or metastatic solid tumors reported to be FAP-positive. PK (AUC) based dosing was implemented to calculate maximum cycles of AVA6000 with the cumulative limit of 550 mg/m2. Results: Forty patients (median age 65 years, range 30-79), with median 3 prior lines of therapy (range, 0-7) received escalating AVA6000 doses from 80mg/m2 (51 mg/m2 doxorubicin; molar equivalent, 0.675) to 385 mg/m2 (260 mg/m2 doxorubicin equivalent). Tumor types included soft tissue sarcoma (30%), colorectal cancer (27.5%), pancreatic cancer (20%), cancers of the biliary tract (7.5%). The safety profile was favorable, the most frequent adverse events (any grade) being fatigue (50%), alopecia (42.5%), and nausea (32.5%), with rare grade 3-4 hematologic toxicities of neutropenia (7.5%), anemia, thrombocytopenia, and WBC decreased (5% each). Grade 3 non-hematologic toxicities included mucositis, fatigue and hematemesis (n=1 each). No grade 4 non-hematologic toxicities were reported. Two DLTs were observed of grade 2 cardiac failure (120 mg/m2; LVEF decrease 61 to 39%) and grade 4 neutropenia/thrombocytopenia (200 mg/m2). AVA6000 distributes rapidly with a t1/2 of 45 min. The Cmax of released doxorubicin was reduced as compared to standard dose doxorubicin (range 78-93% reduction) across dose cohorts. Tumor biopsy data demonstrate concentration of doxorubicin in the TME of mean 860 ng/gm (range 76-2310 ng/gm, n=9). In contrast, blood samples taken with the biopsy demonstrate a circulating free doxorubicin of 8.3 ng/ml (range 2.4-15.9), indicating concentration of doxorubicin in the tumor relative to plasma. Using RECISTv.1.1, one PR (-65%) with duration of 6 months was observed in a patient with undifferentiated pleomorphic sarcoma at 160 mg/m2, and a mixed response in a patient with angiosarcoma (SLD -22%) at 200 mg/m2. The disease control rate was 50%, at 12 wks. In each case, SD > 4 months or PR was associated with high FAP enzyme activity in the on-study tumor biopsy compared to observed disease progression as best response (n=6). Conclusions: AVA6000 delivers high concentration of doxorubicin to the TME relative to plasma which results in antitumor activity in tumors with high FAP activity. A q2w dose escalation arm is ongoing. Citation Format: Udai Banerji, Natalie Cook, Alan Anthoney, Ruth Plummer, William D. Tap, Jeffry T. Evans, Lee D. Cranmer, Christopher Plummer, Paul Loadman, Gezim Lahu, Huw S. Jones, Nelson Kinnersley, Fiona McLaughlin, Chris Twelves. A Phase I trial of AVA6000, a Fibroblast Activation Protein (FAP)-released and tumor microenvironment (TME)-targeted doxorubicin peptide drug conjugate in patients with FAP-positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT188.

Supplementary Table 2 shows the results of Negative binomial regression analysis of total colorectal polyp number according to aspirin use stratified for each SNP

Supplementary Figure 2 shows the distribution of individual total (combined adenomatous and serrated polyp) colorectal polyp counts according to seAFOod trial treatment allocation by factorial margins and ALOX SNP genotypes.

BACKGROUND:The seAFOod randomized controlled trial tested colorectal polyp prevention by the omega-3 (ω-3) highly unsaturated fatty acid (HUFA) eicosapentaenoic acid (EPA) and aspirin. Variable dietary intake of omega-3 HUFAs (also including docosahexaenoic acid [DHA]) and differential EPA capsule compliance could confound analysis of trial outcomes. OBJECTIVE:The objective of this study was to investigate the relationship between total (diet and capsule) daily omega-3 HUFA intake, red blood cell (RBC), and rectal mucosa omega-3 HUFA concentrations, and colorectal polyp outcomes in a secondary analysis of the seAFOod trial. METHODS:Individual-participant dietary omega-3 HUFA intake (mg/d) was derived from food frequency questionnaires using the European Prospective Investigation into Cancer and Nutrition-Norfolk fatty acid nutrient database. Capsule EPA intake (mg/d) was adjusted for compliance (capsule counting). Fatty acids were analyzed by liquid chromatography-tandem mass spectrometry (as % of total fatty acids). HUFA oxidation was measured using the HUFA/saturated fatty acid (SAT) ratio. The colorectal polyp detection rate (PDR; % with ≥1 polyps) and polyp number per participant were analyzed according to the change in RBC EPA concentrations during the trial (ΔEPA), irrespective of treatment allocation. RESULTS:There was a small degree of HUFA degradation over time in RBC samples stored at > -80oC at research sites (r = -0.36, P<0.001 for HUFA/SAT ratio over time), which did not affect analysis of omega-3 HUFA concentrations. Low baseline EPA concentration, as well as allocation to EPA and % compliance, were associated with a high ΔEPA. Individuals with a ΔEPA value >+0.5% points (ΔEPAhigh), irrespective of allocation to EPA or placebo, had a lower PDR than ΔEPAlow individuals (odds ratio: 0.63; 95% confidence interval [CI]: 0.40, 1.01) and reduced colorectal polyp number (incidence rate ratio: 0.74; 95% CI: 0.54, 1.02). CONCLUSIONS:Analysis of the seAFOod trial according to the change in EPA concentration, instead of treatment allocation, revealed a protective effect of EPA treatment on colorectal polyp recurrence (ISRCTN05926847).

The integrin family of cell surface extracellular matrix binding proteins are key to several physiological processes involved in tissue development, as well as cancer proliferation and dissemination. They are therefore attractive targets for drug discovery with cancer and non-cancer applications. We have developed a new integrin antagonist chemotype incorporating a functionalised cyclobutane ring as the central scaffold in an arginine-glycine-aspartic acid mimetic structure. Here, we report the synthesis of cyclobutanecarboxylic acids and cyclobutylamines with tetrahydronaphthyridine and aminopyridine arginine mimetic sidechains and masked carboxylic acid aspartic acid mimetic sidechains of varying length. Effective αvβ3 antagonists and new aspartic acid mimetics were identified in cell-based adhesion and invasion assays. A lead compound selected based on in vitro activity (IC50 < 1 μM), stability (t 1/2 > 80 minutes) and synthetic tractability was well-tolerated in vivo. These results show the promise of this synthetic approach for developing αvβ3 antagonists and provide a firm foundation to progress into advanced preclinical evaluation prior to progression towards the clinic. Additionally, they highlight the use of functionalised cyclobutanes as metabolically stable core structures and a straightforward and robust method for their synthesis. This important contribution to the medicinal chemists' toolbox paves the way for increased use of cyclobutanes in drug discovery.

IntroductionWe examined the relationship between Apolipoprotein E (APOE) genotype and n-3 highly unsaturated fatty acid (HUFA) levels in participants of the seAFOod trial, who were undergoing colonoscopy surveillance after removal of colorectal polyps.MethodsBaseline and on-treatment (eicosapentaenoic acid [EPA] 2 g daily or placebo for 6 months) levels of n-3 HUFAs, and plasma 18-hydroxyeicosapentaenoic acid (HEPE), were analysed according to APOE genotype (based on polymorphisms rs429358 and rs7412) in 584 participants.ResultsBefore treatment, APOE2/2 individuals had lower levels, and APOE4/4 participants had higher levels, of n-3 HUFAs, including EPA, than APOE3/3 counterparts (P < 0.01 for the APOE2/2 versus APOE4/4 comparison). After EPA supplementation, n-3 HUFA levels were not significantly different when stratified by APOE genotype, although APOE4 carriers displayed lower plasma 18-HEPE levels than individuals without an APOE4 allele (P = 0.002).ConclusionsAPOE genotype is associated with differential n-3 HUFA and 18-HEPE levels in individuals with multiple colorectal polyps.

Targeted therapy remains the future of anti-cancer drug development, owing to the lack of specificity of current treatments which lead to damage in healthy normal tissues. ATR inhibitors have in recent times demonstrated promising clinical potential, and are currently being evaluated in the clinic. However, despite the considerable optimism for clinical success of these inhibitors, reports of associated normal tissues toxicities remain a concern and can compromise their utility. Here, ICT10336 is reported, a newly developed hypoxia-responsive prodrug of ATR inhibitor, AZD6738, which is hypoxia-activated and specifically releases AZD6738 only in hypoxic conditions, in vitro. This hypoxia-selective release of AZD6738 inhibited ATR activation (T1989 and S428 phosphorylation) and subsequently abrogated HIF1a-mediated adaptation of hypoxic cancers cells, thus selectively inducing cell death in 2D and 3D cancer models. Importantly, in normal tissues, ICT10336 is demonstrated to be metabolically stable and less toxic to normal cells than its active parent agent, AZD6738. In addition, ICT10336 exhibited a superior and efficient multicellular penetration ability in 3D tumor models, and selectively eradicated cells at the hypoxic core compared to AZD6738. In summary, the preclinical data demonstrate a new strategy of tumor-targeted delivery of ATR inhibitors with significant potential of enhancing the therapeutic index.

Therapeutic microbubbles (thMBs) contain drug-filled liposomes linked to microbubbles and targeted to vascular proteins. Upon the application of a destructive ultrasound trigger, drug uptake to tumour is improved. However, the structure of thMBs currently uses powerful non-covalent bonding of biotin with avidin-based proteins to link both the liposome to the microbubble (MB) and to bind the targeting antibody to the liposome–MB complex. This linkage is not currently FDA-approved, and therefore, an alternative, maleimide–thiol linkage, that is currently used in antibody–drug conjugates was examined. In a systematic manner, vascular endothelial growth factor receptor 2 (VEGFR2)-targeted MBs and thMBs using both types of linkages were examined for their ability to specifically bind to VEGFR2 in vitro and for their ultrasound imaging properties in vivo. Both showed equivalence in the production of the thMB structure, in vitro specificity of binding and safety profiles. In vivo imaging showed subtle differences for thMBs where biotin thMBs had a faster wash-in rate than thiol thMBs, but thiol thMBs were longer-lived. The drug delivery to tumours was also equivalent, but interestingly, thiol thMBs altered the biodistribution of delivery away from the lungs and towards the liver compared to biotin thMBs, which is an improvement in biosafety.

BACKGROUND:The seAFOod polyp prevention trial was a randomised, placebo-controlled, 2 × 2 factorial trial of aspirin 300 mg and eicosapentaenoic acid (EPA) 2000 mg daily in individuals who had a screening colonoscopy in the English Bowel Cancer Screening Programme (BCSP). Aspirin treatment was associated with a 20% reduction in colorectal polyp number at BCSP surveillance colonoscopy 12 months later. It is unclear what happens to colorectal polyp risk after short-term aspirin use.AIM:To investigate colorectal polyp risk according to the original trial treatment allocation, up to 6 years after trial participation.METHODS:All seAFOod trial participants were scheduled for further BCSP surveillance and provided informed consent for the collection of colonoscopy outcomes. We linked BCSP colonoscopy data to trial outcomes data.RESULTS:In total, 507 individuals underwent one or more colonoscopies after trial participation. Individuals grouped by treatment allocation were well matched for clinical characteristics, follow-up duration and number of surveillance colonoscopies. The polyp detection rate (PDR; the number of individuals who had ≥1 colorectal polyp detected) after randomization to placebo aspirin was 71.1%. The PDR was 80.1% for individuals who had received aspirin (odds ratio [OR] 1.13 [95% confidence interval 1.02, 1.24]; p = 0.02). There was no difference in colorectal polyp outcomes between individuals who had been allocated to EPA compared with its placebo (OR for PDR 1.00 [0.91, 1.10]; p = 0.92).CONCLUSION:Individuals who received aspirin in the seAFOod trial demonstrated increased colorectal polyp risk during post-trial surveillance. Rebound elevated neoplastic risk after short-term aspirin use has important implications for aspirin cessation driven by age-related bleeding risk. ISRCTN05926847.

PDF file - 156K, Chromatogram of metabolite profile of ICT2700 incubated with human ex vivo liver microsomes demonstrating there is no evidence for the appearance of the cytotoxic metabolite ICT2740

Supplementary Data from Development of a Novel Tumor-Targeted Vascular Disrupting Agent Activated by Membrane-Type Matrix Metalloproteinases

PDF file - 52K, Lack of CYP1 induction by the chloromethylpyrroloindoline ICT2700

PDF file - 107K, In vivo metabolic profile of tumour and liver after administration of ICT2700 to CYP1A1 tumor-bearing mice

Clinical characteristics of the study cohort of 542 seAFOod trial participants who had a SNP genotype profile and trial colonoscopy outcome data.

Abstract Aspirin and eicosapentaenoic acid (EPA) reduce colorectal adenomatous polyp risk and affect synthesis of oxylipins including prostaglandin E2. We investigated whether 35 SNPs in oxylipin metabolism genes such as cyclooxygenase (PTGS) and lipoxygenase (ALOX), as well as 7 SNPs already associated with colorectal cancer risk reduction by aspirin (e.g., TP53; rs104522), modified the effects of aspirin and EPA on colorectal polyp recurrence in the randomized 2 × 2 factorial seAFOod trial. Treatment effects were reported as the incidence rate ratio (IRR) and 95% confidence interval (CI) by stratifying negative binomial and Poisson regression analyses of colorectal polyp risk on SNP genotype. Statistical significance was reported with adjustment for the false discovery rate as the P and q value. 542 (of 707) trial participants had both genotype and colonoscopy outcome data. Reduction in colorectal polyp risk in aspirin users compared with nonaspirin users was restricted to rs4837960 (PTGS1) common homozygotes [IRR, 0.69; 95% confidence interval (CI), 0.53–0.90); q = 0.06], rs2745557 (PTGS2) compound heterozygote-rare homozygotes [IRR, 0.60 (0.41–0.88); q = 0.06], rs7090328 (ALOX5) rare homozygotes [IRR 0.27 (0.11–0.64); q = 0.05], rs2073438 (ALOX12) common homozygotes [IRR, 0.57 (0.41–0.80); q = 0.05], and rs104522 (TP53) rare homozygotes [IRR, 0.37 (0.17–0.79); q = 0.06]. No modification of colorectal polyp risk in EPA users was observed. In conclusion, genetic variants relevant to the proposed mechanism of action on oxylipins are associated with differential colorectal polyp risk reduction by aspirin in individuals who develop multiple colorectal polyps. SNP genotypes should be considered during development of personalized, predictive models of colorectal cancer chemoprevention by aspirin. Prevention Relevance: Single-nucleotide polymorphisms in genes controlling lipid mediator signaling may modify the colorectal polyp prevention activity of aspirin. Further investigation is required to determine whether testing for genetic variants can be used to target cancer chemoprevention by aspirin to those who will benefit most.