SN-38 Levels Research Articles

A platform technology for ultra-long acting intratumoral therapy

Intratumoral (IT) therapy is a powerful method of controlling tumor growth, but a major unsolved problem is the rapidity that injected drugs exit tumors, limiting on-target exposure and efficacy. We have developed a generic long acting IT delivery system in which a drug is covalently tethered to hydrogel microspheres (MS) by a cleavable linker; upon injection the conjugate forms a depot that slowly releases the drug and “bathes” the tumor for long periods. We established technology to measure tissue pharmacokinetics and studied MSs attached to SN-38, a topoisomerase 1 inhibitor. When MS ~ SN-38 was injected locally, tissues showed high levels of SN-38 with a long half-life of ~ 1 week. IT MS ~ SN-38 was ~ tenfold more efficacious as an anti-tumor agent than systemic SN-38. We also propose and provide an example that long-acting IT therapy might enable safe use of two drugs with overlapping toxicities. Here, long-acting IT MS ~ SN-38 is delivered with concurrent systemic PARP inhibitor. The tumor is exposed to both drugs whereas other tissues are exposed only to the systemic drug; synergistic anti-tumor activity supported the validity of this approach. We propose use of this approach to increase efficacy and reduce toxicities of combinations of immune checkpoint inhibitors such as αCTLA-4 and αPD-1.

Abstract LB439: Intra-tumoral administration of releasable hydrogel microsphere drug conjugates for long acting therapeutics

Abstract Background. Intra-tumoral (IT) injections of therapeutics hold promise for the treatment of solid tumors. However, drugs administered directly in the tumor have short IT residence times potentially limiting their exposure and therefore efficacy. We are developing a platform of novel hydrogel microsphere (MS) conjugates that enables long-acting tumor residence after a single injection of the conjugate. We demonstrate this approach not only solves the problem of short acting intra-tumoral therapy, but also allows for the safe administration of two therapies with overlapping toxicities such as SN-38 and a PARP inhibitor (PARPi). Methods. Hydrogel microsphere (MS) conjugates were prepared and characterized. Using MS conjugates labeled with rhodamine and fluorescein, we established methods to directly measure the IT concentration and half-life of the released drug after IT administration into CT26 tumor bearing mice. We next measured the tissue pharmacokinetics and efficacy of MSs attached to SN-38, a potent topoisomerase 1 inhibitor and immunomodulator, using NSG mice bearing 22Rv1 ATM-/- xenografts. Finally, we measured the anti-tumor efficacy of MS~SN-38 in combination with an oral PARPi, talazoparib, in 22Rv1 ATM-/- xenografts. Results. We successfully established methods to quantify the concentration of released drug and MS bound drug from tumor biopsies using fluorescent surrogates. When applied to MS~SN-38 conjugates, locally injected tissues showed high levels of SN-38 with a long half-life of about one week. In contrast, the systemic half-life of free SN-38 is ~30 minutes. IT MS~SN-38 was ~10-fold more efficacious as an anti- tumor agent than systemic SN-38 from subcutaneous administration of MS~SN-38. Finally, we show high anti-tumor synergy from the SN-38 localized in a tumor by IT MS~SN-38 in combination with systemic administration of the PARPi talazoparib. Conclusions. This microsphere technology allows for sustained delivery of high local concentrations of drugs administered intratumorally. Here, IT MS~SN-38 resulted in an intra-tumoral half-life of ~1 week and marked anti-tumor efficacy in 22Rv1 ATM-/- xenografts. We also demonstrated that long-acting IT injections enables safe use of drug combinations with well-described overlapping toxicities such as SN-38 and PARPi. The microsphere technology may offer an alternative strategy for localizing and sustaining drugs in tumors and may mitigate toxicities from combination therapy where two agents have a similar toxicological profile. Other combinations, including small molecules and immuno-oncology agents, are being explored. Citation Format: John A. Hangasky, Jeff Henise, Deborah Charych, Christopher W. Carreras, Gary Ashley, Daniel V. Santi. Intra-tumoral administration of releasable hydrogel microsphere drug conjugates for long acting therapeutics [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 LB439.

Tight-Binding Small-Molecule Carboxylesterase 2 Inhibitors Reduce Intracellular Irinotecan Activation.

As the primary enzyme responsible for the activatable conversion of Irinotecan (CPT-11) to SN-38, carboxylesterase 2 (CES2) is a significant predictive biomarker toward CPT-11-based treatments for pancreatic ductal adenocarcinoma (PDAC). High SN-38 levels from high CES2 activity lead to harmful effects, including life-threatening diarrhea. While alternate strategies have been explored, CES2 inhibition presents an effective strategy to directly alter the pharmacokinetics of CPT-11 conversion, ultimately controlling the amount of SN-38 produced. To address this, we conducted a high-throughput screening to discover 18 small-molecule CES2 inhibitors. The inhibitors are validated by dose-response and counter-screening and 16 of these inhibitors demonstrate selectivity for CES2. These 16 inhibitors inhibit CES2 in cells, indicating cell permeability, and they show inhibition of CPT-11 conversion with the purified enzyme. The top five inhibitors prohibited cell death mediated by CPT-11 when preincubated in PDAC cells. Three of these inhibitors displayed a tight-binding mechanism of action with a strong binding affinity.

CTNI-10. SACITUZUMAB GOVITECAN FOR BREAST CANCER BRAIN METASTASIS

Abstract Breast cancer brain metastasis (BCBM) is a challenge that significantly impacts patient prognosis and quality of life. Sacituzumab govitecan (SG), an antibody-drug conjugate, has demonstrated antitumor activity and is approved for triple negative and HR+/HER2 negative metastatic breast cancer. It consists of a humanized anti-Trop-2 monoclonal antibody linked to a potent topoisomerase I inhibitor, SN-38. SG activity was tested in a breast cancer intracranial mouse model. We conducted a prospective, single center, window of opportunity trial (NCT03995706) to examine the intra-tumoral concentrations of SN-38 in patients undergoing craniotomy for breast cancer brain metastases (n = 13). We enrolled patients aged ≥18 years diagnosed with brain metastatic breast cancer to receive a single intravenous dose of SG at 10 mg/kg one day before surgical resection. Tumor and corresponding serum were collected during surgery to measure their levels of SN-38. Following recovery, patients resumed SG treatment at 10 mg/kg on days 1 and 8 of 21-day cycles and were assessed for responses by MRI every third cycle using response assessment in neuro-oncology (RANO) criteria. Trop-2 and carbonic anhydrase IX (CAIX) expression was investigated by IHC. SG significantly inhibited tumor growth and increased overall survival in mice. An average of 1953.79 ng/ml in serum and 134.3 ng/g in tissue of SN-38 was quantified in patient samples. Trop-2 expression was observed in 100% of patient tumors. 41% of the samples showed high expression of CAIX. A positive correlation between the tissue:serum [SN-38], Trop-2 and gH2AX staining was observed. Median overall survival of 34.5 months (5.7 - 35.6 months) and progression free survival of 7.4 months (2 – 24.4 months) were observed. Our data suggest that SG has therapeutic potential in patients with breast cancer brain metastasis. A prospective multicenter cooperative trial of SG for BCBM, S2007, is ongoing and an update on accrual will be provided.

CTNI-36. SACITUZUMAB GOVITECAN FOR RECURRENT GLIOBLASTOMA

Abstract Glioblastoma (GBM) is an aggressive and highly malignant brain tumor characterized by its infiltrative growth, rapid progression, and poor prognosis. Sacituzumab govitecan (SG), an antibody-drug conjugate, has demonstrated antitumor activity and acceptable tolerability in patients with advanced epithelial cancers. It consists of a humanized anti-Trop-2 monoclonal antibody linked to a potent topoisomerase I inhibitor, SN-38. We conducted a prospective, single center, window of opportunity trial (NCT03995706) to examine the intra-tumoral concentrations of SN-38 in patients undergoing craniotomy for recurrent GBM (rGBM; n = 12). We enrolled patients aged ≥18 years diagnosed with rGBM to receive a single intravenous dose of SG at 10 mg/kg one day before surgical resection. Tumor and corresponding serum were collected during surgery to measure their levels of SN-38. Following recovery, patients resumed SG treatment at 10 mg/kg on days 1 and 8 of 21-day cycles and were accessed for responses by MRI every third cycle using response assessment in neuro-oncology (RANO) criteria. Trop-2 and carbonic anhydrase IX (CAIX) expression was investigated by IHC. An average of 2850.6 ng/ml in serum and 128.7 ng/g in tissue of SN-38 was quantified in our patient samples. Trop-2 expression was observed in 50% of patient tumors and 38% of the samples showed high expression of hypoxia marker CAIX. A strong positive correlation between the tissue/serum SN-38 and Trop-2 (r = 0.86, p = 0.01) was observed. Median overall survival of 17.5 months (0.9 - 28.5 months) and progression free survival of 2 months (0.5 - 12 months) were observed. A multicenter, phase II clinical trial has been initiated to further investigate the efficacy of SG in patients with breast brain metastasis. Updated data on response rates and accrual to the multicenter trial for rGBM will be presented

A novel dsDNA decamer-based electrochemical biosensor for selective determination of irinotecan active metabolite - SN38

This paper presents a novel biosensor for detecting the active metabolite of irinotecan, SN38, with high selectivity and sensitivity. The biosensor utilizes a dsDNA decamer (5′-GCGTTGTCGC-3′) that selectively interacts with SN38. The biosensor, consisting of a screen-printed graphite electrode with the dsDNA decamer as the recognition layer, demonstrates stability, sensitivity, and selectivity towards SN38 through electrochemical studies. It offers a wide linear range of 1–200 nM, comparable to the high-performance liquid chromatography (HPLC) method, with a sensitivity of 57 nA/nM and a detection limit of 1.06 nM. Compared to a bare graphite electrode, the biosensor exhibits a significant 31-fold increase in sensitivity and a detection limit three orders of magnitude lower. Selectivity studies were conducted using electrochemically active and inactive interferents, including biologically active compounds. Bioreceptor interactions with chosen interferents were examined using HPLC and nuclear magnetic resonance (NMR). Tests of the biosensor was performed using laboratory samples and artificial urine samples spiked with SN38. This disposable biosensor, based on dsDNA, provides an innovative approach for accurately determining SN38 levels in the therapeutic drug concentration range. It holds promise as an alternative method for clinical diagnosis and monitoring of SN38 levels in cancer patients undergoing irinotecan treatment.

Abstract 6716: Preclinical evaluation of nano-liposomal irinotecan in pediatric solid tumor patient-derived xenografts

Abstract Background: Pediatric solid tumors are rare and the use of well characterized preclinical models can be a helpful tool to prioritize novel drug combinations and formulations which are challenging to test in a limited patient population. Irinotecan (IRN) has shown efficacy in diverse pediatric cancers where children are often treated on an extended low-dose schedule rather than a single large dose commonly used in adults. The use of nano-liposomal irinotecan (nal-IRI) is a promising strategy as 95% of the drug remains encapsulated during systemic exposure while maintaining prolonged exposure in the tumor. Adult clinical trials have shown reduced side effects without compromising efficacy but nal-IRI has had limited use thus far in pediatric cancers. Methods: Orthotopic xenograft models of Ewing sarcoma (EWS, 4 models) and rhabdomyosarcoma (RMS, 2 models) were generated by injecting luciferase labeled cells into the femur and hind-leg muscle of nude mice respectively. Pharmacokinetic (PK) studies on normal and tumor-bearing mice were performed for both nal-IRI and standard IRN to determine matched human AUC-guided dosing and schedule of chemotherapy agents. Mice were randomized to single agent and relevant combinations of nal-IRI with PARP inhibitors and temozolomide (EWS) and with vincristine and temozolomide (RMS) and compared to the same regimens using standard IRN. Therapy was given on a clinically relevant schedule with each course consisting of 21 days and tumor response was measured weekly using bioluminescence. Results: PK studies showed that plasma levels of the active metabolite of IRN (SN-38) drop quickly for both standard IRN and nal-IRI single dosed mice while the levels of SN-38 were maintained in the tumor for several days using the nal-IRI formulation. In all 6 models tested, nal-IRI combinations were as or more effective when compared to regimens using standard IRN. No significant toxicity was seen in mice treated with nal-IRI combinations. Conclusions: The substitution of nal-IRI for standard IRN remains a promising strategy in pediatrics with the potential to reduce side effects as well as hospital visits due to the dosing schedule with nal-IRI given once per cycle where standard IRN is often given for 5 or 10 days in children. Our preclinical PK data confirmed the liposomal formulation resulted in sustained SN-38 levels in the tumor and efficacy was maintained or improved in pediatric cancer models. The combination of nal-IRI with temozolomide and talazoparib is now being tested in a multi-center randomized phase I/II trial for children with relapsed solid tumors and this data supports consideration for use in other pediatric cancer trials both in the upfront and relapse setting. Citation Format: Elizabeth Stewart, Kaley Blankenship, Burgess Freeman, Sara Federico, Michael Dyer. Preclinical evaluation of nano-liposomal irinotecan in pediatric solid tumor patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6716.

Abstract P1-14-04: A Phase 0 Clinical trial of Sacituzumab Govitecan in Patients with Breast Cancer Brain Metastases and Recurrent Glioblastoma

Abstract Purpose: Sacituzumab govitecan has shown efficacy and acceptable tolerability in a multicenter phase I/II clinical trial (NCT01631552) in patients with advanced epithelial cancers. Our study was initiated to determine the bioavailability of Sacituzumab govitecan (SG) in breast brain metastasis and glioblastoma. The goals were to evaluate the extent by which SG can penetrate the blood brain barrier and access tumor tissues by testing free SN-38, SN-38G and total SN-38 concentrations in tumor tissue, serum, and CSF. Patients and methods: Patients diagnosed with brain metastatic breast cancer and recurrent glioblastoma were enrolled in a single-center clinical phase 0 study to receive a single intravenous dose of SG at 10 mg/kg one day before surgical resection. Tumor and corresponding serum were collected during surgery to measure their levels of SN-38 and its metabolites. Following recovery, patients resumed SG treatment at 10 mg/kg on days 1 and 8 of 21-day cycles and were assessed for responses by MRI every third cycle using response assessment in neuro-oncology (RANO) criteria. Total Sn-38 levels were quantified in tumor tissue and corresponding serum from the patients. Trop-2 and carbonic anhydrase IX (CAIX) expression was investigated by IHC. SG activity was tested in a breast cancer intracranial mouse model. Results: An average of 2365 ng/ml in serum and 132 ng/g in tissue of total SN-38 was quantified in our patient samples. Trop-2 expression was observed in 90% of patient tumors. 40% of the samples showed high expression of CAIX. SG significantly inhibited tumor growth in vivo and increased overall survival. 20% of patients in the breast cancer arm of the trial demonstrated a complete response by RANO criteria and the remaining 80% of patients demonstrated a partial response. Ultimately, 80% of the patients in the breast cancer metastasis arm survived. Conclusion: Sacituzumab govitecan is an effective drug that crosses the blood brain barrier to inhibit the growth of metastatic breast cancer to the brain after tumor resection surgery. Citation Format: Pegah Ghamasaee, Henriette Balinda, Andrew Brenner, John Floyd. A Phase 0 Clinical trial of Sacituzumab Govitecan in Patients with Breast Cancer Brain Metastases and Recurrent Glioblastoma [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-14-04.

Liposomal Irinotecan Shows a Larger Therapeutic Index than Non-liposomal Irinotecan in Patient-Derived Xenograft Models of Pancreatic Cancer.

Liposomal irinotecan promotes controlled sustained release of irinotecan (CPT-11), therefore, we hypothesize that the therapeutic index (quantitative measurement of the relative efficacy/safety ratio of a drug) will be higher for liposomal than non-liposomal irinotecan. We compared the therapeutic indexes of liposomal and non-liposomal irinotecan in mice bearing subcutaneous patient-derived xenograft (PDX) pancreatic tumors under dosing regimens approximating the clinical setting. Following preliminary drug sensitivity/antitumor activity analyses on three PDX tumor models, one model was selected for analyses of efficacy, biomarker, toxicology, pharmacokinetics in mice receiving liposomal irinotecan (2.5, 10, 50mg/kg/week) or non-liposomal irinotecan (10, 25, 50mg/kg/week). The maximum tolerated dose (MTD) for each treatment was 50mg/kg/week. Using the selected IM-PAN-001 model at the MTD (both treatments, 50mg/kg/week), antitumor activity, phospho-histone gamma-H2AX protein staining in cancer cell nuclei, histological tumor regression, and plasma levels of CPT-11 and its active metabolite SN-38 after 24h were greater with liposomal than non-liposomal irinotecan, but tumor SN-38 levels were similar. At the lowest doses assessed, antitumor activity, histological tumor regression, and jejunum and bone marrow toxicity were similar. Based on these findings, liposomal and non-liposomal irinotecan had therapeutic indexes of 20 and 5, respectively. This non-clinical study showed a fourfold broader therapeutic index with liposomal than non-liposomal irinotecan in mice bearing IM-PAN-001 PDX pancreatic tumors, even at optimal dosing for the two drugs. These findings support the clinical benefit observed with liposomal irinotecan in patients with pancreatic cancer.

Pulmonary delivery of size-transformable nanoparticles improves tumor accumulation and penetration for chemo-sonodynamic combination therapy.

Very little is currently known about how inhaled nanomedicine for lung cancer treatment overcomes biological barriers hampering the tumor availability of drug and nanoparticles. Here, we developed a size-transformable nanocarrier (~ 119nm) in which small-size nanoparticles (~ 28nm) were loaded in the large nanocarrier after the addition of modified hyaluronan and could be released upon size-transformation at tumor tissue. Subsequently, the pulmonary and tumor pharmacokinetics of the two nanocarriers containing 7-ethyl-10-hydroxycamptothecin (SN38) and a covalently linked fluorescent sonosensitizer were comparatively investigated after intratracheal instillation to mice bearing orthotopic Lewis lung carcinoma tumors. The results showed that both instilled nanoparticles seemed to transport drug to tumor by direct access and transcytosis of nanoparticles, and diffusion of the released drug with the latter accounting for a great proportion of the drug tumor bioavailability. Relative to the small-size nanocarrier, the size-transformable counterpart appeared to restrict the mucociliary and absorption clearances from the lung and the clearance from the tumor interstitium to circulation, leading to increases in lung and tumor bioavailability of SN38 by 58.5% and 199%, respectively. In addition, the size-transformable nanoformulation conferred deep tumor penetration and sustained levels of both sonosensitizer and SN38 within tumors and simultaneously exerted sonodynamic- and chemo-therapies. Overall, the pulmonary delivery of size-transformable nanocarrier could co-deliver sonosensitizer and drug to deep tumor sites with enhanced tumor accumulation to realize combination therapy in lung cancer.

Evaluation of UGT1A1 and CYP3A Genotyping and Single-Point Irinotecan and Metabolite Concentrations as Predictors of the Occurrence of Adverse Events in Cancer Treatment.

The variability on irinotecan (IRI) pharmacokinetics and toxicity has been attributed mostly to genetic variations in the UGT1A1 gene, responsible for conjugation of the active metabolite SN-38. Also, CYP3A mediates the formation of inactive oxidative metabolites of IRI. The association between the occurrence of severe adverse events, pharmacokinetics parameters, and UGT1A1 and CYP3A4 predicted phenotypes was evaluated, as the evaluation of [SN-38]/IRI dose ratio as predictor of severe adverse events. Forty-one patients undergoing IRI therapy were enrolled in the study. Blood samples were collected 15min after the end of drug the infusion, for IRI, SN-38, SN-38G, bilirubin concentrations measurements, and UGT1A1 and CYP3A genotype estimation. Data on adverse event was reported. Fifteen patients (36.5%) developed grade 3/4 adverse events. A total of 9.8% (n = 4) of the patients had UGT1A1 reduced activity phenotype, and 48.7% (n = 20) had UGT1A1 and 63.4% (n = 26) CYP3A intermediary phenotypes. Severe neutropenia and diarrhea were more prevalent in patients with reduced UGT1A1 in comparison with functional metabolism (50% and 75% versus 0% and 13%, respectively). SN-38 levels and its concentrations adjusted by IRI dose were significantly correlated to toxicity (rs = 0.31 (p = 0.05) and rs = 0.425 (p < 0.01)). The [SN-38]/IRI dose ratio had a ROC curve of 0.823 (95% CI 0.69-0.956) to detect any severe adverse event and 0.833 (95% CI 0.694-0.973) to detect severe diarrhea. The cut-off of 0.075ngmL-1mg-1 had 100% sensitivity and 65.7% specificity to predict severe diarrhea. Our data confirmed the relevance of the pre-emptive genotypic information of UGT1A1. The [SN-38]/IRI ratio, measured 15min after the end of the IRI infusion, was a strong predictor of severe toxicity and could be applied to minimize the burden of patients after IRI administration.

In vitro metabolic biomodulation of irinotecan to increase potency and reduce dose-limiting toxicity by inhibition of SN-38 glucuronide formation

Colorectal cancer continues to have one of the highest incidents of occurrence with a rising rate of diagnosis among people under the age of 50. Chemotherapy with irinotecan results in severe gastrointestinal dose-limiting toxicity that is caused by the glucuronidated form of the active metabolite (SN-38G). This study evaluates herbal compounds and analogs to biomodulate the metabolism of IR to decrease dose-limiting toxicity while increasing the amount of the active metabolite. In vitro metabolism using human liver microsomes was conducted with white willow bark (WWB) extract, select specific components of WWB, and analogues to evaluate biomodulation of the IR metabolism. Samples were analyzed using liquid chromatography-tandem mass spectrometry to measure metabolites between reactions with and without herbals components. WWB showed an optimal decrease (>80%) in SN-38G and a corresponding increase in SN-38 levels (128%) at a concentration of near 200μg/mL. Tannic acid produced a 75% decrease in SN-38G with a 130% increase in SN-38 at 10μg/mL, whereas the treatment with beta-pentagalloyl glucose and various analogues decreased SN-38G by 70% and increased SN-38 by 20% at 10μg/mL. These results suggest naturally occurring compounds from WWB may have the potential to increase potency by increasing the conversion of IR to SN-38 and decrease dose-limiting toxicity of IR chemotherapy by reducing glucuronidation of SN-38.

Identification of the bioactive components of Banxia Xiexin Decoction that protect against CPT-11-induced intestinal toxicity via UPLC-based spectrum-effect relationship analyses

Ethnopharmacological relevanceIrinotecan (CPT-11) is a valuable chemotherapeutic compound, but its use is associated with severe diarrhea in some patients. The CPT-11 prodrug is converted into the active 7-ethyl-10-hydroxycamptothecin (SN-38) metabolite, which can then be retained for extended periods in the intestine, leading to the onset of diarrhea and related symptoms. Banxia Xiexin Decoction (BXD) is commonly employed for the treatment of gastroenteritis in traditional Chinese medicine (TCM), and in clinical settings, it is used to prevent diarrhea in patients undergoing CPT-11 treatment. To date, however, there have been no studies specifically examining which components of BXD can alleviate the gastrointestinal symptoms associated with CPT-11 administration. AimThis study aimed to identify the main herbal components of BXD associated with protection against CPT-11-induced intestinal toxicity in a murine model system. Materials and methodsSN-38 levels were measured by UPLC-ESI-MS/MS in samples collected from mice subjected to CPT-11-induced diarrhea that had been administered BXD or different components thereof. Pearson correlation and Grey relational analyses were then used to explore spectrum-effect relationships between reductions in intestinal SN-38 levels and specific chemical fingerprints in samples from mice administered particular combinations of BXD component herbs. ResultsWe found that different herbal combinations were associated with significant differences in intestinal SN-38 reductions in treated mice. Our spectrum-effect analysis revealed that BXD components including chrysin 6-C-arabinoside-8-C-glucoside, coptisine, hydroxyl oroxylin A 7-O-glucuronide (hydroxyl wogonoside), baicalin, an isomer of 5,6,7-trihydroxyl-flavanone-7-O-glucuronide, berberine, palmatine, and chrysin-7-O-glucuronide were all directly linked with reductions in intestinal SN-38 levels. We therefore speculate that these compounds are the primary bioactive components of BXD, suggesting that they offer protection against CPT-11-induced diarrhea. ConclusionBy utilizing UPLC to analyze SN-38 levels in mice treated with a variety of herbal combinations, we were able to effectively explore BXD spectrum-effect relationships and to thereby establish the components of this medicinal preparation that were bioactive and capable of preventing CPT-11-induced diarrhea in mice. This and similar spectrum-effect studies represent a robust means of exploring the mechanistic basis for the pharmacological activity of TCM preparations.

Abstract 1715: Anti-cancer activity of a SN-38 nanoparticle, DEP® irinotecan, in human colon cancer xenograft models

Abstract Irinotecan (Camptosar®) is a water-soluble prodrug of the potent topoisomerase I inhibitor SN-38 used clinically to treat multiple cancers. Irinotecan must be metabolized to its active metabolite SN-38 by carboxylesterases in hepatic and tumor cells. This metabolism in humans is inefficient with significant interpatient variability and leads to serious toxicities, notably diarrhea and myelosuppression. The narrow therapeutic window of irinotecan makes it an ideal candidate for improvement using dendrimer nanomedicine delivery. Starpharma's novel dendrimer nanoparticle DEP® platform has broad applicability in drug delivery, enhancing the therapeutic utility of drugs through improved solubility, efficacy and pharmacokinetics, reductions in certain toxicities (e.g. bone marrow toxicity) and providing novel intellectual property. The DEP platform has shown reproducible advantages across a wide range of drug classes including small molecule, peptides and proteins. Starpharma's DEP irinotecan is a PEGylated polylysine dendrimer with SN-38 conjugated to the surface via a hydrolytically labile linker. The incorporation of the active metabolite, SN-38 avoids the need for hepatic conversion and thereby minimizes variability in SN-38 levels. In preclinical studies, DEP irinotecan has shown to be well tolerated in animals. Efficacy assessments have been conducted, using a range of xenograft cancer models [SW620 (colon), HT-29 (colon), MDA-MB-231 (Breast) and CAPAN-1 (pancreatic)]. These studies demonstrated significant efficacy and survival benefits of DEP irinotecan compared to standard irinotecan. DEP irinotecan also showed significant benefit when dosed in combination with current standard of care therapies such as the anti-EGFR antibody, cetuximab, and the PARP inhibitor, olaparib. DEP irinotecan in combination with olaparib, significantly improved anti-tumor efficacy leading to tumor regression and extended median survival by 28 days compared to DEP irinotecan alone (P&amp;lt;0.0001, Mantel Cox Log rank test) whereas olaparib alone had minimal activity. This result highlights the synergistic combination benefits that can be achieved using DEP technology. DEP irinotecan is Starpharma's third internal DEP candidate to enter the clinic. The fourth DEP candidate, AZD0466 is a promising Bcl-2/Bcl-xL inhibitor and is partnered with AstraZeneca. AZD0466, is currently transitioning into clinical trials in the US. In summary, DEP irinotecan is well tolerated and allows for the delivery of SN-38 by successfully utilizing dendrimer drug delivery technology. The dendrimer nanoparticle shows significant efficacy and survival benefits compared to current standard of care therapies when used either as a monotherapy or in combination. DEP irinotecan is currently in a phase 1 / 2 adaptive trial at leading UK hospitals; The Christie, The Royal Marsden and The Newcastle Upon Tyne. Citation Format: Brian D. Kelly, Victoria McLeod, Rachael Walker, Jeannette Schreuders, Susan Jackson, Michael Giannis, Christine Dietinger, Shirley Xia, Anne Cargill, Aynaz Seta, Richard Hufton, Graham Heery, Carleen Cullinane, David J. Owen. Anti-cancer activity of a SN-38 nanoparticle, DEP® irinotecan, in human colon cancer xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1715.

Abstract CT156: Characterization of PEN-866, a Heat Shock Protein 90 (HSP90) binding conjugate of SN-38, in patient plasma and tumors from the first in human study

Abstract Background: Heat Shock Protein 90 (HSP90) is a molecular chaperone that aids in folding and trafficking of proteins and is upregulated in cells under stress. HSP90 is overexpressed in cancer cells and exists in the activated configuration within the tumor microenvironment. PEN-866 is a novel small molecule drug conjugate containing a HSP90 targeting moiety that accumulates in tumors and releases its payload SN-38 through a cleavable linker within the tumor cells. The first in human phase 1/2a PEN-866 study was designed to assess safety, pharmacokinetics (PK), and exploratory biomarkers of PEN-866 related to its mechanism of action. Here we describe the evaluation of PEN-866 and the payload SN-38 in plasma and tumors. Methods: Patients with advanced solid malignancies were enrolled in dose escalation cohorts and received PEN-866 IV on days 1, 8, and 15 in 28-days cycles, over a dose range of 16-228 mg/m2. Plasma samples were collected up to 8 days after the first dose and up to 24 hours after the third dose. Non-compartmental analysis was performed to obtain PK parameters. Optional tumor biopsies were collected from two patients and analyzed for PEN-866 and SN-38 concentrations. Results: PEN-866 plasma concentration profile was biphasic with a rapid concentration decline after end of infusion resulting in an average half-life (t½) of approximately 10 hours across all dose cohorts. PEN-866 maximum plasma concentrations (Cmax) ranged from 0.69 to 54 µg/mL and area under the concentration curve (AUC) from 0.80 to 88 h·µg/mL. The Cmax and AUC increased higher than dose proportionally at the lower dose levels which resulted in higher clearance (CL) and volume of distribution (Vd) variability. Free SN-38 AUC's were generally low demonstrating linker stability in circulation and low systemic exposure. PEN-866 and SN-38 levels in patient tumors confirmed accumulation and retention of the conjugate up to 6 days after PEN-866 dose and the targeted release of SN-38. Conclusion: PEN-866 has an extended plasma half-life in patients with advanced solid malignancies and showed good stability in circulation with limited plasma exposure of the payload SN-38. Analysis of tumor biopsies up to 6 days after dosing demonstrated significant accumulation and extended exposures of PEN-866 and SN-38 within the tumor tissue that exceed plasma exposures. Citation Format: Anish Thomas, Kristina Kriksciukaite, Gerald Falchook, Johanna Bendel, Susanna Ulahannan, Christophe Redon, Laura Mei, Kerry Whalen, Jeffrey Bloss, Mark T. Bilodeau. Characterization of PEN-866, a Heat Shock Protein 90 (HSP90) binding conjugate of SN-38, in patient plasma and tumors from the first in human study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT156.

Abstract 553: Antitumor efficacy of a liposomal formulation of irinotecan in preclinical gastric cancer models: Augmenting its response by antiangiogenic agents

Abstract BACKGROUND: Gastric adenocarcinoma (GAC) remains the 3rd most common cause of cancer-related deaths worldwide. Systemic chemotherapy is commonly a fundamental treatment for metastatic GAC that usually leads to a modest patient benefit, resulting in a 5-year survival rate of 31%. A liposomal formulation of irinotecan (nal-IRI) has shown improved pharmacokinetic and drug biodistribution compared with free irinotecan (IRI) in preclinical studies. Angiogenesis plays a crucial role in the progression and metastasis of GAC. We evaluated the therapeutic efficacy of nal-IRI in combination with nintedanib, a tyrosine kinase inhibitor targeting FGFR, PDGFR and VEGFR, and DC101, a monoclonal antibody targeting VEGFR2 in preclinical models of GAC. METHODS: In vitro cell proliferation was evaluated in three GAC cell lines (MKN-45, KATO-III and SNU-5) by WST-1 assay. Protein expression was measured by Western blot analysis in MKN-45 cell lysates. Animal survival studies were performed using two cell lines MKN-45 and KATO-III cells in peritoneal dissemination models in NOD/SCID mice (n=5-7). Tumor growth and pharmacokinetic studies were performed in GAC cell-derived xenografts. Mechanistic evaluation included IHC and Immunoblot analysis in tumor tissues. RESULTS: Animal survival was increased by nal-IRI (&amp;gt;156%) and IRI (&amp;gt;94%) therapy compared with PBS-treated controls. Importantly, nal-IRI led to a &amp;gt;30% extension of animal survival compared with IRI. The addition of antiangiogenic agents nintedanib or DC101 led to a further improvement (range 3-40%) in animal survival caused by nal-IRI and IRI. In GAC cell-derived subcutaneous xenografts, compared to controls, nal-IRI demonstrated greater tumor growth inhibition (92%) than IRI (71%). Here, the combinations of nal-IRI or IRI with antiangiogenic agents exhibited an additive response. Ki67 staining of tumor sections revealed that nal-IRI was most effective in reducing intratumoral proliferation (by 58%), followed by IRI (39%), nintedanib (33%), and DC101 (25%). Combinations of nal-IRI or IRI with antiangiogenic agents demonstrated an additive effect in reducing tumor cell proliferation. As expected, tumor vasculature (assessed by endomucin staining) was reduced by nintedanib (65%) and DC101 (58%), while nal-IRI and IRI showed no effect. Pharmacokinetic studies revealed that nal-IRI increased the retention, circulation time and tumor levels of IRI and its active metabolite SN-38. The addition of nintedanib or DC101 had no effect on plasma or tumor levels of IRI or SN38. In in vitro cell viability assays in mutationally different GAC cells, SN-38 had a dose-dependent growth inhibitory effect that was more pronounced than single-agent IRI. Importantly, combinations of SN-38 or IRI with antiangiogenic agents demonstrated additive effects on cell proliferation inhibition. CONCLUSION: nal-IRI showed greater antitumor efficacy than IRI, and its antitumor effects can be enhanced by antiangiogenic agents suggesting that this combination has potential for improving clinical GAC therapy. Citation Format: Niranjan Awasthi, Margaret A. Schwarz, Changhua Zhang, Stephan Klinz, Florence Meyer-Losic, Benjamin Beaufils, Arunthathi Thiagalingam, Roderich E. Schwarz. Antitumor efficacy of a liposomal formulation of irinotecan in preclinical gastric cancer models: Augmenting its response by antiangiogenic agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 553.

Phase II study of fluorouracil (FU), leucovorin (LV), and nanoliposomal irinotecan (nal-IRI) in previously treated advanced biliary tract cancer (NAPOLI-2).

TPS593 Background: Biliary tract cancers (BTCs) are rare and aggressive malignancies. The current standard of care for advanced BTC is gemcitabine (GEM) plus cisplatin . Although there is no established second-line treatment, regimens such as FOLFOX, XELOX, FOLFIRI, XELIRI, GEM, and capecitabine have activity. Nal-IRI contains IRI free base encapsulated in liposome nanoparticles which shelter IRI from conversion to its active metabolite (SN-38) and increase intratumoral levels of SN-38 compared with IRI. FU/LV/nal-IRI has shown overall survival benefit and acceptable toxicity in patients (pts) with metastatic pancreatic adenocarcinoma following GEM-based therapy in the NAPOLI-1 trial. Methods: This is a single arm, open label, multicenter phase II study of pts with advanced BTC previously treated with gemcitabine plus platinum chemotherapy. Pts will receive nal-IRI 70 mg/m2 IV over 90 minutes, LV 400 mg/m2 IV over 30 minutes, and FU 2400 mg/m2 over 46 hours, every 14 days. The primary objective is to determine progression-free survival (PFS) rate at 4 months (4mo) using RECIST v. 1.1 criteria and central radiology review. Response assessments will occur using imaging every 8 weeks. All pts who receive at least 1 dose of the study treatment will be eligible for the primary analysis. We will substitute pts who screen fail or do not begin treatment. Median PFS reported for pts receiving second-line 5-FU doublet chemotherapy is 3 months with a PFS4mo of 30%. FU/LV/nal-IRI would be of interest if it could increase the PFS4mo to 50% or higher. We will use a 2-stage Simon Minimax design. Using a one-sided α of 0.05 and 80% power, 39 pts will be required to detect a difference in PFS4mo between 30% and 50%. Assuming a dropout rate of 10%, 44 pts will be enrolled across the 5 study sites. Enrollment began in Q2 2019. Clinical trial information: NCT04005339.

PLX038: a PEGylated prodrug of SN-38 independent of UGT1A1 activity.

The purpose of this study was to determine the importance of UGT1A1 activity on the metabolism and pharmacokinetics of a releasable PEG ~ SN-38 conjugate, PLX038A. Irinotecan (CPT-11) is converted to the topoisomerase 1 inhibitor SN-38 by first-pass hepatic metabolism and is converted to its glucuronide SN-38G by UGT1A1. With diminished UGT1A1 activity, the high liver exposure to SN-38 can cause increased toxicity of CPT-11. In contrast, releasable PEG ~ SN-38 conjugates-such as PLX038-release SN-38 in the vascular compartment, and only low levels of SN-38 are expected to enter the liver by transport through the OATP1B1 transporter. We measured CPT-11 and PLX038A metabolites in plasma and bile, and determined pharmacokinetics of PLX038A in UGT1A-deficient and replete rats. Compared to CPT-11, treatment of rats with PLX038A results in very low levels of biliary SN-38 and SN-38G, a low flux through UGT1A, and a low SN-38G/SN-38 ratio in plasma. Further, the pharmacokinetics of plasma PLX038A and SN-38 in rats deficient in UGT1A is unchanged compared to normal rats. The disposition of PEGylated SN-38 is independent of UGT1A activity in rats, and PLX038 may find utility in full-dose treatment of patients who are UGT1A1*28 homozygotes or have metastatic disease with coincidental or incidental liver dysfunction.

Irinotecan toxicity during treatment of metastatic colorectal cancer: focus on pharmacogenomics and personalized medicine.

Colorectal cancer, one of the most frequent types of cancer worldwide, has a high mortality rate. Irinotecan (CPT-11) has been approved for the treatment of advanced or metastatic disease either as a single agent or, more commonly, as part of combined chemotherapeutic regimens. Treatment with irinotecan is often accompanied by severe toxicity (e.g. neutropenia and diarrhea) that can result in treatment interruption or cessation, thus jeopardizing the patient's prognosis and quality of life. Irinotecan is bioactivated into its metabolite SN-38, which is subsequently detoxified by uridine diphosphate-glucuronosyl transferases (mainly UGT1A1). Further, ABC transporters (i.e. ABCB1, ABCC1-ABCC6, and ABCG2) are responsible for drug efflux into bile and urine whereas OATP transporters (SLCO1B1) enable its influx from blood into hepatocytes. Genetic polymorphisms in these enzymes/pumps may result in increased systemic SN-38 level, directly correlating with toxicity. Contemporary research is focused on the clinical implementation of genetic screenings for validated gene variations prior to treatment onset, allowing tailored individual doses or treatment regimens.

Pharmacological inhibition of bacterial β-glucuronidase prevents irinotecan-induced diarrhea without impairing its antitumor efficacy in vivo.

Irinotecan (CPT-11), a first-line chemotherapy for advanced colorectal cancer, causes serious diarrhea in patients receiving treatment. The underlying mechanism has been shown that the active metabolite of CPT-11, SN-38, is metabolized to the inactive metabolite SN-38 glucuronide (SN-38 G) during hepatic glucuronidation, and subsequently is exported into the intestine, where SN-38 G is hydrolyzed by bacterial β-glucuronidase (βG) to be SN-38, thus leading to intestinal toxicity. Thus, inhibition of the intestinal bacterial βG activity is expected to prevent CPT-11-induced diarrhea. However, the effects of such inhibition on serum pharmacokinetics of SN-38, the key determinant of CPT-11 treatment, are uncertain. Here, we determined the effects of a potent E. coli βG (eβG)-specific inhibitor pyrazolo[4,3-c]quinoline derivative (TCH-3562) for the potential use in preventing CPT-11-induced diarrhea. TCH-3562 exhibited efficacious inhibitory potency of endogenous βG activity in two anaerobes, Eubacteriumsp. and Peptostreptococcus anaerobius. Oral administration of TCH-3562 also effectively reduced the bacterial βG activity in mice intestine. Moreover, pharmacokinetic analysis of TCH-3562 revealed a relatively low amount of TCH-3562 was detected in the plasma whereas the majority of TCH-3562 was found in the feces. Importantly, co-treatment of CPT-11 and TCH-3562 did not decrease active SN-38 level in mice plasma. Finally, we established that TCH-3562 as an adjuvant treatment showed protective effects on CPT-11-induced diarrhea and had no negative effects on the therapeutic efficacy of CPT-11 in tumor-bearing mice. Therefore, inhibition of the intestinal bacterial βG activity by the specific inhibitor, TCH-3562, is promising to prevent CPT-11-induced diarrhea while maintaining its anti-tumor efficacy that may have clinical potentials for the treatment with CPT-11.