DNA minor groove; covalent binder

PM01183 is a newly developed synthetic alkaloid tetrahydroisoquinoline that is used to treat solid tumors. PM01183: Double-strand breaks in living cells are caused by DNA adducts, which lead to the accumulation of S-phase and subsequent cell disinfection. With an average GI50 value of 2.7 nM, PM01183's strong cytotoxic activity was found in a panel of 23 cell lines [2]. In vitro, lurbinectedin significantly inhibits human ovarian clear cell carcinoma (CCC) cells that are both chemically sensitive and robust[1].

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Lurbinectedin exhibited significant antitumor activity toward chemosensitive and chemoresistant CCC cells in vitro. Among the tested combinations, Lurbinectedin (PM01183) plus SN-38 resulted in a significant synergistic effect. This combination also had strong synergistic effects on both the cisplatin-resistant and paclitaxel-resistant CCC cell lines. Everolimus significantly enhanced the antitumor activity of lurbinectedin-based chemotherapies. [1]

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Electrophoretic mobility shift assays demonstrated that Lurbinectedin (PM01183) bound to DNA. Fluorescence-based thermal denaturation experiments showed that the most favourable DNA triplets providing a central guanine for covalent adduct formation are AGC, CGG, AGG and TGG. These binding preferences could be rationalized using molecular modelling. PM01183-DNA adducts in living cells give rise to double-strand breaks, triggering S-phase accumulation and apoptosis. The potent cytotoxic activity of PM01183 was ascertained in a 23-cell line panel with a mean GI(50) value of 2.7 nM[2].

Lurbinectedin effectively suppressed tumor growth in CCC cell xenografts. There is a notable synergistic effect between SN-38 and luerbinectedin [1]. PM01183 markedly suppressed tumor growth in a four-cell xenograft model of human cancer, while lurbinectedin or NSC 119875 combination therapy effectively cured NSC 119875-sensitive and NSC 119875-combined single preclinical ovarian carcinoma tumors in animals. The combined treatment showed the greatest positive results, particularly for NSC 119875. inside tumors. Reduced proliferation, increased aberrant mitotic rates in malignancies, and triggered apoptosis are linked to luerbinectedin growth inhibition [3].

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In vivo growth-inhibitory effects of Lurbinectedin (PM01183) on ovarian CCC [1]
To examine the in vivo growth-inhibitory effects of Lurbinectedin (PM01183) , researchers employed an s.c. xenograft model in which athymic mice were s.c. inoculated with RMG1 cells. Overall, the drug treatment was well tolerated throughout the study and did not cause any apparent toxicities. The changes in the body weights of the mice are shown in Fig 2A. As shown in Fig 2B, the mean RMG1-derived tumor burden in the mice treated with lurbinectedin was 171.9 mm3, whereas it was 537.3 mm3 in the PBS-treated mice. Overall, treatment with lurbinectedin decreased the RMG1-derived tumor burden by 68.0% compared with PBS treatment.

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Researchers performed xenograft studies to test whether the cytotoxicity of Lurbinectedin (PM01183) translates into in vivo anti-tumour activity. NCI-H460 (lung), A2780 (ovary), HT29 (colon) and HGC-27 (gastric) cells were xenografted into the right flank of athymic nu/nu mice. Once the tumours reached c. 150 mm3, the mice were randomized into groups of 10–15 mice each and either vehicle or PM01183 (0.18 mg·kg−1·day−1) was intravenously administered in three consecutive weekly doses. At the drug doses used in the experiment, no significant toxicity or body weight loss was observed in the treated animals (data not shown). As shown in Figure 4, PM01183 presented anti-tumour activity with a statistically significant inhibition of tumour growth in the four tested models [2].

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Researchers showed that single Lurbinectedin (PM01183) or cisplatin-combined therapies were effective in treating cisplatin-sensitive and cisplatin-resistant preclinical ovarian tumor models. Furthermore, the strongest in vivo synergistic effect was observed for combined treatments, especially in cisplatin-resistant tumors. Lurbinectedin tumor growth inhibition was associated with reduced proliferation, increased rate of aberrant mitosis, and subsequent induced apoptosis. Conclusions: Taken together, preclinical orthotopic ovarian tumor grafts are useful tools for drug development, providing hard evidence that lurbinectedin might be a useful therapy in the treatment of EOC by overcoming cisplatin resistance[3].

DNA electrophoretic mobility shift assay [2]
The binding assay was performed with a 250 bp PCR product from the human adiponectin gene. After incubation with appropriate concentrations of the compound at 25°C during 1 h, the DNA was subjected to electrophoresis in a 2% (w/v) agarose/TAE gel, stained with 1 µg·mL−1 ethidium bromide and photographed.

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DNA melting assay [2]
Synthetic oligodeoxynucleotides with one strand 5′-end-labelled with the fluorophore 6-carboxyfluorescein and the complementary strand 3′-end-labelled with the quencher tetramethylrhodamine were synthesized at a CRO cpmpany (Table S1). For the experiments, we followed the methodology previously described (Negri et al., 2007; Casado et al., 2008) using a 7500 Fast Real-Time PCR System. Analyses of the raw data obtained for each oligodeoxynucleotide using an in-house developed Visual Basic Application running on Microsoft Excel yielded estimates of both the increase in melting temperature (ΔTm) brought about by drug binding, relative to the free DNA molecule, and the ligand concentration that produces half the maximal change in melting temperature (C50). The inverse of this latter value (1/C50) was taken as a measure of the relative DNA binding affinity, and the parameter ΔTm(max) was used to reflect the relative stability of the DNA–ligand complexes (Negri et al., 2007).

Cell proliferation assay [1]
The MTS assay was used to analyze the effects of each drug. Cells were plated in 96-well plates and exposed to the drugs at different concentrations. After 48 hours’ incubation, the number of surviving cells was assessed by determining the A490nm of the dissolved formazan product after the addition of MTS for 1 hour, as described by the manufacturer. Cell viability was calculated as follows: Aexp group / Acontrol × 100. The experiments were repeated at least three times, and representative results are shown.

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Cell cycle analysis [1]
CCC cells (2×105) were incubated with Lurbinectedin (PM01183) at the indicated concentrations for 48 hours. The cells were then fixed with 75% ethanol overnight at -20°C and stained with propidium iodide (PI; 50 μg/mL) in the presence of RNase A (100 μg/mL; Roth) for 60 minutes at 4°C. In each experiment, the cell cycle distribution was determined by analyzing 10,000 cells using a FACScan flow cytometer and Cell Quest software (Becton Dickinson, NJ, USA), as reported previously. The experiments were repeated at least three times, and representative results are shown.

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Detection of apoptosis [1]
CCC cells (2~5×105) were treated with Lurbinectedin (PM01183) , SN38 or in combination of the two drugs at the indicated concentrations for 48 hours. Then, the cells were harvested and stained with PI and annexin V using the annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit, according to the manufacturer’s instructions. Fluorescence data were collected using flow cytometry, as reported previously. The sum total of early apoptotic cells (annexin V(+), PI(−) cells) and late apoptotic cells (annexin V(+), PI(+) cells) was defined as the total number of apoptotic cells. The experiments were repeated at least three times, and representative results are shown.

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Isobologram method and the combination index [1]
The isobologram method relies on determining the combined concentrations of D1 (lurbinectedin) and D2 (the drug used in combination with Lurbinectedin (PM01183) ) that result in a fractional kill value of 50%. For each experimental concentration of lurbinectedin, the concentration of D2 that would cause the desired effect when used in combination with lurbinectedin was found by non-linear fitting of the concentration-effect relationship of D2 to the given lurbinectedin concentration. Conversely, for each experimental concentration of D2, the lurbinectedin concentration that would cause the desired effect when used in combination with D2 was found by non-linear fitting of the concentration-effect relationship of lurbinectedin to the particular D2 concentration. In this manner, multiple pairs of drug concentrations that achieved the desired isoeffect were found. For each pair of drug concentrations (DLurbinectedin, DD2) that produced a fractional kill value of 50%, the combination index (CI) was calculated as follows: CI = DLurbinectedin / IC50Lurbinectedin + DD2 / IC50D2. CI values of <1 indicate synergism, CI values of 1 indicate an additive effect, and CI values of >1 indicate antagonism. The significance of the differences between the mean CI values for each combination and 1 was evaluated using a 2-tailed t test. The experiments were repeated at least three times, and representative results are shown.

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Western blot analysis [1]
CCC cells were treated with Lurbinectedin (PM01183) or other agents for appropriate periods of time, washed twice with ice cold phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer. The protein concentrations of the cell lysates were determined using the Bio-Rad protein assay reagent. Equal amounts of protein were applied to 5–20% polyacrylamide gels, and then the electrophoresed proteins were transblotted onto nitrocellulose membranes. After the membranes had been blocked, they were incubated with anti-PARP, anti-cleaved caspase 3, anti-P-gp, or anti-β-actin antibodies. The immunoblots were visualized with horseradish peroxidase-coupled goat anti-rabbit or anti-mouse immunoglobulins, using the enhanced chemiluminescence Western blotting system.

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Cell culture and cytotoxicity [2]
All the tumour cell lines used in this study were obtained from the American Type Culture Collection. For the cytotoxicity experiments, cells were seeded in 96-well trays. Serial dilutions of Lurbinectedin (PM01183) dissolved in dimethyl sulphoxide (DMSO) were prepared and added to the cells in fresh medium, in triplicate. Exposure to the drug was maintained during 72 h and cellular viability was estimated from conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its coloured reaction product, MTT formazan, which was dissolved in DMSO so as to measure absorbance at 540 nm with a POLARStar Omega Reader. Determination of the concentration giving 50% growth inhibition (GI50) values was performed by iterative non-linear curve fitting using the Prism 5.0 statistical software. The data presented are the average of three independent experiments performed in triplicate.

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Fluorescent microscopy [2]
Cells were treated with the appropriate concentration of Lurbinectedin (PM01183) for 6 h, washed and then cultured for 18 additional hours. At the end of this period, they were fixed (4% paraformaldehyde), permeabilized (0.5% Triton X-100) and incubated with the primary anti-γ-H2AX monoclonal antibody for 1 h at 37°C. Thereafter the cells were washed and incubated with the AlexaFluor 594 secondary goat anti-mouse IgG for 30 min at 37°C. Finally, the slides were incubated with Hoechst 33 342 and mounted with Mowiol mounting medium. Pictures were taken with a Leica DM IRM fluorescence microscope equipped with a 100× oil immersion objective and a DFC 340 FX digital camera.

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Cell cycle analysis [2]
For the cell cycle experiments and sub-G1 peak determination, cells were treated with the appropriate amount of Lurbinectedin (PM01183) for 12 h and 24 h, and then stained with 0.4 µg·mL−1 propidium iodide. Samples were analysed with a FACScalibur flow cytometer and the FlowJo7 cytometry analysis software.

Subcutaneous xenograft model [1]
Preliminary experiments were conducted to examine the effects of Lurbinectedin (PM01183) on ovarian CCC. Five- to 7-week-old nude mice (n = 12) had 1×107 RMG1 cells in 150 μL of PBS s.c. injected into their left flanks. When the tumors reached about 50 mm3 in size, the mice were assigned to one of two treatment groups. The first group (n = 6) was i.v. administered PBS, and the second group (n = 6) was i.v. administered lurbinectedin (0.180 mg/kg) each week for 6 weeks. The dose of lurbinectedin (0.180 mg/kg) used was based on that employed in a previous preclinical study of ovarian cancer, in which it showed significant in vivo antitumor activity. A second set of experiments was conducted to examine the antitumor effects of combination treatment involving lurbinectedin and irinotecan. We employed irinotecan in the in vivo experiments because the clinical use of SN-38 is limited by its poor aqueous solubility, and the goal of this study was to identify practical treatments that could be used in the clinical setting. Five- to 7-week-old nude mice (n = 18) had 1×107 RMG1 cells in 150 μL of PBS s.c. injected into their flanks. When the tumors reached about 50 mm3 in size, the mice were assigned to 1 of 3 treatment groups, which received PBS, CPT-11 (50 mg/kg weekly), or lurbinectedin (0.180 mg/kg weekly) plus CPT-11 (50 mg/kg weekly). Caliper measurements of the longest perpendicular diameter of each tumor were obtained twice a week and used to estimate tumor volume according to the following formula: V = L × W × D × π / 6, where V is the volume, L is the length, W is the width, and D is the depth.

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Anti-tumour activity in xenograft murine models [2]
Four to 6-week-old athymic nu/nu mice were s.c. xenografted with NCI-H460 (lung), A2780 (ovary), HT29 (colon) and HGC-27 (gastric) cells into their right flank with c. 3 × 106 cells in 0.2 mL of a mixture (50:50; v:v) of Matrigel basement membrane matrix and serum-free medium. When tumours reached c. 150 mm3, mice were randomly assigned to treatment or control groups. Lurbinectedin (PM01183) was intravenously administered in three consecutive weekly doses (0.18 mg·kg−1·day−1) whereas the control animals received an equal volume of vehicle with the same schedule. Caliper measurements of the tumour diameters were made twice weekly and tumour volumes were calculated according to the following formula: (a·b)2/2, where a and b were the longest and shortest diameters respectively. Animals were humanely killed when their tumours reached 3000 mm3 or if significant toxicity (e.g. severe body weight reduction) was observed. Differences in tumour volumes between treated and control groups were evaluated using the Mann–Whitney U-test. Statistical analyses were performed by LabCat® v8.0 SP1.

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Drug treatment of engrafted cisplatin-sensitive and cisplatin-resistant tumor models [3]
Mice were transplanted with fragments of OVAX1 and OVAX1R tumors, and when tumors reached a homogeneous palpable size were randomly allocated into the treatment groups (n = 8–12/group): i) Placebo; ii) Lurbinectedin (PM01183) (0.18 mg/kg); iii) Cisplatin (3.5 mg/kg); and iv) Lurbinectedin plus cisplatin (0.18 + 3.5 mg/kg). Drugs were i.v. administered once per week for 3 consecutive weeks (days 0, 7, and 14). Seven days after the final dose (day 21), animals were sacrificed, their ovaries dissected out, and weighed. Representative fragments were either frozen in nitrogen or fixed and then processed for paraffin embedding.

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In vivo evaluation of synergism among Lurbinectedin (PM01183) and cisplatin treatments [3]
Female mice were subcutaneously implanted with 107 A2780 cells suspended in a 1:1 solution of RPMI-1640:Matrigel. Mice bearing tumors (ca. 150 mm3) were randomly allocated to 13 treatment groups (see Fig. 3 legend). All treatments were intravenously administered once per week for 2 consecutive weeks (days 0 and 7). Tumor growth was recorded 2 to 3 times per week starting from the first day of treatment (day 0) and tumor volume (in mm3), estimated according to the formula V = (a·b2)/2, (a: length or biggest diameter; b: width or smallest diameter). Antitumor drug activity was measured with respect to the T/C index, and the fraction affected (Fa) by treatment was calculated (Fa = 1−T/C). A CI was determined by the CI-isobol method

Absorption, Distribution and Excretion
Following intravenous administration, the Cmax and AUC0-inf were 107 µg/L and 551 µg*h/L, respectively. No accumulation between dosing intervals (every 3 weeks) has been observed. No significant differences in absorption were found between special populations (e.g. based on age, sex, ethnicity, etc.), but lurbinectedin has not been studied in the setting of severe renal impairment or moderate/severe hepatic impairment.
Approximately 89% of a given dose is recovered in the feces (<0.2% unchanged) and 6% in the urine (1% unchanged).
The steady-state volume of distribution of lurbinectedin is 504 L.
The total plasma clearance of lurbinectedin is approximately 11 L/h.

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Metabolism / Metabolites
Lurbinectedin is metabolized primarily by CYP3A4 _in vitro_, though specific data regarding its biotransformation are lacking. An N-desmethylated metabolite has been identified in canine subjects.

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Biological Half-Life
The terminal half-life of lurbinectedin is 51 hours.

Hepatotoxicity
Elevations in serum aminotransferase levels arise in approximately two-thirds of patients treated with lurbinectedin and elevations above 5 times the upper limit of normal occur in 4% to 5% of patients. Pretreatment with dexamethasone appears to decrease the degree and frequency of enzyme elevations. The elevations arise within 2 to 5 days of the intravenous infusion, rise to maximal levels between 5 and 9 days, and generally fall to baseline values within 2 to 3 weeks. Minor elevations in serum alkaline phosphatase and bilirubin are also common. However, clinically apparent liver injury with jaundice from lurbinectedin is uncommon. On the other hand, patients with underlying liver disease appear to be at increased risk for septicemia and multiorgan failure as a result of chemotherapy, and monitoring of liver tests before and during lurbinectedin therapy is recommended. The severe liver injury typically mimics acute decompensation of an underlying cirrhosis with modest elevations in serum enzymes and worsening jaundice and hepatic synthetic dysfunction. Immunoallergic and autoimmune features are uncommon. Fatalities are generally due to sepsis and multiorgan failure rather than typical acute liver failure.
Likelihood score: D (possible cause of clinically apparent liver injury, generally in the setting of preexisting liver disease and use of high doses).

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Protein Binding
Lurbinectedin is highly protein-bound in plasma (~99%) to both serum albumin and α-1-acid glycoprotein.

[1]. Preclinical Investigations of PM01183 (Lurbinectedin) as a Single Agent or in Combination with Other Anticancer Agents for Clear Cell Carcinoma of the Ovary. PLoS One. 2016 Mar 17;11(3):e0151050.

[2]. PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity. Br J Pharmacol. 2010 Nov;161(5):1099-110.

[3]. Lurbinectedin (PM01183), a new DNA minor groove binder, inhibits growth of orthotopic primary graft of NSC 119875-resistant epithelial ovarian cancer. Clin Cancer Res. 2012 Oct 1;18(19):5399-411.

Lurbinectedin is a DNA alkylating agent that has been investigated in the treatment of a variety of cancers, including mesothelioma, chronic lymphocytic leukemia (CLL), breast cancer, and small-cell lung cancer (SCLC). It is a derivative of the marine-derived agent ecteinascidin ([trabectedin]), an anticancer agent found in extracts of the tunicate Ecteinascidia turbinata, with the primary difference being the substitution of the tetrahydroisoquinoline with a tetrahydro β‐carboline that results in increased antitumour activity of lurbinectedin as compared to its predecessor. On June 15, 2020, the FDA granted accelerated approval and orphan drug designation to lurbinectedin for the treatment of adult patients with metastatic SCLC who have experienced disease progression despite therapy with platinum-based agents. This accelerated approval is based on the rate and duration of therapeutic response observed in ongoing clinical trials and is contingent on the verification of these results in confirmatory trials.
Lurbinectedin is an Alkylating Drug. The mechanism of action of lurbinectedin is as an Alkylating Activity.
Lurbinectedin is an antineoplastic alkylating agent and synthetic derivative of trabectedin that is used to treat refractory, metastatic small cell lung cancer. Lurbinectedin therapy is associated with a high rate of transient serum enzyme elevations during treatment and with occasional instances of clinically apparent liver injury with jaundice.
Lurbinectedin is a synthetic tetrahydropyrrolo [4, 3, 2-de]quinolin-8(1H)-one alkaloid analogue with potential antineoplastic activity. Lurbinectedin covalently binds to residues lying in the minor groove of DNA, which may result in delayed progression through S phase, cell cycle arrest in the G2/M phase and cell death.

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Drug Indication
Lurbinectedin is indicated for the treatment of adult patients with metastatic small-cell lung cancer (SCLC) with disease progression on or after platinum-based chemotherapy.
Treatment of malignant mesothelioma
Treatment of small cell lung cancer

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Mechanism of Action
Lurbinectedin is a DNA alkylating agent. It covalently binds to guanine residues in the DNA minor groove, forming adducts that bend the DNA helix towards the major groove. This process triggers a cascade of events that affect the activity of transcription factors and impairs DNA repair pathways, ultimately leading to double-strand DNA breaks and eventual cell death. Additional mechanism(s) of action include inhibition of RNA-polymerase-II activity, inactivation of Ewing Sarcoma Oncoprotein (EWS-FL11) via nuclear redistribution, and the inhibition of human monocyte activity and macrophage infiltration into tumor tissue.

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Pharmacodynamics
Lurbinectedin exerts its chemotherapeutic activity by covalently binding to DNA, resulting in double-strand DNA breaks and subsequent cell death. Lurbinectedin has been associated with myelosuppression, and patients receiving therapy with this agent should be closely monitored for evidence of cytopenias. Prior to beginning therapy, ensure baseline neutrophil counts are >1,500 cells/mm³ and platelet counts are >100,000/mm³. The supplementary use of granulocyte colony-stimulating factor (G-CSF) should be considered if the neutrophil count falls below 500 cells/mm³. Lurbinectedin has also been associated with hepatotoxicity. Monitor liver function tests at baseline and regular intervals throughout therapy, and consider holding, reducing, or permanently discontinuing therapy based on the severity of observed hepatotoxicity.

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Objective: The objective of this study was to evaluate the antitumor effects of lurbinectedin as a single agent or in combination with existing anticancer agents for clear cell carcinoma (CCC) of the ovary, which is regarded as an aggressive, chemoresistant, histological subtype. Methods: Using human ovarian CCC cell lines, the antitumor effects of lurbinectedin, SN-38, doxorubicin, cisplatin, and paclitaxel as single agents were assessed using the MTS assay. Then, the antitumor effects of combination therapies involving lurbinectedin and 1 of the other 4 agents were evaluated using isobologram analysis to examine whether these combinations displayed synergistic effects. The antitumor activity of each treatment was also examined using cisplatin-resistant and paclitaxel-resistant CCC sublines. Finally, we determined the effects of mTORC1 inhibition on the antitumor activity of lurbinectedin-based chemotherapy. Results: Lurbinectedin exhibited significant antitumor activity toward chemosensitive and chemoresistant CCC cells in vitro. An examination of mouse CCC cell xenografts revealed that lurbinectedin significantly inhibits tumor growth. Among the tested combinations, lurbinectedin plus SN-38 resulted in a significant synergistic effect. This combination also had strong synergistic effects on both the cisplatin-resistant and paclitaxel-resistant CCC cell lines. Everolimus significantly enhanced the antitumor activity of lurbinectedin-based chemotherapies. Conclusions: Lurbinectedin, a new agent that targets active transcription, exhibits antitumor activity in CCC when used as a single agent and has synergistic antitumor effects when combined with irinotecan. Our results indicate that lurbinectedin is a promising agent for treating ovarian CCC, both as a first-line treatment and as a salvage treatment for recurrent lesions that develop after platinum-based or paclitaxel treatment.[1]

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Background and purpose: PM01183 is a new synthetic tetrahydroisoquinoline alkaloid that is currently in phase I clinical development for the treatment of solid tumours. In this study we have characterized the interactions of PM01183 with selected DNA molecules of defined sequence and its in vitro and in vivo cytotoxicity. Experimental approach: DNA binding characteristics of PM01183 were studied using electrophoretic mobility shift assays, fluorescence-based melting kinetic experiments and computational modelling methods. Its mechanism of action was investigated using flow cytometry, Western blot analysis and fluorescent microscopy. In vitro anti-tumour activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the in vivo activity utilized several human cancer models. Key results: Electrophoretic mobility shift assays demonstrated that PM01183 bound to DNA. Fluorescence-based thermal denaturation experiments showed that the most favourable DNA triplets providing a central guanine for covalent adduct formation are AGC, CGG, AGG and TGG. These binding preferences could be rationalized using molecular modelling. PM01183-DNA adducts in living cells give rise to double-strand breaks, triggering S-phase accumulation and apoptosis. The potent cytotoxic activity of PM01183 was ascertained in a 23-cell line panel with a mean GI(50) value of 2.7 nM. In four murine xenograft models of human cancer, PM01183 inhibited tumour growth significantly with no weight loss of treated animals. Conclusions and implications: PM01183 is shown to bind to selected DNA sequences and promoted apoptosis by inducing double-strand breaks at nanomolar concentrations. The potent anti-tumour activity of PM01183 in several murine models of human cancer supports its development as a novel anti-neoplastic agent.[2]

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Purpose: Epithelial ovarian cancer (EOC) is the fifth leading cause of death in women diagnosed with gynecologic malignancies. The low survival rate is because of its advanced-stage diagnosis and either intrinsic or acquired resistance to standard platinum-based chemotherapy. So, the development of effective innovative therapeutic strategies to overcome cisplatin resistance remains a high priority. Experimental design: To investigate new treatments in in vivo models reproducing EOCs tumor growth, we generated a preclinical model of ovarian cancer after orthotopic implantation of a primary serous tumor in nude mice. Further, matched model of acquired cisplatin-resistant tumor version was successfully derived in mice. Effectiveness of lurbinectedin (PM01183) treatment, a novel marine-derived DNA minor groove covalent binder, was assessed in both preclinical models as a single and a combined-cisplatin agent. Results: Orthotopically perpetuated tumor grafts mimic the histopathological characteristics of primary patients' tumors and they also recapitulate in mice characteristic features of tumor response to cisplatin treatments. We showed that single lurbinectedin or cisplatin-combined therapies were effective in treating cisplatin-sensitive and cisplatin-resistant preclinical ovarian tumor models. Furthermore, the strongest in vivo synergistic effect was observed for combined treatments, especially in cisplatin-resistant tumors. Lurbinectedin tumor growth inhibition was associated with reduced proliferation, increased rate of aberrant mitosis, and subsequent induced apoptosis. Conclusions: Taken together, preclinical orthotopic ovarian tumor grafts are useful tools for drug development, providing hard evidence that lurbinectedin might be a useful therapy in the treatment of EOC by overcoming cisplatin resistance.[3]

C41H44N4O10S

784.88

784.278

C, 62.74; H, 5.65; N, 7.14; O, 20.38; S, 4.08

497871-47-3

Lurbinectedin-d3

57327016

White to light yellow solid powder

4.393

4

14

4

56

1530

7

S1C[C@@]2(C3=C(C4C=C(C=CC=4N3)OC)CCN2)C(=O)OC[C@@]2([H])C3=C4C(=C(C)C(=C3[C@]1([H])[C@@]1([H])[C@@]3([H])C5C(=C(C(C)=CC=5C[C@@]([H])([C@@H](N12)O)N3C)OC)O)OC(C)=O)OCO4

YDDMIZRDDREKEP-HWTBNCOESA-N

InChI=1S/C41H44N4O10S/c1-17-11-20-12-25-39(48)45-26-14-52-40(49)41(38-22(9-10-42-41)23-13-21(50-5)7-8-24(23)43-38)15-56-37(31(45)30(44(25)4)27(20)32(47)33(17)51-6)29-28(26)36-35(53-16-54-36)18(2)34(29)55-19(3)46/h7-8,11,13,25-26,30-31,37,39,42-43,47-48H,9-10,12,14-16H2,1-6H3/t25-,26-,30+,31+,37+,39-,41+/m0/s1

[(1R,2R,3R,11S,12S,14R,26R)-5,12-dihydroxy-6,6'-dimethoxy-7,21,30-trimethyl-27-oxospiro[17,19,28-trioxa-24-thia-13,30-diazaheptacyclo[12.9.6.13,11.02,13.04,9.015,23.016,20]triaconta-4(9),5,7,15,20,22-hexaene-26,1'-2,3,4,9-tetrahydropyrido[3,4-b]indole]-22-yl] acetate

PM-01183; 497871-47-3; Tryptamicidin; Zepzelca; PM01,183; zepsyre; PM-01,183; lurbinectedina; PM01183; Lurbinectedin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~20 mg/mL (~25.48 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)