Phase I/II trial of plinabulin in combination with nivolumab and ipilimumab in patients with recurrent small cell lung cancer (SCLC): Big ten cancer research consortium (BTCRC-LUN17-127) study.

BACKGROUND: Plinabulin is a GEF-H1 releasing agent with an immune-enhancing function. We report results from a multicenter Phase I/II study (NCT03575793) assessing plinabulin in combination with nivolumab and ipilimumab for the treatment of recurrent SCLC. METHODS: In Phase I, patients were enrolled using a 3 + 3 design to determine dose-limiting toxicities (DLTs) and recommended Phase 2 dose (RP2D). Patients received nivolumab (1 mg/kg), ipilimumab (3 mg/kg), and plinabulin (in escalating doses) on day 1 of each 21-day cycle for 4 cycles followed by maintenance with plinabulin and nivolumab. In phase II, patients with recurrent PD(L)1 inhibitor resistant SCLC were enrolled. The primary objective was median progression-free survival (PFS). RESULTS: Between 9/2018 and 2/2023, 39 patients were enrolled, and 36 patients received study treatment and were evaluable for safety (16 in Phase I; 20 in Phase II). In the phase I dose-escalation, there were 2 DLTs; grade 3 altered mental status lasting <24 h and grade 3 infusion reaction. The Plinabulin RP2D was determined to be 30 mg/m2. Common TRAEs were vomiting (44 %), nausea (42 %), and infusion reaction (36 %); 6 % of patients had a ≥grade 3 TRAE. Five patients (14 %) had ≥grade 3 irAEs; there were no cases of immune-related pneumonitis. In the efficacy analysis in 27 patients, the median PFS was 1.6 months (95 % CI 1.2 to 2.7) and the trial did not meet the pre-specified target median PFS of 3.5 months. Four patients treated at 30 mg/m2 had PR (confirmed 1, unconfirmed 3); 5 patients had SD with a CBR of 33 %. Two of 8 patients treated in phase I at the lower 20 mg/m2 dose had confirmed PR, with 1 patient on the drug regimen for >90 cycles. The median OS and follow-up time were 5.5 months and 2.5 months respectively. CONCLUSIONS: Plinabulin in combination with nivolumab and ipilimumab was tolerable at the dose of 30 mg/m2. While the clinical responses in PD-1 resistant SCLC were limited, some patients had a long duration of response. The number of ≥grade 3 irAE with the combination were lower than expected.

Co-delivery of Plinabulin and Tirapazamine boosts anti-tumor efficacy by simultaneously destroying tumor blood vessels and killing tumor cells.

It is imperative to optimize chemotherapy for heightened anti-tumor therapeutic efficacy. Unrestrained tumor cell proliferation and sustained angiogenesis are pivotal for cancer progression. Plinabulin, a vascular disrupting agent, selectively destroys tumor blood vessels. Tirapazamine (TPZ), a hypoxia-activated prodrug, intensifies cytotoxicity in diminishing oxygen levels within tumor cells. Despite completing Phase III clinical trials, both agents exhibited modest treatment efficiency due to dose-limiting toxicity. In this study, we employed methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-b-PDLLA) to co-deliver Plinabulin and TPZ to the tumor site, concurrently disrupting blood vessels and eliminating tumor cells, addressing both symptoms and the root cause of tumor progression. Plinabulin was converted into a prodrug with esterase response (PSM), and TPZ was synthesized into a hexyl chain-containing derivative (TPZHex) for effective co-delivery. PSM and TPZHex were co-encapsulated with mPEG-b-PDLLA, forming nanodrugs (PT-NPs). At the tumor site, PT-NPs responded to esterase overexpression, releasing Plinabulin, disrupting blood vessels, and causing nutritional and oxygen deficiency. TPZHex was activated in response to increased hypoxia, killing tumor cells. In treating 4T1 tumors, PT-NPs demonstrated enhanced therapeutic efficacy, achieving a 92.9 % tumor suppression rate and a 20 % cure rate. This research presented an innovative strategy to enhance synergistic efficacy and reduce toxicity in combination chemotherapy.

Design, synthesis, and structure-activity relationship study of novel plinabulin derivatives as anti-tumor agents based on the co-crystal structure.

Plinabulin, a 2, 5-diketopiperazine-type tubulin inhibitor derived from marine natural products, is currently undergoing Phase III clinical trials for the treatment of non-small cell lung cancer (NSCLC) and chemotherapy-induced neutropenia (CIN). To obtain novel 2, 5-diketopiperazine derivatives with higher biological activity, we designed and synthesized two series of 37 plinabulin derivatives at the C-ring, based on the co-crystal structure of compound 1 and tubulin. Their structures were characterized using NMR and HRMS. All compounds were screened in vitro using the lung cancer cell line NCI-H460 using the MTT method, and the compounds with better activity were further screened in BxPC-3 and HT-29 cells. The compounds 16c (IC50 = 2.0, NCI-H460; IC50 = 1.2 nM, BxPC-3; IC50 = 1.97 nM, HT-29) and 26r (IC50 = 0.96, NCI-H460; IC50 = 0.66 nM, BxPC-3; IC50 = 0.61 nM, HT-29) had the best activity. The cytotoxic activity of compound 26r against various tumor cell lines occurred at less than 1 nM.

Development and Validation of a Novel UHPLC-MS/MS Method for the Quantification of Plinabulin in Plasma and Its Application in a Pharmacokinetic Study with Leukopenic Rats.

Plinabulin, a new antitumor drug developed from marine natural products that targets microtubules in cancer cells, is currently being tested in a phase III clinical study. Plinabulin has been clinically proven to be effective on leukopenia. However, to our knowledge, there are no reports investigating the pharmacokinetics of plinabulin in individuals with leukopenia and healthy individuals. In this study, we developed a rapid and sensitive UHPLC-MS/MS method for the detection of plinabulin for the first time. Using a novel cyclophosphamide-induced leukopenia model, we investigated the differences in the pharmacokinetic characteristics of plinabulin between rats with leukopenia and normal rats. Plinabulin and propranolol (IS) peaks were separated by gradient elution for a total run time of 5 min. The methodological validation showed a good accuracy (101.96-109.42%) and precision (RSD ≤ 5.37%) with the lower limit of quantification at 0.5 ng/mL. The recovery of plinabulin was between 91.99% and 109.75% (RSD ≤ 7.92%). The values of the area under the plasma concentration-time curve (AUC0-t) for leukopenia groups and control groups at doses of 0.5 mg/kg, 1 mg/kg, and 3 mg/kg were 148.89 ± 78.74 h·µg/L and 121.75 ± 31.56 h·µg/L; 318.15 ± 40.00 h·µg/L and 272.06 ± 42.85 h·µg/L; and 1432.43 ± 197.47 h·µg/L and 1337.12 ± 193.56 h·µg/L; respectively. The half-lives (t1/2s) of plinabulin were 0.49-0.72 h for leukopenia groups and 0.39-0.70 h for control groups at three doses, and the clearance rates (CLs) of plinabulin were 2.13-3.87 L/h/kg for leukopenia groups and 2.29-4.23 L/h/kg for control groups. Pharmacokinetic results showed that there was no significant pharmacokinetic difference between the normal group and the leukopenia group. Based on the power model, plinabulin exhibits a lack of dose proportionality over the dose range of 0.5-3 mg/kg after intravenous administration. This study provides guidance for the development of plinabulin as a potential candidate for the treatment of chemotherapy-induced leukopenia.

Design, synthesis and anti-tumor evaluation of plinabulin derivatives as potential agents targeting ß-tubulin.

Plinabulin is a promising microtubule destabilizing agent in phase 3 clinical stage for treating non-small cell lung cancer. However, the high toxicity and the poor water solubility of plinabulin limited its use and more plinabulin derivatives need to be explored. Here, two series of 29 plinabulin derivatives were designed, synthesized and evaluated for their anti-tumor effect against three types of cancer cell lines. Most of derivatives exerted obvious inhibition to the proliferation of the cell lines tested. Among them, compound 11c exerted stronger efficiency than plinabulin, and the reason might be the additional hydrogen bond between the nitrogen atom of the indole ring in compound 11c and Gln134 of ß-tubulin. Immunofluorescence assay showed that compound 11c at 10 nM significantly disrupted tubulin structure. Compound 11c also significantly induced G2/M cell cycle arrest and apoptosis in dose dependent manner. These results suggest that compound 11c might be a potential candidate for cancer treatment as antimicrotubule agent.

Study protocol of KeyPemls-004: A phase 2 study of pembrolizumab in combination with plinabulin and docetaxel in previously treated patients with metastatic non-small cell lung cancer and progressive disease (PD) after immunotherapy (PD-1/PD-L1 inhibitor) alone or in combination with platinum-doublet chemotherapy.

INTRODUCTION: Immune checkpoint inhibitor (ICI)-based treatment regimens have become the standard of care for first-line treatment of metastatic epidermal growth factor receptor (EGFR)/anaplastic lymphoma kinase (ALK) wild-type non-small cell lung cancer (NSCLC). Nevertheless, most patients inevitably develop disease progression, and the mechanisms of resistance to first-line immunotherapy are not clear. ICIs in combination with agents targeting other pathways may serve as second-line therapy options. Plinabulin is a selective immunomodulating microtubule-binding agent which inhibits the polymerization of tubulin monomers, with multiple mechanisms to inhibit tumor growth. Clinical studies have demonstrated preliminary the antitumor efficacy of this agent. Therefore, we hypothesize that a combination of plinabulin with programmed death 1 (PD-1) inhibitor and docetaxel may result in higher efficacy and fewer side effects leading to better tolerance. METHODS: In this investigator-initiated, single-arm, open-label, phase II trial, metastatic NSCLC patients who acquired resistance to first-line immunotherapy-based therapy will be enrolled. Participants will receive pembrolizumab 200 mg D1, plinabulin 30 mg/m2 D1 and D8, and docetaxel 75 mg/m2 D1 intravenously for a 21-day cycle. The study intervention will be given until disease progression, intolerable toxicity, informed consent withdrawal or investigator decision. The primary endpoint is investigator-based objective response rate per Response Evaluation Criteria in Solid Tumors, version 1.1. The secondary endpoints are progression-free survival, overall survival, duration of response, and safety. DISCUSSION: This trial will provide evidence of the benefit and safety of pembrolizumab in combination with plinabulin and docetaxel in metastatic NSCLC patients who have been exposed and developed resistance to first-line PD-1/PD-L1 inhibitor either as monotherapy or in combination with chemotherapy.

Conformation and energy investigation of microtubule longitudinal dynamic instability induced by natural products.

The natural products plinabulin, docetaxel, and vinblastine are microtubule targeting agents (MTAs). They have been used alone or in combination in cancer treatment. However, the exact nature of their effects on microtubule (MT) polymerization dynamics is poorly understood. To elucidate the longitudinal conformational and energetic changes during MT dynamics, a total of 140 ns molecular dynamic simulations combined with binding free energy calculations were performed on seven tubulin models. The results indicated that the drugs disrupted MT polymerization by altering both MT conformation and binding free energy of the neighboring tubulin subunits. The combination of plinabulin and docetaxel destabilized MT polymerization due to bending MT and weakening the polarity of tubulin polymerization. The new combination of docetaxel and vinblastine synergistically enhanced MT depolymerization and bending, while plinabulin and vinblastine had no synergistic inhibitory effects. The results were verified by the tubulin assembly assay. Our study obtained a comprehensive understanding of the action mechanisms of three natural drugs and their combinations on MT dynamic, provided theoretical guidance for new MTA combinations, and would promote the optimal use of MTA and contribute to developing new MTAs as anticancer agents.

A self-activating nanoized vascular disrupting agent for selective anti-tumor therapy.

Vascular disrupting agents (VDAs) have great potential in antitumor therapy, while the efficiency is limited by cardiovascular toxicity. In this study, a self-activating nanoized plinabulin (poly (l-glutamic acid) grafted Azo-Plinabulin, AzoP-NP) was constructed. The AzoP-NPs can selectively be activated to an amino derivative of plinabulin (AmP) by intrinsic tumor hypoxia, disrupting tumor vessels and amplifying hypoxia, whilst be activated by self-amplified tumor hypoxia, then selectively inhibit tumor growth. In 4T1 tumor model, the AzoP-NPs had a selective biodistribution in tumor, as the free AmP in tumors at 24 h after AzoP-NPs treatment was 18.6 fold of that after AmP treatment and significantly higher than that in other tissues. Accordingly, AzoP-NPs resulted in no obvious acute cardiovascular toxicity (plasma von Willebrand factor in PBS, AzoP-NPs and AmP group: 143.1, 184.0 and 477.6 ng/mL) and a significantly stronger tumor inhibition than AmP. And the sustained release of drug in AzoP-NPs led to a higher maximum tolerated dose (MTD) (MTD of AzoP-NPs and AmP: > 80 vs 20 mg/kg). In addition, AzoP-NPs amplified tumor hypoxic, and synergized the anti-tumor effect of Tirapazamine (TPZ), a hypoxia-activated drug in clinical trials, with an inhibition rate of 97.7% and Q value of 1.89. Therefore, our findings provide new insights into next generation VDAs and their application in tumor therapy.

From the North Sea to Drug Repurposing, the Antiseizure Activity of Halimide and Plinabulin.

PharmaSea performed large-scale in vivo screening of marine natural product (MNP) extracts, using zebrafish embryos and larvae, to identify compounds with the potential to treat epilepsy. In this study, we report the discovery of two new antiseizure compounds, the 2,5-diketopiperazine halimide and its semi-synthetic analogue, plinabulin. Interestingly, these are both known microtubule destabilizing agents, and plinabulin could have the potential for drug repurposing, as it is already in clinical trials for the prevention of chemotherapy-induced neutropenia and treatment of non-small cell lung cancer. Both halimide and plinabulin were found to have antiseizure activity in the larval zebrafish pentylenetetrazole (PTZ) seizure model via automated locomotor analysis and non-invasive local field potential recordings. The efficacy of plinabulin was further characterized in animal models of drug-resistant seizures, i.e., the larval zebrafish ethyl ketopentenoate (EKP) seizure model and the mouse 6 Hz psychomotor seizure model. Plinabulin was observed to be highly effective against EKP-induced seizures, on the behavioral and electrophysiological level, and showed activity in the mouse model. These data suggest that plinabulin could be of interest for the treatment of drug-resistant seizures. Finally, the investigation of two functional analogues, colchicine and indibulin, which were observed to be inactive against EKP-induced seizures, suggests that microtubule depolymerization does not underpin plinabulin's antiseizure action.

Plinabulin ameliorates neutropenia induced by multiple chemotherapies through a mechanism distinct from G-CSF therapies.

PURPOSE: Chemotherapy-induced neutropenia (CIN) increases the risk of infections and mortality in cancer patients. G-CSF therapies are approved for the treatment of CIN, but non-G-CSF therapies are needed to increase efficacy and minimize side effects. Plinabulin is an inhibitor of tubulin polymerization that ameliorates CIN caused in patients by the microtubule stabilizer docetaxel. The present study evaluates the potential of plinabulin to reduce neutropenia induced by chemotherapies of different classes in a manner not dependent on increasing G-CSF. METHODS: The anti-CIN benefits of plinabulin were tested in rodents co-treated with docetaxel, cyclophosphamide or doxorubicin. Effects on G-CSF levels were evaluated in tissues by immunoassay. Flow cytometry was utilized to test treatment effects on femur bone marrow cell counts from immunocompetent mice-bearing orthotopic 4T1 breast cancer tumors. RESULTS: Plinabulin alleviated neutropenia induced by microtubule stabilizing, DNA cross-linking and DNA intercalating chemotherapies, yet did not affect bone marrow or blood G-CSF levels. The number of lineage-/Sca1+/c-Kit+ (LSK) hematopoietic stem/progenitor cells (HSPC) in murine bone marrow collected 2 days after treatment was not affected by docetaxel monotherapy despite increased plasma G-CSF in this group. LSK cell number was, however, increased when plinabulin was combined with docetaxel, without affecting G-CSF. CONCLUSIONS: Results support the clinical testing of plinabulin as a non-G-CSF-based treatment for CIN associated with chemotherapies of different mechanisms. Results also support HSPC as a focal point for future mechanism-of-action work aimed at understanding the ability of plinabulin to reduce this serious side effect of cytotoxic therapy in cancer patients.

Development of novel phenoxy-diketopiperazine-type plinabulin derivatives as potent antimicrotubule agents based on the co-crystal structure.

The co-crystal structure of Compound 6b with tubulin was prepared and solved for indicating the binding mode and for further optimization. Based on the co-crystal structures of tubulin with plinabulin and Compound 6b, a total of 27 novel A/B/C-rings plinabulin derivatives were designed and synthesized. Their biological activities were evaluated against human lung cancer NCI-H460 cell line. The optimum phenoxy-diketopiperazine-type Compound 6o exhibited high potent cytotoxicity (IC50 = 4.0 nM) through SAR study of three series of derivatives, which was more potent than plinabulin (IC50 = 26.2 nM) and similar to Compound 6b (IC50 = 3.8 nM) against human lung cancer NCI-H460 cell line. Subsequently, the Compound 6o was evaluated against other four human cancer cell lines. Both tubulin polymerization assay and immunofluorescence assay showed that Compound 6o could inhibit microtubule polymerization efficiently. Furthermore, theoretical calculation of the physical properties and molecular docking were elucidated for these plinabulin derivatives. The binding mode of Compound 6o was similar to Compound 6b based on the result of molecular docking. The theoretical calculated LogPo/w and PCaco of Compound 6o were better than Compound 6b, which could enhance its cytostatic activity. Therefore, Compound 6o might be developed as a novel potent anti-microtubule agent.

GEF-H1 Signaling upon Microtubule Destabilization Is Required for Dendritic Cell Activation and Specific Anti-tumor Responses.

Dendritic cell (DC) activation is a critical step for anti-tumor T cell responses. Certain chemotherapeutics can influence DC function. Here we demonstrate that chemotherapy capable of microtubule destabilization has direct effects on DC function; namely, it induces potent DC maturation and elicits anti-tumor immunity. Guanine nucleotide exchange factor-H1 (GEF-H1) is specifically released upon microtubule destabilization and is required for DC activation. In response to chemotherapy, GEF-H1 drives a distinct cell signaling program in DCs dominated by the c-Jun N-terminal kinase (JNK) pathway and AP-1/ATF transcriptional response for control of innate and adaptive immune responses. Microtubule destabilization, and subsequent GEF-H1 signaling, enhances cross-presentation of tumor antigens to CD8 T cells. In absence of GEF-H1, anti-tumor immunity is hampered. In cancer patients, high expression of the GEF-H1 immune gene signature is associated with prolonged survival. Our study identifies an alternate intracellular axis in DCs induced upon microtubule destabilization in which GEF-H1 promotes protective anti-tumor immunity.

Marine-Derived Anticancer Agents: Clinical Benefits, Innovative Mechanisms, and New Targets.

The role of the marine environment in the development of anticancer drugs has been widely reviewed, particularly in recent years. However, the innovation in terms of clinical benefits has not been duly emphasized, although there are important breakthroughs associated with the use of marine-derived anticancer agents that have altered the current paradigm in chemotherapy. In addition, the discovery and development of marine drugs has been extremely rewarding with significant scientific gains, such as the discovery of new anticancer mechanisms of action as well as novel molecular targets. Approximately 50 years since the approval of cytarabine, the marine-derived anticancer pharmaceutical pipeline includes four approved drugs and eighteen agents in clinical trials, six of which are in late development. Thus, the dynamic pharmaceutical pipeline consisting of approved and developmental marine-derived anticancer agents offers new hopes and new tools in the treatment of patients afflicted with previously intractable types of cancer.

Plinabulin, an inhibitor of tubulin polymerization, targets KRAS signaling through disruption of endosomal recycling.

Constitutive activation of Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most common oncogenic event in certain types of human cancer and is associated with poor patient survival. Small molecule signaling inhibitors have improved the clinical outcomes of patients with various cancer types but attempts to target KRAS have been unsuccessful. Plinabulin represents a novel class of agents that inhibit tubulin polymerization with a favorable safety profile in clinical trials. In the present study, the potency of plinabulin to inhibit tubulin polymerization and growth of KRAS-driven cancer cells was characterized. In vivo efficacy of plinabulin was tested in two different mouse models; one being the RCAS/t-va gene transfer system and the other being a xenograft model. In vitro cell culture tubulin polymerization assays were used to complement the mouse models. There was improved survival in a KRAS-driven mouse gene transfer glioma model, but lack of benefit in a similar model, without constitutively active KRAS, which supports the notion of a KRAS-specific effect. This survival benefit was mediated, at least in part, by the ability of plinabulin to inhibit tubulin polymerization and disrupt endosomal recycling. It was proposed a mechanism of compromised endosomal recycling of displaced KRAS through targeting microtubules that yields inhibition of protein kinase B, but not extracellular signal regulated kinase (ERK) signaling, therefore lending rationale to combination treatments of tubulin- and ERK-targeting agents in KRAS-driven cancer.

Design, synthesis and biological evaluation of anti-pancreatic cancer activity of plinabulin derivatives based on the co-crystal structure.

Based on the co-crystal structures of tubulin with plinabulin and Compound 1 (a derivative of plinabulin), a total of 18 novel plinabulin derivatives were designed and synthesized. Their biological activities were evaluated against human pancreatic cancer BxPC-3 cell lines. Two novel Compounds 13d and 13e exhibited potent activities with IC50 at 1.56 and 1.72 nM, respectively. The tubulin polymerization assay indicated that these derivatives could inhibit microtubule polymerization. Furthermore, the interaction between tubulin and these compounds were elucidated by molecular docking. The binding modes of Compounds 13d and 13e were similar to the co-crystal structure of Compound 1. H-π interaction was observed between the aromatic hydrogen of thiophene moiety with Phe20, which could enhance their binding affinities.

Click strategy using disodium salts of amino acids improves the water solubility of plinabulin and KPU-300.

Plinabulin and KPU-300 are promising anti-microtubule agents; however, the low water solubility of these compounds (<0.1µg/mL) has limited their pharmaceutical advantages. Here, we developed five water-soluble derivatives of plinabulin and KPU-300 with a click strategy using disodium salts of amino acids. The mother skeleton, diketopiperazine (DKP), was transformed into a monolactim-type alkyne and a copper-catalyzed alkyne azide cycloaddition (CuAAC) combined azides that was derived from amino acids as a water-solubilizing moiety. The conversion of carboxyl groups into disodium salts greatly improved the water solubility by 0.8 million times compared to the solubility of the parent molecules. In addition, the α-amino acid side chains of the water-solubilizing moieties affected both the water solubility and the half-lives of the compounds during enzymatic hydrolysis. Our effort to develop a variety of water-soluble derivatives using the click strategy has revealed that the replaceable water-solubilizing moieties can alter molecular solubility and stability under enzymatic hydrolysis. With this flexibility, we are approaching to the in vivo study using water-soluble derivative.

Synthesis of deuterium-enriched and fluorine-substituted plinabulin derivatives and evaluation of their antitumor activities.

Deuterium-enriched and fluorine-substituted compounds have been widely applied in drug discovery due to their advantages in the studies of clinical pharmacokinetics and metabolic profiles. Herein we synthesized a library of deuterated and fluorine-substituted plinabulin derivatives, and all 15 D- or F-compounds were characterized by MS, [Formula: see text] NMR and [Formula: see text]. Their antitumor activities were evaluated against human Jurkat T lymphocyte cells.

Development of MBRI-001, a deuterium-substituted plinabulin derivative as a potent anti-cancer agent.

Plinabulin, a drug targeting microtubule of cancer cells, has been currently tried in its phase III clinical study. However, low efficacy caused by poor pharmacokinetic (PK) properties has been considered to be the main obstacle to approved by the Food and Drug Administration. Herein, we introduced a deuterium atom as an isostere in its structure to become a new compound named (MBRI-001, No. 9 in a series of deuterium-substituted compounds). The structure of MBRI-001 was characterized by HRMS, NMR, IR and a single crystal analysis. MBRI-001 exhibited better pharmacokinetic characteristics than that of plinabulin. Additionally, its antitumor activity is in a low nanomolar level for a variety of cancer cell lines and high activity against human NCI-H460 xenograted in mice intravenous administration. Importantly, continuous administration of MBRI-001 exhibited lower toxicity compared to docetaxel. We thus suggest that MBRI-001 could be developed as a promising anti-cancer agent in near future.