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We report that the self-assembly of drug amphiphiles, Evans blue conjugated camptothecin prodrug (EB-CPT), can be modulated by another anticancer drug paclitaxel (PTX), resulting in ultrahigh quality of nanovesicles (NVs) with uniform shape and diameters of around 80 nm with the EB-CPT:PTX weight ratio of 1:1, 1:2, and 1:3, denoted as ECX NVs. Significantly, the co-assembly of EB-CPT and PTX without adding other excipients has nearly 100 % drug loading efficiency (DLE) and ultrahigh drug loading content (DLC) of PTX alone of up to 72.3±1.7 wt % which, to our best knowledge, is among the highest level reported in literature. Moreover, the ECX NVs with the EB-CPT:PTX weight ratio of 1:2 showed remarkable combination index of 0.59 at a level of 50 % efficacy against HCT116 cells in vitro and greatly improved tumor inhibition effect in vivo compared with two clinically approved CPT- and PTX-based anticancer nanomedicines (Onivyde and Abraxane) individually and their combinations., (© 2021 Wiley-VCH GmbH.)

Nanomedicine developed to date by means of directly encapsulating cytotoxins suffers from crucial drawbacks, including premature release and detoxification prior to arrival at pharmaceutics targets. To these respects, redox-responsive polymeric prodrugs of platinum (Pt) and camptothecin (CPT), selectively and concomitantly activated in the cytoplasm, were elaborated in manufacture of dual prodrug nanomedicine. Herein, multiple CPTs were conjugated to poly(lysine) (PLys) segments of block copolymeric poly(ethylene glycol) (PEG)-PLys through the redox responsive disulfide linkage [PEG-PLys(ss-CPT)] followed by reversible conversion of amino groups from PLys into carboxyl groups based on their reaction with cis -aconitic anhydride [PEG-PLys(ss-CPT&CAA)]. On the other hand, Pt(IV) in conjugation with dendritic polyamindoamine [(G3-PAMAM-Pt(IV)] was synthesized for electrostatic complexation with PEG-PLys(ss-CPT&CAA) into dual prodrug nanomedicine. Subsequent investigations proved that the elaborated nanomedicine could sequentially respond to intracellular chemical potentials to overcome a string of predefined biological barriers and facilitate intracellular trafficking. Notably, PEG-PLys(ss-CPT&CAA) capable of responding to the acidic endosomal microenvironment for transformation into endosome-disruptive PEG-PLys(ss-CPT), as well as release of G3-PAMAM-Pt(IV) from nanomedicine, prompted transclocation of therapeutic payloads from endosomes into cytosols. Moreover, concurrent activation and liberation of cytotoxic CPT and Pt(II) owing to their facile responsiveness to the cytoplasmic reducing microenvironment have demonstrated overwhelming cytotoxic potencies. Eventually, systemic administration of the dual prodrug construct exerted potent tumor suppression efficacy in treatment of intractable solid breast adenocarcinoma, as well as an appreciable safety profile. The present study illustrated the first example of nanomedicine with a dual prodrug motif, precisely and concomitantly activated by the same subcellular stimuli before approaching pharmaceutic action targets, thus shedding important implication in development of advanced nanomedicine to seek maximized pharmaceutic outcomes.

Tumor cell-targeting drug delivery systems are of great importance to anti-tumor therapy in clinics. Owing to the overexpression of the asialoglycoprotein receptor (ASGPR) on the membrane of hepatoma carcinoma cells, the conjugation of lactose on the surface of drug delivery systems has already shown significant advantages for targeting tumor cells. In this study, a disulfide bond-conjugated prodrug targeting delivery system consisting of camptothecin (CPT) and lactose (LA) was synthesized, which was denoted as CPT-S-S-LA. Camptothecin and lactose act as the chemotherapy drug and targeting ligand in the drug delivery system, respectively. Since CPT-S-S-LA is an amphiphilic compound, it can self-assemble into nanoparticles with a diameter of around 110 nm. The CPT-S-S-LA nanoparticles displayed controllable drug release behavior in the physiological environment. Unlike the free CPT, the CPT-S-S-LA nanoparticles firstly assembled at the tumor sites via the enhanced permeability and retention (EPR) effect, and then were phagocytized by the tumor cells with ASGP receptor-mediated endocytosis. Finally, the antitumor agent CPT was released for killing tumor cells, which have a high glutathione (GSH) concentration environment. The nanoparticles displayed favorable ability to target hepatoma carcinoma cells rather than the normal HUVEC cells in vitro. Both the in vitro and in vivo studies demonstrated that the CPT-S-S-LA nanoparticles display enhanced antitumor ability and reduced side effects. Thus, active targeting prodrug delivery systems should be a promising strategy for liver tumor therapy.

Low loading capacity, poor accumulation rate and weak permeability at tumor sites have been identified as the critical barriers for anti-cancer nanomedicines (ANMs). We herein reported a reactive oxygen species (ROS)-activatable ANM of dextran-b-P(CPTMA-co-OEGMA) (DCPT). It aimed to meet the above challenges for improving the therapeutic efficiency of chemotherapy. In this system, camptothecin (CPT) was selected as a chemotherapy drug and poly(ethylene glycol)methyl ether methacrylate (OEGMA) played the role of a hydrophilic block to enhance the water solubility of polyprodrug micelles. At high ROS levels in the tumor microenvironment, the micelles could be disassembled, and simultaneously, the anti-cancer drug of CPT would be released from the DCPT micelles. The 4T1-tumor growth would be greatly inhibited by these two DCPT polyprodrugs, with outstanding in vivo biosafety. The results of both in vitro and in vivo studies indicated the superior therapeutic effects of DCPT. The rational design of polyprodrug nanomedicines may serve as a promising strategy for the development of tumor microenvironment-responsive ANMs, thus improving chemotherapy efficacy.

A novel enzyme-responsive supramolecular polysaccharide assembly composed of disulfide linked adamantane-naphthalimide fluorescent camptothecin prodrug (AdaCPT) and β-CD modified hyaluronic acid (HACD) was constructed, possessing low cellular cytotoxicity and exhibiting targeted cellular imaging and controlled drug release at specific sites while providing a concurrent means for the real-time tracking of drug delivery.

Camptothecin (CPT) is an important topoisomerase I enzyme (Topo I) targeting anti-cancer drug, but its oral administration is limited by poor bioavailability and severe side effects. In this study, a redox sensitive CPT prodrug loaded solid lipid nanoparticles (SLN) system for oral delivery was developed. First of all, CPT-palmitic acid conjugate via a cleavable disulfide bond linker (CPT-SS-PA) was synthesized and encapsulated into SLN. The drug release of SLN was evaluated in neutral environment, simulated gastrointestinal fluid and reductive solution. The results indicated that CPT-SS-PA SLN maintained chemical structural stability in simulated physiological environment but exhibited quick reduction-response release of CPT in the presence of dithiothreitol. Furthermore, in vitro cytotoxicity of CPT-SS-PA SLN was tested against cancer cell lines, and the cellular uptake behavior for oral delivery was checked by confocal laser scanning microscopy (CLSM) using Caco-2 cells model. From the data, CPT-SS-PA SLN revealed high anti-cancer activity and enhanced Caco-2 cell absorption. Finally, the oral bioavailability and intestinal safety of CPT-SS-PA SLN were preliminary evaluated by in vivo pharmacokinetic and histopathological study, respectively. This study demonstrated that CPT-SS-PA SLN could be developed as an effective CPT oral delivery system due to its enhanced oral bioavailability and reduced intestinal side effect., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: A liposome-based siRNA-drug combination was evaluated as a potential therapeutic strategy to improve the curative effect., Methods: A topoisomerase inhibitor SN38 prodrug was combined with a survivin siRNA through codelivery using transferrin (Tf)-L-SN38/P/siRNA. In this combination, SN38 was conjugated to the cell penetrating peptide TAT through a polyethylene glycol (PEG) linker to synthesize TAT-PEG-SN38. The amphiphilic TAT-PEG-SN38 was used as an ingredient of liposomes to improve the cellular uptake. Protamine was added to form an electrostatic complex with siRNA in the core of the liposomes. Tf was introduced to enable tumor cell targeting of liposomes (Tf-L-SN38/P/siRNA)., Results: Tf-L-SN38/P/siRNA exhibited a particle size of 148 nm and a ζ-potential of +7.8 mV. The cellular uptake and antitumor activity were dependent on Tf receptor targeting, TAT-PEG-SN38, and siRNA codelivery. Tf-L-SN38/P/siRNA was shown to be considerably more effective than liposomes carrying individual components. This combination induced potent tumor inhibition (76.8%) in HeLa cell xenograft tumor-bearing nude mice., Conclusion: These data indicated that Tf-L-SN38/P/siRNA was an effective system for codelivery of SN38 and a survivin siRNA and that its therapeutic potential deserved further evaluation., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Camptothecin plays an important role in clinical cancer treatment, and its derivatives are a favorite of pharmaceutical chemists. Herein, we have designed a series of camptothecin prodrugs that exhibit histone deacetylase (HDAC) inhibition activity based on the synergy effect between HDAC inhibitors and camptothecin derivatives. With the evaluation of stability in buffers or plasma from human or mouse model, an appropriate linker was found, so the active drug can be released efficiently and compound 21a exhibited strong antiproliferative activity in A549 and HCT-116 cell lines. These results indicated that the well-designed prodrug can be promising in cancer treatment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Camptothecin (CPT) is a cytotoxic quinoline alkaloid that is used clinically as an anticancer drug. However, the clinical application of CPT is limited due to its low solubility as well as serious and unfathomable side-effects. In the present study, we created a novel 10-hydroxy CPT prodrug, ZBH-ZM‑06. Its cellular cytotoxic activity was analyzed in terms of cellular viability, acetylcholinesterase (AchE) inhibition, DNA relaxation, cellular cycling and apoptosis properties. Our results showed that the AchE inhibition rate of 10 µmol/l ZBH-ZM-06 was 12.5%, compared to 96.5% for carbonyl-oxycamptothecin (CPT-11). In a chemical stability assay, only 4.9% of ZBH-ZM-06 remained after 4 h at pH 7.4. In addition, 10 µmol/l ZBH-ZM-06 significantly inhibited the tumor cell viability of nine tumor cell lines, compared to CPT-11 and the CPT active ingredient, 7-ethyl-10-hydroxy-camptothecin (SN38) (p<0.01-0.05). In the apoptosis assay, ZBH-ZM-06 increased the ratio of annexin V+/propidium iodide (PI)-/+ cells by flow cytometric analysis (p<0.05). Moreover, ZBH-ZM-06 activated caspase-3 and poly(ADP-ribose)polymerase (PARP) expression by immunoblotting. Furthermore, ZBH-ZM-06 induced a greater G2/M phase arrest ratio, compared to CPT-11 and SN38. These results indicated that ZBH-ZM-06 had higher antitumor activity than CPT-11 and SN38, which was shown by its: i) release of the effective ingredient; ii) growth inhibition of a broad spectrum of tumor cells; iii) inhibition of DNA topoisomerase (Topo-1); and iv) promotion of apoptosis through an intrinsic signaling pathway. Thus, ZBH-ZM-06 may be applied in the preclinic study for cancer treatment.

Supramolecular construction of a targeted and stimuli-responsive drug delivery system is still a challenging task. Herein, GSH-responsive supramolecular prodrug nanoparticles were constructed by the host-guest complexation between a β-d-galactose-functionalized water-soluble pillar[5]arene (GalP5) and a disulfide bond containing camptothecin prodrug (G). The obtained prodrug nanoparticles were stable under physiological conditions, whereas efficient drug release was triggered in a simulated tumor environment with high GSH concentration. In vitro studies revealed that these prodrug nanoparticles preferentially entered asialoglycoprotein receptor-overexpressing HepG2 cells due to the active targeting effect of galactose units. This active targeting effect resulted in the maximization of anticancer efficacy and reduction of the undesirable side effects to normal cells.

A novel amphiphilic camptothecin prodrug, CPT-ss-Ir, consisting of CPT, Ir and a disulfide bond linker, was synthesized, and it could self-assemble into nanowires in aqueous solution. Upon intracellular triggering, active CPT and Ir species were released to exert a considerable anticancer effect.

We report here a tumor-targeting masked phototherapeutic agent 1 (PT-1). This system contains SN-38-a prodrug of the topoisomerase I inhibitor irinotecan. Topoisomerase I is a vital enzyme that controls DNA topology during replication, transcription, and recombination. An elevated level of topoisomerase I is found in many carcinomas, making it an attractive target for the development of effective anticancer drugs. In addition, PT-1 contains both a photo-triggered moiety (nitrovanillin) and a cancer targeting unit (biotin). Upon light activation in cancer cells, PT-1 interferes with DNA re-ligation, diminishes the expression of topoisomerase I, and enhances the expression of inter alia mitochondrial apoptotic genes, death receptors, and caspase enzymes, inducing DNA damage and eventually leading to apoptosis. In vitro and in vivo studies showed significant inhibition of cancer growth and the hybrid system PT-1 thus shows promise as a programmed photo-therapeutic ("phototheranostic").

Malignant tumor cells have been found to have high levels of reactive oxygen species such as hydrogen peroxide (H2O2), supporting the hypothesis that a prodrug could be activated by intracellular H2O2 and lead to a potential antitumor therapy. In this study, the 7-ethyl-10-boronic acid camptothecin (B1) was synthesized for the first time as prodrug of SN-38, by linking a cleavable aryl carbon-boron bond to the SN-38. Prodrug B1 selectively activated by H2O2, converted rapidly to the active form SN-38 under favorable oxidative conditions in cancer cells with elevated levels of H2O2. The cell survival assay showed that prodrug B1 was equally or more effective in inhibiting the growth of six different cancer cells, as compared to SN-38. Unexpectedly, prodrug B1 displayed even more potent Topo I inhibitory activity than SN-38, suggesting that it was not only a prodrug of SN-38 but also a typical Topo I inhibitor. Prodrug B1 also demonstrated a significant antitumor activity at 2.0 mg/kg in a xenograft model using human brain star glioblastoma cell lines U87MG., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Chemical modification of small molecule hydrophobic drugs is a clinically proven strategy to devise prodrugs with enhanced treatment efficacy. While this prodrug strategy improves the parent drug's water solubility and pharmacokinetic profile, it typically compromises the drug's potency against cancer cells due to the retarded drug release rate and reduced cellular uptake efficiency. Here we report on the supramolecular design of self-assembling prodrugs (SAPD) with much improved water solubility while maintaining high potency against cancer cells. We found that camptothecin (CPT) prodrugs created by conjugating two CPT molecules onto a hydrophilic segment can associate into filamentous nanostructures in water. Our results suggest that these SAPD exhibit much greater efficacy against primary brain cancer cells relative to that of irinotecan, a clinically used CPT prodrug. We believe these findings open a new avenue for rational design of supramolecular prodrugs for cancer treatment.

Novel redox-responsive nanomedicines have been synthesized by conjugating camptothecin prodrugs ((pyridine-2-yldisulfanil)alkyl carbonate derivatives) to hybrid porous silica nanoparticles through disulfide bond. After disulfide reduction, camptothecin may be released by an intramolecular cyclization mechanism or by carbonate bond hydrolysis. Samples have been characterized by physico-chemical techniques, and stability and drug release in PBS and human serum have been determined. Moreover, cell uptake was studied by fluorescence microscopy and flow cytometry, whilst cytotoxic activity was validated by MTT test. Obtained results indicate that prodrug side chain carbon number (n=1,2,3) determines material hydrophobic properties and, as a consequence, its stability in aqueous medium. When n value increases, the negative surface charge decreases dramatically due to a shielding effect provoked by hydrophobic ligands, which promotes particle aggregation and favors cell internalization. Furthermore, the n value determines the type of products released and, subsequently, the cytotoxic activity. Full disulfide bridge reduction takes place in all cases, but quick delivery of the free drug by intramolecular cyclization is only possible with the shortest linker (n=1), whereas other nanomedicines only present slow discharge of camptothecin by carbonate hydrolysis. Overall, the drug precursor incorporated to the inorganic nanoplatform modulates both cell uptake rate and cytotoxicity according to the different functionalization., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In an attempt to improve the antitumor activity and reduce the side effects of irinotecan (2), novel prodrugs of SN-38 (3) were prepared by conjugating amino acids or dipeptides to the 10-hydroxyl group of SN-38 via a carbamate linkage. The synthesized compounds completely generated SN-38 in pH 7.4 buffer or in human plasma, while remaining stable under acidic conditions. All prodrug compounds demonstrated much greater in vitro antitumor activities against HeLa cells and SGC-7901 cells than irinotecan. The most active compounds, 5h, 7c, 7d, and 7f, exhibited IC50 values that were 1000 times lower against HeLa cells and 30 times lower against SGC-7901 cells than those of irinotecan, and the inhibitory activities of these prodrugs against acetylcholinesterase (AchE) were significantly reduced, with IC50 values more than 6.8 times greater than that of irinotecan. In addition, compound 5e exhibited the same level of tumor growth inhibitory activity as irinotecan (CPT-11) in a human colon xenograft model in vivo.

We have recently reported a chemical approach for half-life extension that utilizes β-eliminative linkers to attach amine-containing drugs or prodrugs to macromolecules. The linkers release free drug or prodrug over periods ranging from a few hours to over 1 year. We adapted these linkers for use with phenol-containing drugs. Here, we prepared PEG conjugates of the irinotecan (CPT-11) active metabolite SN-38 via a phenyl ether that release the drug with predictable long half-lives. Pharmacokinetic studies in the rat indicate that, in contrast to other SN-38 prodrugs, the slowly released SN-38 shows a very low C(max), is kept above target concentrations for extended periods, and forms very little SN-38 glucuronide (the precursor of enterotoxic SN-38). The low SN-38 glucuronide is attributed to low hepatic uptake of SN-38. These macromolecular prodrugs have unique pharmacokinetic profiles that may translate to less intestinal toxicity and interpatient variability than the SN-38 prodrugs thus far studied.

The introduction of a methylenthiol group at position 7 of camptothecin was carried out in four steps. This preparation also yielded the corresponding disulfide, which behaves as a prodrug due to its reactivity with glutathione. Assessment of their antiproliferative activities, investigations of their mechanism of action, and molecular modeling analysis indicated that the 7-modified camptothecin derivatives described herein maintain the biological activity and drug-target interactions of the parent compound., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The objective of the present study was to identify a camptothecin (CPT) prodrug with optimal release and cytotoxicity properties for immobilization on a passively targeted microparticle delivery system. A series of alpha-amino acid ester prodrugs of CPT were synthesized, characterized, and evaluated. Four CPT prodrugs were synthesized with increasing aliphatic chain length (glycine (Gly) (2a), alanine (Ala) (2b), aminobutyric acid (Abu) (2c), and norvaline (Nva) (2d)). Prodrug reconversion was studied at pH 6.6, 7.0, and 7.4 corresponding to tumor, lung, and extracellular/physiological pH, respectively. Cytotoxicity was evaluated in A549 human lung carcinoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The hydrolytic reconversion rate to parent CPT increased with decreasing side chain length as well as increasing pH. The Hill slope of 2d was significantly less than CPT and the other prodrugs tested, indicating a higher cell death rate at lower concentrations. These results suggest that 2d is the best candidate for a passively targeted sustained release lung delivery system.

CH0793076 (1) is a novel hexacyclic camptothecin analog showing potent antitumor activity in various human caner xenograft models. To improve the water solubility of 1, water-soluble prodrugs were designed to generate an active drug 1 nonenzymatically, thus expected to show less interpatient PK variability than CPT-11. Among the prodrugs synthesized, 4c (TP300, hydrochloride) having a glycylsarcosyl ester at the C-20 position of 1 is highly water-soluble (>10mg/ml), stable below pH 4 and rapidly generates 1 at physiological pH in vitro. The rapid (ca. <1min) generation of 1 after incubation of TP300 with plasma (mouse, rat, dog and monkey) was also demonstrated. TP300 showed a broader antitumor spectrum and more potent antitumor activity than CPT-11 in various human cancer xenograft models.

Purpose: Irinotecan is a prodrug converted to the active cytotoxic molecule SN38 predominantly by the action of liver carboxylesterases. The efficacy of irinotecan is limited by this hepatic activation that results in a low conversion rate, high interpatient variability, and dose-limiting gastrointestinal toxicity. The purpose of this study was to evaluate a novel peptidic prodrug of SN38 (DTS-108) developed to bypass this hepatic activation and thus reduce the gastrointestinal toxicity and interpatient variability compared with irinotecan., Experimental Design: SN38 was conjugated to a cationic peptide (Vectocell) via an esterase cleavable linker. The preclinical development plan consisted of toxicity and efficacy evaluation in a number of different models and species., Results: The conjugate (DTS-108) is highly soluble, with a human plasma half-life of 400 minutes in vitro. Studies in the dog showed that DTS-108 liberates significantly higher levels of free SN38 than irinotecan without causing gastrointestinal toxicity. In addition, the ratio of the inactive SN38-glucuronide metabolite compared with the active SN38 metabolite is significantly lower following DTS-108 administration, compared with irinotecan, which is consistent with reduced hepatic metabolism. In vivo efficacy studies showed that DTS-108 has improved activity compared with irinotecan. A significant dose-dependent antitumoral efficacy was observed in all models tested and DTS-108 showed synergistic effects in combination with other clinically relevant therapeutic agents., Conclusions: DTS-108 is able to deliver significantly higher levels of SN38 than irinotecan, without the associated toxicity of irinotecan, resulting in an increased therapeutic window for DTS-108 in preclinical models. These encouraging data merit further preclinical and clinical investigation.

A folate receptor targeted camptothecin prodrug was synthesized using a hydrophilic peptide spacer linked to folate via a releasable disulfide carbonate linker. The conjugate was found to possess high affinity for folate receptor-expressing cells and inhibited cell proliferation in human KB cells with an IC(50) of 10nM. Activity of the prodrug was completely blocked by excess folic acid, demonstrating receptor-mediated uptake.

The alpha-amino acid ester prodrugs of the antitumor agent camptothecin and a more potent, lipophilic silatecan analogue, DB-67, have been shown by NMR spectroscopy and quantitative kinetic analyses to undergo quantitative conversion to their pharmacologically active lactones via a nonenzymatic mechanism that at pH 7.4 is favored over direct hydrolysis. The alternate pathway involves the reversible intramolecular nucleophilic amine attack at the camptothecin E-ring carbonyl to generate a lactam (I) followed by a second intramolecular reaction to produce a bicyclic hemiortho ester (I'). The intermediates were isolated and shown to exist in an apparent equilibrium dominated by the hemiortho ester in DMSO using NMR spectroscopy. The conversion of prodrugs of camptothecin or DB-67 containing either alpha-NH(2) or alpha-NHCH(3) and their corresponding hemiortho esters were monitored versus time in aqueous buffer (pH 3.0 and 7.4) at 37 degrees C, and the kinetic data were fit to a model based on the proposed mechanism. The results indicated that while the prodrugs are relatively stable at pH 3, facile lactone release occurs from both the prodrugs and their corresponding hemiortho ester intermediates under physiological conditions (pH 7.4). The glycinate esters and their hemiortho esters were found to be more cytotoxic than the N-methylglycinates or their corresponding hemiortho ester intermediates in vitro using a human breast cancer cell line (MDA-MB-435S), consistent with their more rapid conversion to active lactone. The pH dependence of the nonenzymatic pathway for conversion of these alpha-amino acid ester prodrugs suggests that they may be useful for tumor-targeting via liposomes, as they can be stabilized in an acidic environment in the core of liposomes and readily convert to the active lactone following their intratumoral release.

Purpose: The novel fluoro-substituted camptothecin analog, BMS-286309, and its prodrug, BMS-422461, were evaluated for their pharmacologic, toxicologic, metabolic and pharmacokinetic developmental potential., Methods: In vitro and in vivo assays were used to assess the compounds for topoisomerase I activity, antitumor activity, gastrointestinal (GI) toxicity, and pharmacokinetic parameters., Results: BMS-286309-induced topoisomerase I-mediated DNA breaks in vitro and was similar in potency to camptothecin. Both BMS-286309 and -422461 were comparable to irinotecan regarding preclinical antitumor activity assessed in mice bearing distal site murine and human tumors. BMS-422461 was also found to be orally active. Both analogs were >100-fold more potent in vivo than irinotecan and both were superior to irinotecan with respect to toxicological assessment of GI injury in mice. The generation of parent compound from BMS-422461 was qualitatively similar in mouse, rat and human blood and liver S9 fractions. The percentage of BMS-286309 remaining as the active lactone form at equilibrium was comparable in mouse and human plasma. The pharmacokinetic profile in rat blood demonstrated that BMS-422461 was rapidly cleaved to BMS-286309., Conclusions: The favorable in vivo metabolic activation of BMS-422461, and the pharmacokinetic characteristics of BMS-286309, suggest that the good efficacy of BMS-422461 is derived from robust in vivo release of BMS-286309 in rodents and the likelihood that this biotransformation will be preserved in humans. The comparable antitumor activity of BMS-422461 to irinotecan, as well as reduced preclinical GI toxicity, make this novel camptothecin analog attractive for clinical development.

We designed, synthesized, and evaluated in vitro and in vivo a novel targeted anticancer polymeric prodrug containing multiple copies of tumor targeting moiety [synthetic luteinizing hormone-releasing hormone (LHRH) peptide, analog of LHRH] and anticancer drug (camptothecin). One, two, or three molecules of the targeting peptide and anticancer drug were covalently conjugated with bis(2-carboxyethyl) polyethylene glycol polymer using citric acid as a multivalent spacer. We showed that LHRH peptide was bound to extracellular receptors and localized in plasma membrane of cancer cells. The designed tumor-targeted prodrug increased the solubility of anticancer drug and offered cytoplasmic and/or nuclear delivery of drug to cancer cells expressing LHRH receptors. The multicomponent prodrug containing three copies of the targeting peptide and drug was almost 100 times more cytotoxic and substantially had enhanced antitumor activity compared with the analogous nontargeted prodrug and prodrugs containing one or two copies of active components.

Several water-soluble derivatives (CPT3, CPT3a-d) of camptothecin (CPT) were synthesized, among which CPT3 bearing an N,N'-dimethyl-1-aminoethylcarbamate side-chain was further conjugated with reductively eliminating structural units of indolequinone, 4-nitrobenzyl alcohol and 4-nitrofuryl alcohol to produce novel prodrugs of camptothecin (CPT4-6). All CPT derivatives were of lower cytotoxicity than their parent compound of CPT. In contrast, CPT4 and CPT6 showed higher hypoxia selectivity of cytotoxicity towards tumor cells than CPT. A mechanism by which a representative prodrug CPT4 is activated in the presence of DT-diaphorase to release CPT was also discussed. The bioreduction activated CPT prodrugs including CPT4 and CPT6 are identified to be promising for application to the hypoxia targeting tumor chemotherapy.

In an effort to improve the stability of labile lactone ring of camptothecins, the bis and mono-alkyl carbonate prodrugs of 10-hydroxycamptothecins were synthesized and their chemical and enzymatical stability as well as antitumor activity were studied. The in vitro evaluation of the stability of these carbonates indicates that the 10,20-biscarbonates are firstly hydrolyzed to afford the stable 20-monocarbonates. And the 10-carbonates are not stable in human plasma, mouse plasma and pH7.4 phosphate buffer, while the 20-carbonates are relatively stable in the three media and can be readily cleaved by porcine liver esterase. The overall toxicity of the tested carbonate against mice bearing S180 sarcoma is much lower when compared with the parent compound, and the antitumor activity is maintained.

Despite the high antitumor activity of camptothecins, few derivatives have been developed and tested for human treatment of solid tumors, due to unpredictable toxicity mainly connected to their poor water solubility. We report the conjugation of the antitumor agent 10-amino-7-hydroxy camptothecin (SN-392) to linear or branched poly(ethylene glycol)s (PEGs) of different loading capacity through a tri- or tetrapeptide spacer selectively cleaved by lysosomal enzymes (cathepsins). A synthetic strategy based on the chemoselective acylation of the aromatic amino group in the presence of the unprotected C20 tertiary alcohol allowed high overall yields. Two conjugates demonstrated good stability at physiological pH and in mouse plasma (nonspecific proteases) but slowly released the drug payload in the presence of the lysosomal enzyme cathepsin B1. Compound 3, selected for in vivo experiments, was very active against P388, P388/ADM leukaemia, and Meth A fibrosarcoma cell lines, scoring T/C% values comparable with the camptothecin derivative CPT-11. Pharmacokinetic studies indicated that 3 acts as a reservoir of 10-amino-7-ethylcamptothecin, as the mean residence time (MRT) is about 3-fold higher than that of the free drug.

Two PEG prodrugs utilizing conjugation of PEG through the C-21 acid functionality as well as the C-17 OH group of CPT hydroxy-amide open forms were synthesized and characterized. Both of these open lactone tripartate prodrugs were shown to be water soluble and highly effective in MX-1 mouse xenograph studies. Indirect evidence implies that the initial ester or carbonate bond breaking is esterase mediated in the first step of the cascade of CPT release.

In situ binding of thiol-reactive prodrugs to the cysteine-34 position of circulating albumin is a new approach in drug delivery. Therefore, five maleimide-bearing derivatives of the anticancer drug camptothecin (CPT) were developed as albumin-binding prodrugs. These compounds were synthesized by reacting heterobifunctional cross-linkers based on oligo(ethylene glycols) [3-6 (O-CH(2)-CH(2)) units] bearing a maleimide group on one end and a carboxylic acid group on the other with camptothecin 20-O-glycinate. Incorporating oligo(ethylene glycol) chains into the prodrugs enhanced their water-solubility when compared to the parent compound (up to 27-fold). HPLC studies showed that the prodrugs react almost quantitatively with the cysteine-34 position of endogenous albumin within a few minutes after incubation of the CPT derivatives with human blood plasma. The therapeutic potential of two of the prodrugs was assessed in nude mice bearing a colon xenograft (HT-29). Both albumin-binding derivatives of camptothecin were well-tolerated and showed enhanced antitumor efficacy when compared to CPT.

Five (20S)-10-hydroxycamptothecin derivatives carrying the long-chain fatty acid esters were prepared for the development of a new class of prodrug-type agents. In vitro experiments using three kinds of purified carboxylesterase isozymes from the liver microsomes of rat, pig, and human demonstrated that these derivatives were efficiently metabolized by enzymes compared with CPT-11.