Doxorubicin Derivative Research Articles

Multifunctional Polymeric Micelles for Enhanced Intracellular Delivery of Doxorubicin to Metastatic Cancer Cells

To develop multifunctional RGD-decorated poly(ethylene oxide)-b-poly(ester) based micelles and assess their pH-triggered core degradation and targeted drug release in tumor cells that overexpress RGD receptors. Novel poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PEO-b-PCL) based copolymers modified with RGD ligands on PEO and pendent functional groups on PCL, i.e., GRGDS-PEO-b-poly(alpha-benzylcarboxylate-epsilon-caprolactone) (GRGDS-PEO-b-PBCL) and GRGDS-PEO-b-poly(alpha-carboxyl-epsilon-caprolactone) (GRGDS-PEO-b-PCCL), were synthesized. Chemical conjugation of doxorubicin (DOX) to PCCL core produced GRGDS-PEO-b-P(CL-DOX) micellar conjugates, while GRGDS-PEO-b-PBCL were used to physically encapsulate DOX. For both systems, micellar core degradation, drug release, intracellular drug uptake/disposition, and cytotoxicity against B16F10 metastatic cells were investigated. The PBCL and P(CL-DOX) cores were found resistant to degradation in pH 7.2, but showed 10% and 40% loss in core molecular weight in pH 5.0 within 144 h, respectively. Preferential release of DOX and DOX derivatives from PBCL and P(CL-DOX) cores was noted in pH 5.0, respectively. The GRGDS-modified micelles showed enhanced cellular internalization through endocytosis, increased intracellular DOX release, nuclear localization, and improved cytotoxicity against metastatic B16F10 cells compared to their unmodified counterparts. The results clearly suggest a promise for the development of multifunctional polymeric micelles with RGD ligand decorated shell and endosomal pH-triggered degradable core for selective DOX delivery to metastatic cancer cells.

In Vitro and in Vivo Evaluation of Doxorubicin Conjugates with the Divalent Peptide E-[c(RGDfK)2] that Targets Integrin αvβ3

Integrins, especially integrin alpha vbeta3, are attractive receptors for vascular targeting strategies. Recently, a divalent RGD peptidomimetic, E-[c(RGDfK)2], has been described that demonstrates increased uptake in human ovarian carcinoma OVCAR-3 xenograft tumors. Inspired by these results, we set out to develop doxorubicin conjugates with E-[c(RGDfK)2] by binding two different maleimide derivatives of doxorubicin to E-[c(RGDfK)2] that was thiolated with iminothiolane. In this way, two water-soluble derivatives were obtained, E-[c(RGDfK)2]-DOXO-1 and E-[c(RGDfK)2]-DOXO-2. In E-[c(RGDfK)2]-DOXO-1, doxorubicin was bound to the peptide through a stable amide bond, and in E-[c(RGDfK)2]-DOXO-2, a MMP-2/MMP-9 cleavable octapeptide was introduced between doxorubicin and the peptide. The rationale for a MMP-2/MMP-9-cleavable linker was that MMP-2 and MMP-9 bind to integrin alpha vbeta3 and both are overexpressed in tumor vasculature. In addition, analogous control doxorubicin-containing peptides bearing c(RADfK) that does not bind to integrin alpha vbeta3 were synthesized, i.e., c(RADfK)-DOXO-1 and c(RADfK)-DOXO-2. Whereas E-[c(RGDfK) 2]-DOXO-2 was cleaved effectively by MMP-2 and in OVCAR-3 tumor homogenates releasing a doxorubicin-tetrapeptide or doxorubicin as the final cleavage product, no release of doxorubicin was observed for E-[c(RGDfK)2]-DOXO-1. Proliferation of HUVEC in the presence of MMP-2-cleavable doxorubicin-containing peptides exhibited 6- to 10-fold increased inhibition compared to the amide-linked doxorubicin-containing peptides. In addition, inhibition of HUVEC sprouting during a 24 h exposure was approximately 3-fold stronger for E-[c(RGDfK) 2]-DOXO-2 and 20-fold stronger for the reference peptide conjugate c(RADfK)-DOXO-2 than for doxorubicin alone. In vivo studies in an OVCAR-3 xenograft model demonstrated no or only moderate antitumor efficacy for either E-[c(RGDfK)2], E-[c(RGDfK)2]-DOXO-1, E-[c(RGDfK)2]-DOXO-2, or c(RADfK)-DOXO-2, even at doses of 3 x 24 mg/kg doxorubicin equivalents, compared to an improved antitumor effect for doxorubicin at 2 x 8 mg/kg.

Albumin as a drug carrier: Design of prodrugs, drug conjugates and nanoparticles

Albumin is playing an increasing role as a drug carrier in the clinical setting. Principally, three drug delivery technologies can be distinguished: coupling of low-molecular weight drugs to exogenous or endogenous albumin, conjugation with bioactive proteins and encapsulation of drugs into albumin nanoparticles. The accumulation of albumin in solid tumors forms the rationale for developing albumin-based drug delivery systems for tumor targeting. Clinically, a methotrexate-albumin conjugate, an albumin-binding prodrug of doxorubicin, i.e. the (6-maleimido)caproylhydrazone derivative of doxorubicin (DOXO-EMCH), and an albumin paclitaxel nanoparticle (Abraxane) have been evaluated clinically. Abraxane has been approved for treating metastatic breast cancer. An alternative strategy is to bind a therapeutic peptide or protein covalently or physically to albumin to enhance its stability and half-life. This approach has been applied to peptides with antinociceptive, antidiabetes, antitumor or antiviral activity: Levemir, a myristic acid derivative of insulin that binds to the fatty acid binding sites of circulating albumin, has been approved for the treatment of diabetes. Furthermore, Albuferon, a fusion protein of albumin and interferon, is currently being assessed in phase III clinical trials for the treatment of hepatitis C and could become an alternative to pegylated interferon. This review gives an account of the different drug delivery systems which make use of albumin as a drug carrier with a focus on those systems that have reached an advanced stage of preclinical evaluation or that have entered clinical trials.

Anthracycline antibiotics and their derivatives, inhibitors of topoisomerase I

The relationship between the structure of new semisynthetic derivatives of doxorubicin, daunorubicin, and carminomycin and their ability to inhibit topoisomerase 1 were studied. The new derivatives inhibit the activity of topoisomerase 1 at low concentrations, induce the death of K-562 leukemia cells in culture, and produce an antitumor effect in experimental animals with P388 leukemia.

DNA repair in response to anthracycline–DNA adducts: A role for both homologous recombination and nucleotide excision repair

Doxorubicin, a widely used anthracycline anticancer agent, acts as a topoisomerase II poison but can also form formaldehyde-mediated DNA adducts. This has led to the development of doxorubicin derivatives such as doxoform, which can readily form adducts with DNA. This work aimed to determine which DNA repair pathways are involved in the recognition and possible repair of anthracycline–DNA adducts. Cell lines lacking functional proteins involved in each of the five main repair pathways, mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end-joining (NHEJ) were examined for sensitivity to various anthracycline adduct-forming treatments. The treatments used were doxorubicin, barminomycin (a model adduct-forming anthracycline) and doxoform (a doxorubicin–formaldehyde conjugate). Cells with deficiencies in MMR, BER and NHEJ were equally sensitive to adduct-forming treatments compared to wild type cells and therefore these pathways are unlikely to play a role in the repair of these adducts. Some cells with deficiencies in the NER pathway (specifically, those lacking functional XPB, XPD and XPG), displayed tolerance to adducts induced by both barminomycin and doxoform and also exhibited a decreased level of apoptosis in response to adduct-forming treatments. Conversely, two HR deficient cell lines were shown to be more sensitive to barminomycin and doxoform than HR proficient cells, indicating that this pathway is also involved in the repair response to anthracycline–DNA adducts. These results suggest an unusual damage response pathway to anthracycline adducts involving both NER and HR that could be used to optimise cancer therapy for tumours with either high levels of NER or defective HR. Tumours with either of these characteristics would be predicted to respond particularly well to anthracycline–DNA adduct-forming treatments.

Phase I and Pharmacokinetic Study of the (6-Maleimidocaproyl)Hydrazone Derivative of Doxorubicin

The (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH) is an albumin-binding prodrug of doxorubicin with acid-sensitive properties that shows superior antitumor efficacy in murine tumor models and a favorable toxicity profile in mice, rats, and dogs compared with doxorubicin. The purpose of the phase I study was to characterize the toxicity profile of DOXO-EMCH, establish a recommended dose for phase II studies, and assess potential anticancer activity. A starting dose of 20 mg/m2 doxorubicin equivalents was chosen. Forty-one patients with advanced cancer disease were treated with an i.v. infusion of DOXO-EMCH once every 3 weeks at a dose level of 20 to 340 mg/m2 doxorubicin equivalents. Treatment with DOXO-EMCH was well tolerated up to 200 mg/m2 without manifestation of drug-related side effects. Myelosuppression (grade 1-2) and mucositis (grade 1-2) were the predominant adverse effects at dose levels of 260 mg/m2 and myelosuppression (grade 1-3) as well as mucositis (grade 1-3) were dose limiting at 340 mg/m2. No cardiac toxicity was observed. Of 30 of 41 evaluable patients, 12 patients (40%) had progressive disease, 15 patients (57%) had stable disease, and 3 patients (10%) had a partial remission. DOXO-EMCH showed a good safety profile and was able to induce tumor regressions in tumor types known to be anthracycline-sensitive tumors, such as breast cancer, small cell lung cancer, and sarcoma. The recommended doxorubicin equivalent dose for phase II studies is 260 mg/m2.

DOXO-EMCH (INNO-206): the first albumin-binding prodrug of doxorubicin to enter clinical trials

The (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH) is an albumin-binding prodrug of doxorubicin with acid-sensitive properties that demonstrates superior antitumor efficacy in murine tumor models and a favorable toxicity profile in mice, rats and dogs, including significantly reduced cardiotoxicity. After intravenous administration, DOXO-EMCH binds rapidly to the Cys-34 position of circulating albumin and accumulates in solid tumors due to passive targeting. In a clinical Phase I study, the dose of doxorubicin could be increased by a factor of 4.5 – 340 mg/m² when 75 mg/m² of free doxorubicin is considered to be the dose that can be administered as a single agent concomitant with the typical spectrum of side effects (i.e., myelotoxicity and mucositis). DOXO-EMCH was able to induce tumor regressions in anthracycline-sensitive tumors (i.e., breast cancer, small cell lung cancer and sarcoma). Phase II studies will be initiated at the beginning of 2007.

Role of mtDNA lesions in anthracycline cardiotoxicity

Doxorubicin (adriamycin) is an effective drug in the treatment of many malignancies. Its prolonged use is, however, limited by an irreversible, dose-dependent and progressive cardiomyopathy, which may become evident even years after completion of therapy. Data from rats and humans show that oxidative phosphorylation is impaired rapidly after acute doxorubicin-exposure. Such respiratory chain dysfunction is known to enhance the production of reactive oxygen species and may lead to quantitative and qualitative injury of mitochondrial DNA (mtDNA) and its encoded respiratory chain subunits. MtDNA depletion, mtDNA mutations and respiratory defects then accumulate with time also in the absence of continued anthracycline exposure. Chronic cardiotoxicity then manifests, when the bioenergetic capacity of the organelles is severely impaired. The mitochondrial damage in late-onset doxorubicin cardiomyopathy is heart specific and not found in skeletal muscle. DOXO-EMCH, a 6-maleimidocaproyl hydrazone derivative of doxorubicin has evolved from the search for less cardiotoxic anthracyclines. At equieffective antitumor doses, DOXO-EMCH has a substantially lower heart toxicity than free doxorubicin.

Enhancement of Death Receptor 4 Mediated Apoptosis and Cytotoxicity in Renal Cell Carcinoma Cells by Subtoxic Concentrations of Doxorubicin

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) triggers apoptosis in various tumor cells by engaging death receptors 4 and 5. We investigated the effect of chemotherapeutic agents on death receptor 4 mediated apoptosis in human renal cell carcinoma cells using HGS-ETR1, which is a human monoclonal agonistic antibody specific for death receptor 4. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Synergy was assessed by isobolographic analysis. Treatment of the ACHN human renal cell carcinoma cell line with HGS-ETR1 combined with 5-fluorouracil, vinblastine or gemcitabine did not overcome resistance to these agents. However, treatment with HGS-ETR1 combined with doxorubicin had a synergistic cytotoxic effect. Synergy was also achieved in another human renal cell carcinoma cell line, Caki-1, and in 5 freshly derived renal cell carcinoma cell cultures. A synergistic effect was also observed with HGS-ETR1 combined with the doxorubicin derivatives epirubicin, pirarubicin or amrubicin. The synergy achieved in cytotoxicity with HGS-ETR1 and doxorubicin was also achieved in apoptosis. Sequential treatment with doxorubicin followed by HGS-ETR1 induced significantly more cytotoxicity than reverse treatment or simultaneous treatment (p<0.05). Doxorubicin remarkably increased the cell surface expression of death receptor 4 in renal cell carcinoma cells. The combination of doxorubicin and HGS-ETR1 significantly activated the caspase cascade, including caspase-8, 9, 6 and 3, which are the downstream molecules of death receptors. These findings indicate that doxorubicin sensitizes renal cell carcinoma cells to death receptor 4 mediated apoptosis through the induction of death receptor 4 and the activation of caspases, suggesting that combination therapy of doxorubicin and HGS-ETR1 might be effective as renal cell carcinoma therapy.

Acute and repeat-dose toxicity studies of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH), an albumin-binding prodrug of the anticancer agent doxorubicin

The (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH) is an albumin-binding prodrug of doxorubicin with acid-sensitive properties that demonstrates superior antitumor efficacy in murine tumor models, and has been evaluated in a phase I study. In order to establish the toxicity profile of this prodrug, acute and repeat-dose toxicity studies were performed with DOXO-EMCH in CD1-mice, Sprague-Dawley rats and Beagle dogs. Although the objective of the acute toxicity studies was not the determination of LD50 values, the LD50 of DOXO-EMCH was >60 mg/kg doxorubicin equivalents in both male and female mice (the LD50 of doxorubicin in CD-1 mice is -12 mg/kg). In Sprague-Dawley rats, the LD50 was 23.4 and 45.9 mg/kg doxorubicin equivalents for males and females, respectively. For comparison, the LD50 of doxorubicin in Sprague-Dawley rats is -10.5 mg/kg. The major clinical sign noted following intravenous administration of DOXO-EMCH in mice and rats was a dose-dependent peripheral neuropathy which, in general, developed as a delayed toxicity 1-3 weeks after application. The observed neurotoxicity has been well documented for Sprague-Dawley rats treated with doxorubicin at a dose of 5 and 10 mg/kg. In Beagle dogs, LD10 was not reached for DOXO-EMCH at 4.5 mg/kg doxorubicin equivalents. A four-cycle intravenous study with DOXO-EMCH at dose levels of 4 x 2.5, 5.0 or 7.5 mg/kg doxorubicin equivalents in rats revealed approximately three-fold less side effects on the hemolymphoreticular system when compared to 4 x 2.5 mg/kg doxorubicin dose, whereas effects on the testes/oligospermia seem to be comparable between both drugs at equitoxic dose. A No Observable Adverse Effect Level (NOAEL) for DOXO-EMCH of 4 x 2.5 mg/kg doxorubicin equivalents was established in this study. This dose is equivalent to the maximum tolerated dose (MTD) of doxorubicin in rats. In a two-cycle study over a period of 6 weeks in Beagle dogs (intravenous administration of DOXO-EMCH at dose levels of 1.5, 3.0 or 4.5 mg/kg doxorubicin equivalents), dose-related systemic histamine-like reactions within the first 3 hours after injection were noted in all treated groups. Only transient and temporary effects on hematology, urinary function, as well as on histopathology in mid- and/or high-dose animals, were observed. The low dose of 2 x 1.5 mg/kg was considered to be the NOAEL in this study, which is equivalent to twice the MTD o f doxorubicin i nBeagle dogs. In summary, the toxicity studies with DOXO-EMCH in mice, rats or dogs have not identified any other special toxicity when compared to the toxicity data for doxorubicin. Preclinical tolerance of DOXO-EMCH was higher in mice, rats and dogs compared to doxorubicin. A dose of 20 mg/m2 doxorubicin equivalents was recommended as the starting dose for a phase I study with DOXO-EMCH.

The 6‐maleimidocaproyl hydrazone derivative of doxorubicin (DOXO‐EMCH) is superior to free doxorubicin with respect to cardiotoxicity and mitochondrial damage

Doxorubicin causes a chronic cardiomyopathy in which genetic and functional lesions of mitochondria accumulate in the long-term and explain in part the delayed onset of heart dysfunction. DOXO-EMCH a 6-maleimidocaproyl hydrazone derivative of doxorubicin, is an albumin binding prodrug which has entered clinical trials because of its superior antitumor and toxicological profile. In the present work, we examined the chronic cardiotoxicity of DOXO-EMCH in direct comparison with doxorubicin. Rats (11 weeks of age) were treated with intravenous doxorubicin (0.8 mg/kg weekly for 7 weeks), an equimolar dose of DOXO-EMCH (1.1 mg/kg), or with 3.3 mg/kg of DOXO-EMCH. Controls received saline. Animals were euthanized at 48th week. Rats exposed to doxorubicin had a severe clinical, and histopathological cardiomyopathy with depressed myocardial activity of cytochrome c-oxidase (COX, 26% of controls), reduced expression of the mtDNA-encoded COX II subunit, decreased mtDNA copy numbers (46% of controls), and high levels of malondialdehyde and superoxide (787% of controls). All parameters were highly correlated with myocardial damage. Both DOXO-EMCH groups did not differ from controls with regard to clinical symptomatology, mortality and mitochondrial enzymes, although the myocardia of the high-dose group had slightly increased histopathological abnormalities, depressed mtDNA copies (74% of controls) and elevated superoxide levels (347% of controls). Doxorubicin-exposed hearts and to a lesser extent the myocardia of both DOXO-EMCH groups contained mtDNA-deletions. In summary both DOXO-EMCH doses were superior over doxorubicin with respect to clinical and histopathological evidence of cardiomyopathy, myocardial COX-activity, COX II expression, mtDNA-content, mtDNA mutation loads and superoxide production in rats.

Development of elastin-like polypeptide for thermally targeted delivery of doxorubicin

The chemotherapeutic drug doxorubicin (Dox) is widely used as an antitumor agent in hematological malignancies and solid tumors. However, one of the limitations of its clinical use is that systemic administration of an effective dose of Dox results in nonselective cardiac toxicity and myelosuppression. In order to minimize this nonspecific toxicity, Elastin-like polypeptide (ELP) was examined for its ability to serve as a macromolecular carrier for thermally targeted delivery of Dox. The ELP-based doxorubicin delivery vehicle (Tat-ELP-GFLG-Dox) consists of: (1) a peptide derived from the HIV-1 Tat protein to facilitate its cellular uptake, (2) ELP to allow thermal targeting, and (3) the lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer and a cysteine residue conjugated to a thiol reactive doxorubicin derivative. Cytotoxicity of Tat-ELP-GFLG-Dox in MES-SA uterine sarcoma cells was enhanced 20-fold when aggregation of ELP was induced with hyperthermia. The ELP delivered doxorubicin displayed a cytoplasmic distribution and induced temperature dependent caspase activation.

Accumulation of γ-globin mRNA and induction of irreversible erythroid differentiation after treatment of CML cell line K562 with new doxorubicin derivatives

Human chronic myelogenous leukemia (CML) cell line K562 can be chemically induced to differentiate and express embryonic and fetal globin genes. In this study, the effects of doxorubicin (DOX), an inducer of K562 cell erythroid differentiation, with those of epidoxorubicin (EDOX) as well as newly synthesized derivatives of both drugs (DOXM, DOXH, and EDOXM) on cell growth and differentiation were compared. Our results revealed that DOX, EDOX and their derivatives caused irreversible differentiation of K562 cells into more mature hemoglobin-containing cells. This phenomenon was linked to time-dependent inhibition of cell proliferation. Considering the impact of the structure of newly synthesized anthracyclines on their cellular activity, our data clearly indicated that among tested anthracyclines DOXM, a morpholine derivative of DOX exerted the highest antiproliferative and differentiating activity. An increase of γ-globin mRNA level caused both by high transcription rate and by mRNA stabilization, as well as an enhancement of expression but not activity of erythroid transcription factor GATA-1 were observed. Therefore, a high level of hemoglobin-containing cells in the presence of DOXM resulted from transcriptional and post-transcriptional events on γ-globin gene regulation. The same morpholine modification introduced to EDOX did not cause, however, similar effects on cellular level. Characterization of new powerful inducers of erythroid differentiation may contribute to the development of novel compounds for pharmacological approach by differentiation therapy to leukemia or to β-globin disorder, β-thalassemia.

Identification of Doxorubicin and an Imine Derivative from Liquid and Solid Samples Utilizing Liquid Chromatography

Doxorubicin is an anthracycline glycoside used to treat a variety of cancer conditions from breast cancer to Wilms' tumor. The appearance of doxorubicin resistance has initiated studies of doxorubicin derivatives. The formation of the doxorubicin imine derivative is shown with means of identification by HPLC. Both the parent doxorubicin and imine derivative can be extracted from aqueous samples into 2‐pentanol or 2‐methyl‐2‐butanol by utilizing aggressive salting‐out techniques. Both forms of doxorubicin are bright red in color. A mixture of methanol/water or straight ethanol can be used to solubilize from solid samples. Determination of molecular properties indicated the imine group substantially increases lipophilicity and decreases aqueous solubitlity. Doxorubicin and its imine derivative are separated using Alltech Altima 18C 5µ column, UV/Vis detection (200 nm), and a mobile phase of methanol:water (80%/20%) at 1.0 mL/minute. Utilizing non‐destructive detection allows the collection of the eluted drug. The resolution achieved was 1.81 minutes, with 784 plates for doxorubicin separation, and 6834 plates for the imine derivative. The lipophilic substituent constant of the imine group is 3.48, indicating increased lipophilicity. The imine derivative is stable for weeks when stored dry at −10°C.

An Intramolecular Cyclization Reaction Is Responsible for the in Vivo Inefficacy and Apparent pH Insensitive Hydrolysis Kinetics of Hydrazone Carboxylate Derivatives of Doxorubicin

Contradictory reports concerning the pH sensitive hydrolysis kinetics of certain hydrazone carboxylates of doxorubicin have appeared in this journal (Kaneko et al., Bioconjugate Chem. 1991, 2, 133. Padilla De Jesús et al., Bioconjugate Chem. 2002, 13, 453). Since the pH stability of the drug-carrier linkage in macromolecular prodrugs has a significant bearing on pharmacological efficacy, the hydrolysis kinetics of low molecular weight and polymeric doxorubicin hydrazone carboxylates were therefore reinvestigated. As observed previously, the conjugates readily release native doxorubicin at pH 5. Unexpectedly, in neutral buffer the hydrazone carboxylate conjugates do not release native doxorubicin, but instead rapidly release a doxorubicin derivative substituted at C-9 by 3,6-dihydro-1,3,4-oxadiazin-2-one with first-order kinetics (t(1/2) = 2.5 h). The proposed intramolecular cyclization reaction involving doxorubicin's C-14 hydroxyl and the carboxylate-substituted hydrazone rationalizes the seemingly anomalous hydrolysis kinetics seen for hydrazone carboxylate linked doxorubicin, and provides a possible explanation for the poor antitumor activity exhibited by polymer-doxorubicin conjugates utilizing this specific type of linkage.

Targeted chemotherapy with cytotoxic bombesin analogue AN-215 can overcome chemoresistance in experimental renal cell carcinomas: Keller G, Schally AV, Nagy A, Halmos G, Baker B, Engel JB, Endocrine, Polypeptide, and Cancer Institute, Veterans Affairs Medical Center, New Orleans, LA

Multidrug resistance (MDR) mediated by membrane transporters, such as P-glycoprotein (MDR-1) and MDR-associated protein (MRP), remains a challenge in the therapy of renal cell carcinoma (RCC). Chemotherapy targeted to hormone receptors may provide a new approach to overcome chemoresistance. The cytotoxic analogue of bombesin/gastrin-releasing peptide (GRP), AN-215, consists of a superactive derivative of doxorubicin, AN-201, which is linked to a bombesin analogue carrier: RC-3094. The authors examined the expression of bombesin/GRP receptors in 3 human RCC cell lines (A-498, ACHN. and 786–0) by using reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis and radioligand-binding assays. They also evaluated the effects of AN-215 and its cytotoxic radical AN-201 in the same RCC models in vivo, and they studied the effects of AN-215 and AN-201 on the expression levels of MDR-1 and subtype 1 of MRP (MRP-1) by using real-time PCR. A N-215 significantly (P < 0.05) inhibited the growth of A-498, ACHN, and 786–0 RCC xenografted into nude mice by 59.2–67.6%, whereas the cytotoxic radical AN-201 alone had no significant antitumor effects. The efficacy of AN-215 was independent of the expression patterns of MDR-1 and MRP-1 in these RCC cell lines. The induction of MDR-1 by AN-215 was similar (Experiment 2) or weaker (Experiment 1) compared with AN-201. Both AN-215 and AN-201 caused only a minor induction of MRP-1. The current findings indicated that targeted chemotherapy with cytotoxic bombesin/GRP analogue AN-215 can inhibit the growth of RCC, providing a new treatment modality for patients with advanced RCC.

Recent Patents Therapeutic Agents for Cancer

Cancer is one of the most dreaded diseases with a complex pathogenesis, which threats human life greatly. Multidisciplinary scientific investigations are making best efforts to combat this disease and put to the identification of novel anticancer agents. Patent anticancer agents registered in China are therefore increasing dramatically during the past ten years, which will be reviewed briefly in this article. platinum complexes anthracycline analogs (including doxorubicin derivatives) quinoline analogs podophyllotoxins analogs taxane analogs camptothecin (CPT) analogs.

Targeted cytotoxic bombesin analog AN-215 effectively inhibits experimental human breast cancers with a low induction of multi-drug resistance proteins

The cytotoxic analog of bombesin (BN)/gastrin releasing peptide (GRP) AN-215 consisting of 2-pyrrolinodoxorubicin (AN-201), a superactive derivative of doxorubicin linked to a bombesin analog carrier, displays a high affinity to BN/GRP receptors and can be targeted to tumors that express these receptors. We evaluated the antitumor effect and the toxicity of AN-215 in 5 human breast cancer cell lines xenografted into nude mice. In addition, we measured the mRNA expression of multi drug resistance protein 1 (MDR-1), multi drug resistance related protein 1 (MRP-1) and breast cancer resistance protein (BCRP) by real-time PCR analysis after treatment with AN-215. All five cell lines expressed BN/GRP receptors, and AN-215 significantly (P < 0.05) inhibited tumor growth in all models, while its cytotoxic radical AN-201 had no significant effect in four models. In MX-1 tumors, AN-201 had a significantly weaker antitumor effect than AN-215. The effect of AN-215 was nullified by a blockade of BN/GRP receptors with a bombesin antagonist. Low or no induction of MDR-1, MRP-1 and BCRP occurred after treatment with AN-215. In conclusion, targeted chemotherapy with the cytotoxic BN/GRP analog AN-215 strongly inhibits breast cancers that express BN/GRP receptors and might provide a new treatment modality for mammary carcinoma.

Dipeptide-based highly potent doxorubicin antibody conjugates

Highly potent and novel derivatives of doxorubicin were linked to monoclonal antibodies (mAbs) for site-specific drug delivery. Drug linker 5 consisted of a dipeptide linker attached directly to the daunosamine nitrogen of the n-butyldiacetate doxorubicin derivative 2a. Upon hydrolysis of the peptide linker and acetate groups, the free daunosamine nitrogen is able to form the highly potent 2-pyrrolinodoxorubicin ( 3a). The second approach involved the use of an oxazolidine carbamate ( 13) to mask an activating aldehyde group until proteolytic hydrolysis releases 3a. Both drug linkers were shown to be substrates for the lysosomal enzyme cathepsin B. Each molecule was conjugated to the mAbs c1F6 (anti-CD70) and cAC10 (anti-CD30) to give potent drug conjugates against renal cell carcinoma and anaplastic large cell lymphoma cell lines, respectively. The activities were immunologically selective, since antigen negative cell lines were much less sensitive to treatment with the drug conjugates. The approaches described here for attaching highly potent doxorubicin derivatives to mAbs are novel and allow for control of drug stability while covalently bound to the delivery agent.

Targeted chemotherapy with cytotoxic bombesin analogue AN‐215 can overcome chemoresistance in experimental renal cell carcinomas

Multidrug resistance (MDR) mediated by membrane transporters, such as P-glycoprotein (MDR-1) and MDR-associated protein (MRP), remains a challenge in the therapy of renal cell carcinoma (RCC). Chemotherapy targeted to hormone receptors may provide a new approach to overcome chemoresistance. The cytotoxic analogue of bombesin/gastrin-releasing peptide (GRP), AN-215, consists of a superactive derivative of doxorubicin, AN-201, which is linked to a bombesin analogue carrier: RC-3094. The authors examined the expression of bombesin/GRP receptors in 3 human RCC cell lines (A-498, ACHN. and 786-0) by using reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis and radioligand-binding assays. They also evaluated the effects of AN-215 and its cytotoxic radical AN-201 in the same RCC models in vivo, and they studied the effects of AN-215 and AN-201 on the expression levels of MDR-1 and subtype 1 of MRP (MRP-1) by using real-time PCR. A N-215 significantly (P < 0.05) inhibited the growth of A-498, ACHN, and 786-0 RCC xenografted into nude mice by 59.2-67.6%, whereas the cytotoxic radical AN-201 alone had no significant antitumor effects. The efficacy of AN-215 was independent of the expression patterns of MDR-1 and MRP-1 in these RCC cell lines. The induction of MDR-1 by AN-215 was similar (Experiment 2) or weaker (Experiment 1) compared with AN-201. Both AN-215 and AN-201 caused only a minor induction of MRP-1. The current findings indicated that targeted chemotherapy with cytotoxic bombesin/GRP analogue AN-215 can inhibit the growth of RCC, providing a new treatment modality for patients with advanced RCC.