Doxorubicin Derivative Research Articles

Abstract 4440: Anti-tumor activity of a thermally responsive polypeptide-doxorubicin conjugate in a syngeneic mouse breast cancer model

Abstract A thermally responsive macromolecular carrier based on elastin-like polypeptide (ELP) was conjugated to (6-maleimidocaproyl) hydrazone derivatives of doxorubicin (Dox) via three terminal cysteine residues on ELP. ELPs undergo phase transition at a critical temperature known as the inverse transition temperature (Tt). At temperatures below Tt, ELPs remain soluble in aqueous solutions; however, at temperatures above Tt, they aggregate and separate from solution. By changing the molecular composition of ELP, which influences its transition temperature, we have genetically engineered an ELP molecule that is soluble at physiological temperature and aggregates in response to mild hyperthermia at 42°C. The ELP-drug conjugate can therefore be actively targeted to the tumor region by the application of localized heat, and passively targeted through the enhanced retention and permeability effect, making it less likely to be toxic to normal tissues. In this study, ELP was modified at the N-terminus by the addition of the SynB1 cell penetrating peptide to enhance intratumor and intracellular delivery. The doxorubicin prodrug contains a pH-sensitive hydrazone linker that is cleaved in the acidic environment of endosomes/lysosomes allowing a controlled intracellular release of the drug. The antitumor activity of the thermally responsive polypeptide-doxorubicin conjugate, SynB1-ELP1-Dox3 was evaluated in a mouse breast cancer syngeneic model. Tumor bearing animals were intravenously treated with either free Dox or SynB1-ELP1-Dox3 at 5 mg Dox equivalent/kg body weight for 3 alternate days. In the heat treatment group, hyperthermia was applied immediately after injection using an infrared light emitting diode device. Tumor growth was monitored up to 14 days after the initial treatment. Hyperthermia enhanced the antitumor activity of SynB1-ELP1-Dox3 by almost 2-fold, which was comparable to free Dox. Relative to the pre-treatment weight, mice treated with SynB1-ELP1-Dox3 showed no weight loss compared to 5% weight loss seen in mice treated with free Dox. The drug-free carrier SynB1-ELP1 as well as the thermally insensitive control, SynB1-ELP2-Dox3 did not exhibit any anti-tumor effect either in the presence or absence of hyperthermia indicating that hyperthermia enhanced potency of SynB1-ELP1-Dox3 is specific to the phase transition of ELP1. HPLC was used to evaluate the plasma clearance and biodistribution of SynB1-ELP1-Dox3 with and without heat and was compared to free drug. Additional in vivo studies at higher doses of SynB1-ELP1-Dox3 to examine the optimal antitumor efficacy as well as the reduction in cardiac toxicity are underway to validate the therapeutic potential of this thermally responsive doxorubicin conjugate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4440. doi:10.1158/1538-7445.AM2011-4440

Fatty acyl amide derivatives of doxorubicin: Synthesis and in vitro anticancer activities

Doxorubicin is extensively used in anticancer therapy. Doxorubicin is highly hydrophilic, has short half-life, and its use is associated with severe side effects at high doses. Fatty acyl amide derivatives of doxorubicin were synthesized with the expectation to improve the lipophilicity and anticancer activity of the drug. The lipophilicity was enhanced with the increase in chain length of fatty acyl moiety. Conjugation of 4′-amino group with fatty acids through an amide bond reduced the anticancer activity in leukemia, breast, ovarian, and colon cancer cell lines, suggesting that the presence of free amino group is required for anticancer activity of doxorubicin. Dodecanoyl-doxorubicin derivative was consistently the most effective among the synthesized derivatives and inhibited the proliferation of colon (HT-29) and ovarian (SK-OV-3) cancer cells by 64% and 58%, respectively, at a concentration of 1 μM after 96 h incubation.

INNO-206 (DOXO-EMCH), an Albumin-Binding Prodrug of Doxorubicin Under Development for Phase II Studies

The (6-maleimidocaproyl)hydrazone derivative of doxorubicin (INNO-206, formerly DOXO-EMCH) is a prodrug of the anticancer agent doxorubicin which is selectively bound to the cysteine-34 position of endogenous albumin within a few minutes after intravenous administration planned for 2011. Preclinically as well as clinically, the albuminbound form of INNO-206 has a large AUC, a small volume of distribution and low clearance compared to doxorubicin, uptake in solid tumors being mediated by the pathophysiology of tumor tissue, characterized by angiogenesis, hypervasculature, a defective vascular architecture, and an impaired lymphatic drainage. The prodrug contains an acid-sensitive hydrazone linker allowing doxorubicin to be released either extracellularly in the slightly acidic environment often present in tumor tissue or intracellularly in acidic endosomal or lysosomal compartments after cellular uptake of the albumin conjugate by the tumor cell. INNO-206 shows significantly superior antitumor efficacy over free doxorubicin in a spectrum of preclinical tumor models. In a phase I study, INNO-206 showed a good safety profile at doses up to 260 mg/m2 doxorubicin equivalents. Although not the primary end point of the phase I study, INNO-206 was able to induce tumor regressions in breast cancer, small cell lung cancer and sarcoma. Phase II studies against gastric cancer, pancreatic cancer and sarcoma are planned for the end of 2010.

Assessment of late cardiotoxicity of pirarubicin (THP) in children with acute lymphoblastic leukemia

Pirarubicin (tetrahydropyranyl-adriamycin: THP) is a derivative of doxorubicin with reportedly less cardiotoxicity in adults. However no studies of cardiotoxicity in children treated with THP have been reported. This study was performed to assess the THP-induced cardiotoxicity for children with acute lymphoblastic leukemia (ALL). This study comprised 61 asymptomatic patients aged from 7.6 to 25.7 years old. Median follow-up time after completion of anthracycline treatment was 8.1 years (range: 1.7-12.5). The cumulative dose of THP ranged from 120 to 740 mg/m(2) with a median of 180 mg/m(2) . Patients underwent electrocardiogram (ECG), echocardiography, the 6-min walk test (6MWT), and measurements of serum brain natriuretic peptide (BNP) before and after exercise. All subjects showed normal left ventricular function assessed by echocardiography. Ventricular premature contraction in Holter ECG and reduced exercise tolerance in the 6MWT were detected in 2/46 (3.3%) and 5/41(12.2%), respectively. Abnormal BNP levels were detected in 6/60 (10%) both before and after exercise. The cumulative dose of THP was significantly correlated with BNP levels after exercise (r = 0.27, P = 0.03), but not with any other cardiac measurements. Further analysis revealed that subjects with a high cumulative dose ≧300 mg/m(2) had significantly higher BNP levels after exercise compared with subjects with a low cumulative dose <300 mg/m(2) (P = 0.04). No significant cardiac dysfunction was detected in long-term survivors who received THP treatment. The use of post-exercise BNP level to indicate high cardiotoxicity risk should be verified by further study.

Abstract P5-10-13: Phase I Dose Escalation Study of Pirarubicin in Combination with Cyclophosphamide in Breast Cancer

Abstract Background: Doxorubicin containing combination chemotherapy regimens are widely used for treatment of breast and other cancers. However, these regimens are associated with significant toxicities including myocardialdysfunction and alopecia. Analogues of doxorubicin are being developed to reduce these side effects. Pirarubicin is a more lipophilic derivative of doxorubicin, with a higher uptake rate of cells, lower cardiotoxicity and better antitumor efficacy in preclinical models. Purpose: We conducted a single-institution phase I clinical trial to determine the maximum-tolerated dose (MTD) and define the toxic effects and recommended dose (RD) of pirarubicin in combination with cyclophosphamide in patients with breast cancer. Patients and Methods: Patients who had received prior anthracycline therapy were excluded. Cohorts of three patients with breast cancer were treated with escalating doses of pirarubicin (40 to 70 mg/m2) intravenously administered every three weeks in combination with cyclophosphamide (60 mg/m2) for 4 or more cycles. Results: Eleven patients of stage I/II operable breast cancer received a total of 46 cycles of pirarubicin and cyclophosphamide as post-operative adjuvant chemotherapy. The most frequently reported treatment-related grade 2 adverse events were constipation (36%) and nausea (27%). There were no grade 3/4 events. Grade 2 leukocytopenia and grade 2 fatigue were dose-limiting at 70 mg/m2, the maximum-tolerated dose was 60 mg/m2. Grade 2 alopecia was reported in 60 and 70 mg/m2 pirarubicin group. Conclusion: At the MTD of 60 mg/m2 every 3 weeks, pirarubicin in combination with cyclophosphamide was associated with mild, reversible toxicity. The recommended phase II dose is pirarubicin 50 mg/m2 and cyclophosphamide 60 mg/m2 on day 1 every 21 days. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-10-13.

Targeted core-crosslinked polymeric micelles with controlled release of covalently entrapped doxorubicin

Doxorubicin (DOX) is clinically applied in cancer therapy, but its use is associated with dose limiting severe side effects. Core-crosslinked biodegradable polymeric micelles composed of poly(ethylene glycol)-b-poly(N-(2-hydroxypropyl) methacrylamide-lactate) (mPEG-b-p(HPMAm-Lacn)) diblock copolymers have shown prolonged circulation in the blood stream upon intravenous administration and enhanced tumor accumulation through the enhanced permeation and retention (EPR) effect. However a (physically) entrapped anticancer drug (paclitaxel) was previously shown to be rapidly eliminated from the circulation, likely because the drug was insufficiently retained in the micelles. To fully exploit the EPR effect for drug targeting, a DOX methacrylamide derivative (DOX-MA) was covalently incorporated into the micellar core by free radical polymerization. The structure of the doxorubicin derivative is susceptible to pH-sensitive hydrolysis, enabling controlled release of the drug in acidic conditions (in either the intratumoral environment and/or the endosomal vesicles). 30e40% w/w of the added drug was cova- lently entrapped, and the micelles with covalently entrapped DOX had an average diameter of 80 nm. The entire drug payload was released within 24 h incubation at pH 5 and 37 � C, whereas only around 5% release was observed at pH 7.4. DOX micelles showed higher cytotoxicity in B16F10 and OVCAR-3 cells compared to DOX-MA, likely due to cellular uptake of the micelles via endocytosis and intracellular drug release in the acidic organelles. The micelles showed better anti-tumor activity than free DOX in mice bearing B16F10 melanoma carcinoma. The results presented in this paper show that mPEG-b-p(HPMAm- Lacn) polymeric micelles with covalently entrapped doxorubicin is a system highly promising for the targeted delivery of cytostatic agents.

Down-regulation of the Nucleotide Excision Repair gene XPG as a new mechanism of drug resistance in human and murine cancer cells

BackgroundDrug resistance is one of the major obstacles limiting the activity of anticancer agents. Activation of DNA repair mechanism often accounts for increase resistance to cancer chemotherapy.ResultsWe present evidence that nemorubicin, a doxorubicin derivative currently in clinical evaluation, acts through a mechanism of action different from classical anthracyclines, requiring an intact nucleotide excision repair (NER) system to exert its activity. Cells made resistant to nemorubicin show increased sensitivity to UV damage. We have analysed the mechanism of resistance and discovered a previously unknown mechanism resulting from methylation-dependent silencing of the XPG gene. Restoration of NER activity through XPG gene transfer or treatment with demethylating agents restored sensitivity to nemorubicin. Furthermore, we found that a significant proportion of ovarian tumors present methylation of the XPG promoter.ConclusionsMethylation of a NER gene, as described here, is a completely new mechanism of drug resistance and this is the first evidence that XPG gene expression can be influenced by an epigenetic mechanism. The reported methylation of XPG gene could be an important determinant of the response to platinum based therapy. In addition, the mechanism of resistance reported opens up the possibility of reverting the resistant phenotype using combinations with demethylating agents, molecules already employed in the clinical setting.

Core-crosslinked polymeric micelles with controlled release of covalently entrapped doxorubicin

Doxorubicin (DOX) is clinically applied in cancer therapy, but its use is associated with dose limiting severe side effects. Core-crosslinked biodegradable polymeric micelles composed of poly(ethylene glycol) -b-poly[ N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG- b-p(HPMAm-Lac n)) diblock copolymers have shown prolonged circulation in the blood stream upon intravenous administration and enhanced tumor accumulation through the enhanced permeation and retention (EPR) effect. However a (physically) entrapped anticancer drug (paclitaxel) was previously shown to be rapidly eliminated from the circulation, likely because the drug was insufficiently retained in the micelles. To fully exploit the EPR effect for drug targeting, a DOX methacrylamide derivative (DOX-MA) was covalently incorporated into the micellar core by free radical polymerization. The structure of the doxorubicin derivative is susceptible to pH-sensitive hydrolysis, enabling controlled release of the drug in acidic conditions (in either the intratumoral environment and/or the endosomal vesicles). 30–40% w/w of the added drug was covalently entrapped, and the micelles with covalently entrapped DOX had an average diameter of 80 nm. The entire drug payload was released within 24 h incubation at pH 5 and 37 °C, whereas only around 5% release was observed at pH 7.4. DOX micelles showed higher cytotoxicity in B16F10 and OVCAR-3 cells compared to DOX-MA, likely due to cellular uptake of the micelles via endocytosis and intracellular drug release in the acidic organelles. The micelles showed better anti-tumor activity than free DOX in mice bearing B16F10 melanoma carcinoma. The results presented in this paper show that mPEG- b-p(HPMAm-Lac n) polymeric micelles with covalently entrapped doxorubicin is a system highly promising for the targeted delivery of cytostatic agents.

In vitro and in vivo evaluation of tumor targeting styrene‐maleic acid copolymer‐pirarubicin micelles: Survival improvement and inhibition of liver metastases

Pirarubicin is a derivative of doxorubicin with improved intracellular uptake and reduced cardiotoxicity. We have prepared a micellar formulation of pirarubicin using styrene-maleic acid copolymer (SMA) of mean molecular weight of 1.2 kDa, which exhibits a mean diameter of 248 nm in solution. Being a macromolecule, SMA-pirarubicin micelles exhibit excellent tumor targeting capacity due to the enhanced permeability and retention (EPR) effect. Here we report the antitumor activity of SMA-pirarubicin micelles on human colon and breast cancer cell lines in vitro, and a murine liver metastasis model in vivo. Metastatic tumor microvasculature, necrosis, apoptosis, proliferation, and survival were also investigated using immunohistochemistry for Ki-67, active caspase-3, and CD34, respectively. Drug cytotoxicity in vitro was assessed using MTT (3-[4,5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide) assay. In vivo, SMA-pirarubicin was administered at 100, 150, or 200 mg/kg (pirarubicin equivalent). Tumor microvasculature was also assessed using scanning electron microscopy. Styrene-maleic acid copolymer (SMA)-pirarubicin micelles were toxic against human colorectal and breast cancer cells in vitro. IC(50) was at or below 1 muM, free pirarubicin equivalent. In vivo, SMA-pirarubicin at 100 mg/kg reduced tumor volume by 80% and achieved a survival rate of 93% at 40 days after tumor inoculation. Styrene-maleic acid copolymer (SMA)-pirarubicin micelles demonstrated potent antitumor activity in this liver metastases model, contributing to prolonged survival. Histological examination of tumor nodules showed significant reduction and proliferation of tumor cells (>90%). The present results suggest that investigation of the effect of multiple dosing at later time points to further improve survival is warranted.

Synergistic Action of Doxorubicin Bound to the Polymeric Carrier Based on N-(2-Hydroxypropyl)methacrylamide Copolymers through an Amide or Hydrazone Bond

The cytostatic effects of polymeric conjugates based on N-(2-hydroxypropyl)methacrylamide copolymers (PHPMA) and containing doxorubicin bound through amide and hydrazone bonds (mixed conjugates) were compared with the cytostatic effects of monoconjugates containing drug bound through an amide or hydrazone bond. One group of mixed conjugates was formed from two comonomers containing doxorubicin bound to the methacryloyl group through a spacer and an amide (DOX(AM)) or hydrazone (DOX(HYD)) bond via copolymerization with HPMA. A second group of mixed conjugates was formed from two different interconnected HPMA copolymers, one containing DOX(AM) and the other DOX(HYD), forming a high-molecular-weight branched structure. The third mixed polymeric system was a simple mixture of monoconjugates DOX(AM)-PHPMA and DOX(HYD)-PHPMA. Simultaneous treatment with all mixed forms of the polymeric derivatives of doxorubicin significantly increased antitumor efficacy after application of monoconjugates, suggesting a synergizing effect that could be used in designing new doxorubicin-containing therapeutic systems.

Abstract LB-37: In vitro and in vivo studies of novel acid-sensitive prodrugs derived from dendritic polyglycerol

Abstract Research in the development of tumor-specific nanocarriers is currently one of the major sources of innovation for improved anticancer chemotherapy. Due to their low degree of molecular weight dispersity, their flexible design and their biocompatible nature, dendritic polyglycerols (PGs) have a broad range of potential applications in medicine and pharmacology. Recently, we demonstrated that drugs could be bound to the thiolated hyperbranched polymers incorporating enzymatically cleavable spacers that were efficiently released by the target enzyme cathepsin B. These initially developed dug carriers with PGs, however, did not have an optimal molecular weight for EPR-mediated tumor targeting and remaining amino groups on the surface of the dendritic scaffold showed acute toxicity properties. As a consequence, we set out to improve the conjugation method and fine-tune the architecture of the polyglycerol drug conjugates for optimal tumor targeting. A logically consistent option was to mask remaining and potentially toxic amino groups with the polymer polyethylene glycol which would have the additional advantage of increasing the molecular weight of drug polyglycerol conjugate to &amp;gt; 40 kDa which is a pre-requisite for passive targeting. Thus, we developed a macromolecular prodrug derived from hyperbranched amino-bearing polyglycerol that in a first step was partially thiolated with iminothiolane followed by a Michael addition to the maleimide group of the acid-sensitive 6-maleimidocaproyl(hydrazone) derivative of doxorubicin, and in a second step the maleimide group of 2 and 5 kDa PEG chains were reacted with the remaining sulfhydryl groups. Thus, the drug was covalently attached to the hyperbranched polyglycerol core and surrounded by a “shell” of long, solubilizing PEG chains which radiate from the core. By varying the amount of iminothiolane, of doxorubicin prodrug and of PEG-maleimide, drug PG conjugates with different drug loading (1 to 10) were isolated. Analysis by dynamic light scattering measurements (DLS) and z-potential showed that average diameters were in the range of 12 to 16 nm and the conjugates showed a slightly negative surface charge. The in vitro release studies showed that the release of doxorubicin was minimal at pH 7.4 after 22 h (less than 10%), while at acidic pH half-lives were below 4 h. Although free doxorubicin was more active than the PG doxorubicin conjugates against A2780 ovarian carcinoma cells, in the respective xenograft model the pegylated PG doxorubicin conjugates with 5 to 10 bound molecules of doxorubicin showed a distinct increase in tolerability and antitumor efficacy in comparison to the parent drug producing excellent antitumor effects with complete and transient tumor remissions (at 24 mg/kg doxorubicin equivalents). Doxorubicin treated animals only showed a moderate antitumor effect at its optimal dose of 2 × 8 mg/kg. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-37.

Abstract 2653: Thermally targeted delivery of an acid-labile derivative of doxorubicin by elastin-like polypeptide

Abstract Doxorubicin (Dox) is an anthracycline used in the treatment of many types of cancer. It intercalates into the strands of DNA to inhibit the progression of topoisomerase II, stopping the cell from replicating. The side effects, however, are severe and include neutropenia, alopecia, nausea, myelosuppression, and cardiotoxicity. The drug is nonspecific; as a result, high doses are needed to treat cancer cells. These high doses are inadvertently harmful to normal cells. Therefore, there is a need for an efficient drug delivery system in order to deliver doxorubicin to the tumor site. Our lab has developed a macromolecular carrier that can provide this targeted delivery. This elastin-like polypeptide (ELP) is thermally responsive as it remains in solution at physiological temperature and aggregates upon heating at 42 °C. The N-terminus of ELP was modified with three different cell penetrating peptides (CPPs) - Bac, Tat, and SynB1. These peptides are derived from the bactenecin family of antimicrobial peptides, truncated version of the HIV-1 Tat protein, and porcine leukocytes, respectively. This study compared the efficiency of these three CPP-ELP carriers to deliver doxorubicin to the cells. ELP was attached to a hydrazone derivative of doxorubicin (DOXO) by conjugation of a 6-maleimidocaproyl moiety on the drug with a terminal cysteine residue on ELP. This linker allows the drug to be cleaved at acidic pH in the cell. The three CPP-ELP-DOXO conjugates had transition temperatures within the range of 38-42°C. All three carriers of DOXO inhibited MCF-7 breast cancer cell proliferation 2-fold more at 42 °C than at 37 °C, indicating that the hyperthermia induced phase transition of ELP does increase the local concentration of the drug. Inhibition of cell proliferation may be due to apoptosis, since apoptosis was enhanced 2.3-fold in MCF-7 cells when SynB1-ELP-DOXO aggregation was induced with hyperthermia. CPP-ELP-DOXO accumulated in cells to the same level as free Dox. Confocal imaging showed that when cells were treated with any of the three CPP-ELP-DOXO derivatives, Dox localized in the nucleus, similar to the free Dox. The results suggest that CPP-ELP-DOXO conjugates may provide a means to thermally target solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2653.

Interaction between double helix DNA fragments and the new antitumor agent sabarubicin, Men10755

Among the disaccharide derivatives of the antitumor anthracycline doxorubicin, sabarubicin (Men10755) is more active and less cytotoxic than doxorubicin. It showed a strong in vivo antitumor activity in all preclinical models examined, in conjunction with a better tolerability, and is now in phase II clinical trials.The interaction of sabarubicin and Men10749 (a similar disaccharide with a different configuration at C-4′ of the proximal sugar) with the hexanucleotides d(CGTACG)2 and d(CGATCG)2 was studied by a combined use of 2D-1H and 31P NMR techniques. Both 1H and 31P chemical shifts of imino protons and phosphates allowed to established the intercalation sites between the CG base pairs, as it occurs for other anthracyclines of the series. The dissociation rate constants (koff) of the slow step of the intercalation process were measured for Men10755 and Men10749, by NMR NOE-exchange experiments. The increase of koff , with respect of doxorubicin, showed that the intercalation process is significantly faster for both drugs, leading to an average residence time for sabarubicin into d(CGTACG)2 sixfold shorter than for doxorubicin. This could give account of both higher cytoplasmic/nuclear ratio and lower cellular uptake of sabarubicin in comparison with doxorubicin and accordingly of the lower cytotoxicity of these disaccharide analogues.A relevant number of NOE interactions allowed the structure of the complexes in solution to be derived through restrained MD calculations. NMR-DOSY experiments were performed with several drug/oligonucleotide mixtures in order to determine the structure and the dimension of the aggregates.

The Immunohistochemical Pattern of Indoleamine 2,3-Dioxygenase in Tumor Tissue Indicates the Prognosis of Patients with Diffuse Large B-Cell Lymphoma.

Abstract 2924Poster Board II-900 Introduction :Indoleamine 2,3-dioxygenase (IDO) is an enzyme that degrades the essential amino acid tryptophan along the kynurenine pathway. Pro-inflammatory cytokines, such as IFN-g, induce IDO during the inflammatory response in many human cell types. The induction of IDO is synergistic in the presence of TNF-a, IL-1 or IL-6, and might be mediated by a signaling pathway from NF-κB and/or MAPKs. Furthermore, some metabolites derived from tryptophan by IDO, such as L-kynurenine, block antigen-driven specific T-cell proliferation and induce T-cell death. Thus, IDO activity might play an important role in regulation of the immune response exerted by antigen presenting cells and also provide transformed cells with a potent tool to help escape from assault by the immune system. Indeed, we have previously reported that high serum L-kynurenine level is associated with poor prognosis of diffuse large B-cell lymphoma (DLBCL) (ASH 2008 abstract 2812). Here, we investigated the IDO expression of patients with DLBCL. Patients and methods :The study protocol comprised a prospective, consecutive entry design that was approved by our Institutional Review Board. We investigated 119 patients between December 2003 and June 2008 who were histologically diagnosed with DLBCL according to the WHO classification. We performed immunohistochemical (IHC) analysis for IDO expression by mouse anti-human IDO monoclonal antibody. Patients aged <70 y received 8 cycles of either R-CHOP or R-THP-COP therapy. Each regimen consisted of rituximab (R: 375 mg/m2), cyclophosphamide (CPA: 750 mg/m2), doxorubicin (DOX) or tetrahydropyranyl-adriamycin (THP; 50 mg/m2), vincristine (VCR; 1.4 mg/m2, maximal dose 2.0 mg), and prednisolone (PSL; 100 mg daily). The R-THP-COP regimen included THP, an anthracycline derivative of DOX. Patients aged ≥70 y received 6 cycles of R-CHOP or R-THP-COP therapy. The chemotherapy cycles were repeated at 14-day intervals in patients aged <70 y, and at 21-day intervals in patients aged ≥70 y. Patients with bulky disease received radiotherapy ranging from 30 to 40 Gy. Responses to treatment were categorized as defined by Cheson et al. Results :The median age was 65.2 year (range, 24 - 88 y) and the median follow-up was 22.9 month (range, 0.60 – 55.4 mo). The IDO expression patterns were classified into 3 categories; diffuse positive, focal positive and negative patterns. The diffuse positive IDO expression in tumor tissue was found in 38 cases (32%). The focal positive and negative expression of IDO was 16 cases (13.4%) and 65 cases (54.6%), respectively. The diffuse IDO positive cells were lymphoma cells and the focal IDO positive cells were dendritic cells (DC) confirmed by IHC analysis. The CR rates of patients with diffuse positive IDO expression, focal positive and negative were 55.3%, 62.5% and 83.1%, respectively (P<0.05). The 3-year overall survival rates for patients with diffuse positive, focal positive and negative were 49.8%, 66.3% and 81.4%, respectively (p=0.001). IDO expression was not significantly associated with the classification of germinal center (GC) type nor non-GC type. Discussion :A poor prognosis of patients with positive IDO expression might suggest that local immunity in tumor tissue is depressed by increasing L-kynurenine levels. Hence, IDO expression contributes to refractory to chemotherapy for DLBCL. Interestingly, expression pattern of IDO was significantly related with response to the treatment and prognosis of DLBCL. In conclusion, IDO activity might play an important role in DLBCL and the cells which express IDO are important for the response to treatment and prognosis of this malignancy. IDO, therefore, might be a candidate of therapeutic targets for DLBCL patients who are resistance to chemotherapy. Disclosures:No relevant conflicts of interest to declare.

3081 Reduced therapy-related fatigue in mice with nab-paclitaxel as compared with Cremophor-based paclitaxel

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.

Characterisation of the conjugate of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin with lactosaminated human albumin by 13C NMR spectroscopy

In order to improve the efficacy of doxorubicin (DOXO) in the treatment of hepatocellular carcinomas, the drug was conjugated with lactosaminated human albumin (L-HSA), a hepatotropic drug carrier. Conjugation was performed using the (6-maleimidocaproyl)hydrazone derivative of the drug (DOXO-EMCH). The maleimide group of DOXO-EMCH reacts with the aminoacidic residues of the carrier forming stable bonds, whereas the hydrazone bond is rapidly hydrolysed in the acidic endosomal and lysosomal compartments of the cells allowing the intracellular release of DOXO. To identify the amino acids of L-HSA involved in the bond with DOXO-EMCH, in the present study we synthesized this compound with the 2,3 carbon atoms of the maleimide moiety enriched in the 13C isotope and used this labelled DOXO-EMCH to prepare two types of L-HSA conjugate. Type I was prepared in analogy to those studied in the anticancer experiments using tris(2-carboxyethyl)phosphine (TCEP) to reduce l-cysteine disulfides and make the sulfhydryl groups available for the reaction with DOXO-EMCH; type II was synthesized omitting TCEP. By 13C NMR spectroscopy we could demonstrate that in type I conjugate cysteine was the only amino acid residue that reacted with DOXO-EMCH, whereas in type II conjugate lysine was the only amino acid in the reaction with DOXO-EMCH. When hydrolysed in an acidic medium to cleave the hydrazone bond, type I conjugate released only DOXO, whereas type II conjugate also released a derivative of the drug.

INNO-206, the (6-maleimidocaproyl hydrazone derivative of doxorubicin), shows superior antitumor efficacy compared to doxorubicin in different tumor xenograft models and in an orthotopic pancreas carcinoma model

The (6-maleimidocaproyl)hydrazone derivative of doxorubicin (INNO-206) is an albumin-binding prodrug of doxorubicin with acid-sensitive properties that is being assessed clinically. The prodrug binds rapidly to circulating serum albumin and releases doxorubicin selectively at the tumor site. This novel mechanism may provide enhanced antitumor activity of doxorubicin while improving the overall toxicity profile. Preclinically, INNO-206 has shown superior activity over doxorubicin in a murine renal cell carcinoma model and in breast carcinoma xenograft models. In this work, we compared the antitumor activity of INNO-206 and doxorubicin at their respective maximum tolerated doses in three additional xenograft models (breast carcinoma 3366, ovarian carcinoma A2780, and small cell lung cancer H209) as well as in an orthotopic pancreas carcinoma model (AsPC-1). INNO-206 showed more potent antitumor efficacy than free doxorubicin in all tumor models and is thus a promising clinical candidate for treating a broad range of solid tumors.

A Phase II Study of the Combination Chemotherapy Consisted of Rituximab and THP-COP Regimen as First-Line Treatment for Patients with Diffuse Large B-Cell Lymphoma

Background: A combination of rituximab and CHOP (R-CHOP) is currently the standard treatment for diffuse large B-cell lymphoma (DLBCL). We previously reported the utility of THP-COP regimen consisted of cyclophosphamide (CPA), pirarubicin (tetrahydropyranyl adriamycin: THP), vincristine (VCR), and prednisolone (PSL) in patients with DLBCL (Tsurumi H. et al. Hematol Oncol 2007). THP, a derivative of doxorubicin (DOX), is another anthracycline that has been reported to be less cardiotoxic than DOX. We conducted this phase II study to verify untreated patients with CD20 positive DLBCL.Patients and Methods: The primary objective was to assess the efficacy of R-THP-COP with response rates. Secondary objectives were to assess the overall survival and drug safety. The study was conducted with local ethics committee approval, and all patients gave written, informed consent prior to enrollment. Adverse effects were graded according to the National Cancer Institute Common Toxicity Criteria version 2.0. Over 18 were eligible for inclusion in the study if they had a diagnosis of untreated CD20 positive DLBCL. DLBCL was confirmed by biopsy. All patients had DLBCL at clinical stage (CS) II, III, or IV. Patients aged <70 (young patients) received 8 cycles of R-THP-COP therapy. The regimen consisted of R (375mg/m2), CPA (750mg/m2), THP (50mg/m2), VCR (1.4mg/m2, maximal dose 2.0mg), and PSL (100mg daily). Patients aged ≥70 (elderly patients) received 6 cycles of R-THP-COP therapy. The R-THP-COP chemotherapy cycles were repeated at 14-day intervals in patients aged <70, and the THP-COP chemotherapy cycles were repeated at 21-day intervals in patients aged ≥70. Patients with bulky disease received radiotherapy ranging from 30 to 40 Gy. Responses to treatment were categorized as defined by Cheson et al.Results: A total of 100 patients (55 males and 45 females) were enrolled in the study between December 2003 and December 2006. The median age was 59 years (range 24 – 69) in young patients (n = 48), and 75 years (range 70 – 83) in elderly patients (n = 52). 44 (92%) reached CR or CRu in young patients and 33 (63%) in elderly patients. Median follow-up was 29.4 months. The OS rate was 80.8 % after 3 years in young patients and 69.9% in elderly patients. The PFS rate was 74.7% after 3 years and 61.0% in elderly patients. OS rates did not differ significantly in accordance with IPI. There were 16 (35%), 35 (78%) and 7 (15%) patients who developed grade 3 or 4 anemia, neutropenia and thrombocytopenia in young patients, respectively. There were 12 (29%), 35 (85%) and 6 (14%) patients who developed grade 3 or 4 anemia, neutropenia and thrombocytopenia in elderly patients, respectively. Non-hematologic adverse effects were tolerable. Treatment regimen-related death was not observed in this study.Discussion: In this study, the efficacy, feasibility, and toxicity of the THP-COP regimen was not inferior to those previously reported for CHOP. The CR rate was 92%, this CR rate was not inferior to our previous study and other previous reported studies. About the survival rate, the 3-year OS in our R-THP-COP regimen for patients with DLBCL was equal to or higher than that of our previous study. We concluded that R-THP-COP regimen in not inferior to R-CHOP about curative effect at least. About the safety, it is not thought that the R-THPCOP regimen has more adverse events than the R-CHOP regimen either. The results of our clinical trial prove that it is worth performing the randomized trial of the R-THP-COP regimen and the R-CHOP regimen as phase III trial. We designed this phase III trial and the trial is ongoing.

Serum Concentration of L-Kynurenine Can Determine Clinical Outcome of Patients with Diffuse Large B-Cell Lymphoma Treated with R-CHOP

Background: Indoleamine 2,3-dioxygenase (IDO) exerts powerful immunomodulatory effects through its enzymatic activity that leads to catabolism of the essential amino acid L-tryptophan. Pro-inflammatory cytokines such as IFN-g induce IDO during the inflammatory response in many human cell types. The induction of IDO is synergistic in the presence of TNF-a, IL-1 or IL-6, and might be mediated by a signaling pathway from NF-kB and/or MAPKs. Furthermore, some metabolites derived from tryptophan generated by IDO such as L-kynurenine, block Ag-driven specific T-cell proliferation and induce T-cell death. Increments in IDO activity correlate with tumor progression in various malignancies. Therefore, IDO activity might play an important role in regulation of the immune response exerted by antigen presenting cells and also provide transformed cells with a potent tool to help escape from assault by the immune system. The activity of IDO can be estimated by measuring the serum concentration of kynurenine. Here, we investigated the relevance of L-kynurenine as a prognostic factor for DLBCL with R-CHOP therapy.Patients and methods: The study protocol comprised a prospective, consecutive entry design that was approved by our Institutional Review Board. We investigated 75 patients between December 2002 and March 2007 who were histologically diagnosed with DLBCL according to the WHO classification. We measured L-kynurenine by high-performance liquid chromatography using a spectrophotometric or a fluorescence spectrometric detector. Patients aged <70 y received 8 cycles of either R-CHOP or R-THP-COP therapy. Each regimen consisted of rituximab (R: 375 mg/m2), cyclophosphamide (CPA: 750 mg/m2), doxorubicin (DOX) or tetrahydropyranyl-adriamycin (THP; 50 mg/m2), vincristine (VCR; 1.4 mg/m2, maximal dose 2.0 mg), and prednisolone (PSL; 100 mg daily). The R-THP-COP regimen included THP, an anthracycline derivative of DOX. Patients aged ≥70 y received 6 cycles of R-THP-COP therapy, which is often applied to elderly patients with NHL. The regimen consisted of rituximab (R: 375 mg/m2), CPA (650 mg/m2), THP (40 mg/m2), VCR (1.4 mg/m2; maximal dose, 2.0 mg), and PSL (40 mg daily). The R-CHOP and R-THP-COP chemotherapy cycles were repeated at 14-day intervals in patients aged <70 y, and the THP-COP chemotherapy cycles were repeated at 21-day intervals in patients aged ≥70 y. Patients with bulky disease received radiotherapy ranging from 30 to 40 Gy. Responses to treatment were categorized as defined by Cheson et al.Results: The median age was 68 y (range, 24 – 82 y) and the median follow-up was 20.6 mo (range, 0.6 – 42.3 mo). The median serum L-kynurenine level was 1.575 mM (range, 0.537 – 9.588). We found no significant correlations between L-kynurenine and gender, LDH, or sIL-2R. However, L-kynurenine significantly correlated with age (r = 0.279, P <0.05) and a high serum level was significantly associated with advanced disease, and many extranodal involvements (P <0.05). W established the cut-off value of L-kynurenine at 1.5 mM. The CR rates of patients with L-kynurenine <1.5 and ≥1.5 mM were 80.6% and 64.1%, respectively (P <0.05). Other factors associated with a significantly worse outcome were many extranodal involvements, advanced clinical stage, B-symptoms, elevated sIL-2R, and unfavorable IPI. The 2-year OS rates for patients with L-kynurenine <1.5 and ≥1.5 mM were 89.2% and 44.8%, respectively (P <0.05).Discussion: The increase in serum L-kynurenine levels is thought to be caused by enhanced L-TRP catabolism, which inhibits tumoral local immunity. The result of a poor prognosis among patients with high L-kynurenine levels suggests that depressed local immunity due to enhanced IDO activity contributes to DLBCL becoming refractory to treatment.

STI571 and morpholine derivative of doxorubicin collaborate in inhibition of K562 cell proliferation by inducing differentiation and mitochondrial pathway of apoptosis

Differentiation therapy is considered as a supplementary approach to the currently applied treatments for leukemia. We have previously shown that a morpholine derivative of doxorubicin (DOXM) appeared to be a more efficient inducer of erythroid differentiation of K562 cells than the parent drug [Czyz, M., Szulawska, A., Bednarek, A.K., Duchler, M., 2005. Effects of anthracycline derivatives on human leukemia K562 cell growth and differentiation. Biochem. Pharmacol. 70, 1431–1442.; Szulawska, A., Arkusinska, J., Czyz, M., 2007. Accumulation of gamma-globin mRNA and induction of irreversible erythroid differentiation after treatment of CML cell line K562 with new doxorubicin derivatives. Biochem. Pharmacol. 73, 175–184.]. In the current study we used this compound in combination with STI571, a front-line drug in therapy of chronic myelogenous leukemia (CML), to evaluate possible benefits of the combined treatment on the cellular level. Using K562 cells, we analyzed the response of CML cells to low concentrations of DOXM when Bcr-Abl activity was reduced to various levels by its specific inhibitor, STI571. Differentiation was significantly enhanced with the combination of 150 nM STI571 and 100 nM DOXM as compared to the levels obtained with either drug alone. A higher concentration of STI571 was required to diminish Bcr-Abl activity to the level which was sufficient to stimulate apoptotic cell death pathway in K562. Apoptosis induced by 250 nM STI571 was markedly enhanced by DOXM in the combined treatment. Mitochondrial transmembrane potential dissipation and translocation of phosphatydylserine to the outer plasma membrane were increased by 50%. Our results clearly indicate that differentiation and apoptosis, both reducing cellular proliferation, could be substantially enhanced by the combined treatment. We provide experimental evidence implicating that the diversification of cellular effects obtained in the combined treatment employing non-toxic approaches to enhance efficacy of STI571 might be considered as an alternative therapeutic strategy against CML, especially for apoptosis-reluctant cells.