Encapsulation Of Doxorubicin Research Articles

Abstract 5511: Multifunctional CREKA peptide conjugated lipid nanocarriers of synergistically acting Noscapine and Doxorubicin for breast cancer therapy

Abstract Estrogen receptor negative (ER−) breast cancers (∼40%) are clinically aggressive with poor clinical outcome. Combination of different mechanism based antimicrotubular Noscapine and DNA intercalating Doxorubicin may lead to additive/synergistic activity against ER− breast cancer. Clinical utility of safer oral Noscapine (poor bioavailability and short half life) and Doxorubicin (cardiotoxicity and myelosuppression) has been limited. Encapsulation of Noscapine and Doxorubicin in nanocarriers whose surface is modified with pegylated CREKA peptide (MNCs) will significantly deliver nanocarriers to tumors by homing to tumor stroma, vessel wall and thereby releasing both drugs in controlled manner to exert anticancer activity. The purpose of this study was to encapsulate synergistically acting Noscapine and Doxorubicin in nanocarrier and modify the nanocarrier surface with CREKA and evaluate for anticancer activity in MDA-MB 231 ER− breast cancer cells. Isobolographic method and TUNEL assay were used to study Noscapine (10, 20 and 30 µM) and Doxorubicin interaction in MDA-MB 231 cells. For preparation of MNCs, Noscapine and Doxorubicin (molar ratio of 1:400) were dissolved in lipophilic phase composed of Miglyol (6% w/v), Compritol (3% w/v) and DOGS-NTA-Ni (0.2 % w/v). Lipophilic phase was poured to aqueous phase containing Polaxamer 188 (1.2 % w/v in water) and subjected to high-pressure homogenization to yield DOGS-NTA-Ni engrafted nanocarriers. Six-Histidine tagged PEG (1K)-CREKA (0.01-0.04 % w/v) was incubated with nanocarriers for 30 min for conjugation of DOGS-NTA-Ni engrafted nanocarrier with Histidine tagged peptide to yield MNCs. MNCs were characterized for size, drug release, antiproliferative and clot binding efficiency. Noscapine and Doxorubicin alone showed IC50 of 42 ± 4 µM and 0.25 ± 0.02 µM against MDA-MB cells respectively. In presence of Noscapine solution (20 µM), the IC50 of Doxorubicin solution was reduced to 0.05 µM (5-fold). Further, the combination Index values (< 0.6) and higher apoptotic cells (P 96 % of encapsulation and controlled release of both drugs (8 hr∼15 % and 48 hr∼ 60 %). A significantly (P < 0.01) higher binding of MNCs (CREKA concentration 0.045 % w/v) to the clotted plasma proteins showed the targeting ability of nanocarriers. MNCs showed similar IC50 values (20 µM Noscapine + 0.05 µM Doxorubicin) to that of solution combination. In conclusion, Noscapine acts synergistically with Doxorubicin and combination delivery of Noscapine and Doxorubicin using nanocarriers conjugated with CREKA showed significant increase in cytotoxicity with controlled drug release and significant binding efficiency. Multifunctional nanocarriers can effectively inhibit breast cancer and may reduce limitations associated with chemotherapy. 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 5511.

Magnetically Sensitive Alginate-Templated Polyelectrolyte Multilayer Microcapsules for Controlled Release of Doxorubicin

Magnetically sensitive alginate-templated polyelectrolyte multilayer microcapsules were successfully synthesized by a novel process combining emulsification and layer-by-layer self-assembly techniques. The as-synthesized microcapsules (2.67 μm in diameter) were superparamagnetic with a saturation magnetization of 14.2 emu·g−1, which contained approximately 30 wt % maghemite nanoparticles. The drug (doxorubicin) encapsulation efficiency was 56.4% and loading content was 3.5%. The in vitro release behavior under a high-frequency magnetic field (HFMF) indicated that the applied HFMF accelerated significantly the drug release from the microcapsules, which might be related to the microcapsules’ magnetic properties. Moreover, the in vitro cytotoxicity of doxorubicin-loaded alginate-templated polyelectrolyte multilayer microcapsules and the doxorubicin released from the microcapsules were investigated.

Reduction‐Sensitive Self‐Aggregates as a Novel Delivery System

AbstractMethoxy PEG amine with molecular weight of 5k and ε‐caprolactone with molecular weight of 1 960 were conjugated to a peptide comprising three cysteine residues. The shift of peak molecular weight and narrow molecular weight distribution in GPC trace without any noticeable shoulder as well as 1H NMR analysis confirmed the successful synthesis of the copolymer. A modified O/W dialysis system was employed to prepare self‐aggregates having the size around 210 nm. During the dialysis, stabilized aggregates were obtained by intermolecular disulfide bonds via oxidation. Critical aggregate concentration (CAC) of the copolymer was determined as 0.07 mg · mL−1 and disulfide‐stabilized self‐aggregates remained stable regardless of the concentration without displaying CAC. Doxorubicin‐loading amount and efficiency was 8.7 and 26.0%, respectively. Release profile of doxorubicin below CAC at 37 °C showed a sustained release and the addition of D,L‐dithiothreitol (DTT) after 24 h triggered a burst release of doxorubicin. Intermolecular disulfide bonds via oxidation stabilized the polymeric aggregates even in the diluted condition similar to that in the bloodstream and addition of DTT destabilized the aggregates to burst encapsulated doxorubicin in the reductive condition.magnified image

A phase I/II study of neoadjuvant liposomal doxorubicin, paclitaxel, and hyperthermia in locally advanced breast cancer

Purpose: The prognosis for locally advanced breast cancer (LABC) patients continues to be poor, with an estimated five-year survival of only 50–60%. Preclinical data demonstrates enhanced therapeutic efficacy with liposomal encapsulation of doxorubicin combined with hyperthermia (HT). Therefore this phase I/II study was designed to evaluate the safety and efficacy of a novel neoadjuvant combination treatment of paclitaxel, liposomal doxorubicin, and hyperthermia.Materials and methods: Eligible patients received four cycles of neoadjuvant liposomal doxorubicin (30–75 mg/m2), paclitaxel (100–175 mg/m2), and hyperthermia. They subsequently underwent either a modified radical mastectomy or lumpectomy with axillary node dissection followed by radiation therapy and then eight cycles of CMF (cyclophosphamide, methotrexate, 5-fluorouracil) chemotherapy.Results: Forty-seven patients with stage IIB-III LABC were enrolled and 43 patients were evaluable. Fourteen patients (33%) had inflammatory breast cancer. Combined (partial + complete) clinical response rate was 72% and combined pathological response rate was 60%. Four patients achieved a pathologically complete response. Sixteen patients were eligible for breast-conserving surgery. The cumulative equivalent minutes (CEM 43) at T90 (tenth percentile of temperature distribution) was significantly greater for those with a pathological response. Four-year disease-free survival was 63% (95% CI, 46%–76%) and the four-year overall survival was 75% (95% CI, 58–86%).Conclusions: Neoadjuvant therapy using paclitaxel, liposomal doxorubicin and hyperthermia is a feasible and well tolerated treatment strategy in patients with LABC. The thermal dose parameter CEM 43 T90 was significantly correlated with attaining a pathological response.

In vitro evaluation of novel polymer-coated magnetic nanoparticles for controlled drug delivery

Previously uncharacterized poly( N-isopropylacrylamide-acrylamide-allylamine)-coated magnetic nanoparticles (MNPs) were synthesized using silane-coated MNPs as a template for radical polymerization of N-isopropylacrylamide, acrylamide, and allylamine. Properties of these nanoparticles such as size, biocompatibility, drug loading efficiency, and drug release kinetics were evaluated in vitro for targeted and controlled drug delivery. Spherical core-shell nanoparticles with a diameter of 100 nm showed significantly lower systemic toxicity than did bare MNPs, as well as doxorubicin encapsulation efficiency of 72%, and significantly higher doxorubicin release at 41°C compared with 37°C, demonstrating their temperature sensitivity. Released drugs were also active in destroying prostate cancer cells (JHU31). Furthermore, the nanoparticle uptake by JHU31 cells was dependent on dose and incubation time, reaching saturation at 500 μg/mL and 4 hours, respectively. In addition, magnetic resonance imaging capabilities of the particles were observed using agarose platforms containing cells incubated with nanoparticles. Future work includes investigation of targeting capability and effectiveness of these nanoparticles in vivo using animal models. From the Clinical Editor In this paper, previously uncharacterized magnetic nanoparticles were synthesized using silane-coated MNPs as a template for radical polymerization of N-isopropylacrylamide, acrylamide, and allylamine. Various properties of these nanoparticles were evaluated in vitro for targeted drug delivery.

Anti-Tumour Effect of a Chitosan Delivery System for Doxorubicin

Used against a variety of tumours, doxorubicin (DOX), causes multiple side-effects in patients, especially in high doses required to control tumour growth. A drug delivery system (DDS) was developed to deliver DOX. Through DOX encapsulation into chitosan DDS, novel DOX microparticles (DMPs) were formed. Multiple optimisation steps produced DMPs which caused tumour cell death. Treatment of mice bearing tumours with DMP decreased tumour growth and spread, with no visible side-effects. There are plans to evaluate this delivery vehicle more closely towards clinical development and testing.

Temperature-sensitive liposomes for doxorubicin delivery under MRI guidance

Local drug delivery of doxorubicin holds promise to improve the therapeutic efficacy and to reduce toxicity profiles. Here, we investigated the release of doxorubicin and [Gd(HPDO3A)(H 2O)] from different temperature-sensitive liposomes for applications in temperature-induced drug delivery under magnetic resonance image guidance. In particular, two temperature-sensitive systems composed of DPPC:MPPC:DPPE-PEG2000 (low temperature-sensitive liposomes, LTSL) and DPPC:HSPC:cholesterol:DPPE-PEG2000 (traditional temperature-sensitive liposomes, TTSL) were investigated. The co-encapsulation of [Gd(HPDO3A)(H 2O)], a clinically approved MRI contrast agent, did not influence the encapsulation and release of doxorubicin. The LTSL system showed a higher leakage of doxorubicin at 37 °C, but a faster release of doxorubicin at 42 °C compared to the TTSL system. Furthermore, the rapid release of both doxorubicin and the MRI contrast agent from the liposomes occurred near the melting phase transition temperature, making it possible to image the release of doxorubicin using MRI.

The performance of doxorubicin encapsulated in chitosan–dextran sulphate microparticles in an osteosarcoma model

Osteosarcoma (OS) is the most common primary bone cancer affecting children and adolescents. It is potentially debilitating and fatal due to pulmonary metastasis. A common management strategy, chemotherapy, has a 10-year disease-free survival of approximately 60%. However, a targeted approach to OS tumor inhibition is still lacking, calling for improved management strategies. A frontline drug for OS, doxorubicin (DOX), causes multiple side-effects (example myelosuppression, heart failure, hepatic toxicity, alopecia) in patients, especially in high doses required to control tumor growth. A drug delivery system (DDS) was developed to deliver DOX specifically to tumor sites. Through DOX encapsulation into chitosan DDS via the complex coacervation method with dextran sulphate, novel DOX microparticles (DMPs), with a DOX loading content of more than 99%, were formed. Multiple optimisation steps produced DMPs which caused OS cell death through apoptosis, necrosis and autophagic cell death. Treatment of mice bearing orthotopic OS with DMP decreased tumor volume, decreased bone lysis, and reduced secondary metastasis to the lungs. DMP-treated mice also maintained their weight and did not appear to suffer from any visible side-effects such as heart failure or dry skin. Thus, DMP may prove to be a useful DDS platform clinically provided further studies are performed to rigorously validate this technology.

Poly(ε-caprolactone)/Poly(ethylene glycol)/Poly(ε-caprolactone) Nanoparticles: Preparation, Characterization, and Application in Doxorubicin Delivery

Biodegradable poly(epsilon-caprolactone)/poly(ethylene glycol) (PCL/PEG) copolymer nanoparticles showed potential application in drug delivery systems. In this article, monodisperse poly(epsilon-caprolactone)/poly(ethylene glycol)/poly(epsilon-caprolactone) (PCL/PEG/PCL, PCEC) nanoparticles, approximately 40 nm, were prepared by solvent extraction method using acetone as the organic solvent. These PCL/PEG/PCL nanoparticles did not induce hemolysis in vitro and did not show toxicity in vitro or in vivo. The prepared PCL/PEG/PCL nanoparticles were employed to load doxorubicin by a pH-induced self-assembly method. In vitro release study indicated that doxorubicin release from nanoparticles at pH 5.5 was faster than that at pH 7.0. The encapsulation of doxorubicin in PCL/PEG/PCL nanoparticles enhanced the cytotoxicity of doxorubicin on a C-26 cell line in vitro. Meanwhile, compared with free doxorubicin, doxorubicin in nanoparticles could more efficiently treat mice bearing subcutaneous C-26 tumors. The doxorubicin-loaded PCL/PEG/PCL nanoparticles might be a novel doxorubicin formulation for cancer therapy.

Pegylated liposomal doxorubicin in ovarian cancer

The encapsulation of doxorubicin in a pegylated liposomal matrix led to a reformulated agent with a different toxicity profile and improved clinical utility. Liposomal doxorubicin is devoid of the cardiac toxicity associated with doxorubicin, but is associated with predictable muco-cutaneous toxicity. The liposomal formulation leads to improved delivery to the target tumor tissue, allowing enhanced uptake by cancer cells. These properties translate into clinical utility in recurrent ovarian cancer as demonstrated by phase II and III trials, this proven clinical efficacy leading to FDA approval in second-line therapy for ovarian cancer. New combinations with cytotoxics, in particular with carboplatin, have demonstrated an acceptable toxicity profile and clinical utility in platinum-sensitive ovarian cancer. A favorable toxicity profile renders liposomal doxorubicin an ideal partner for combination regimens with other cytotoxics, and more recently with biological agents. Such combinations are the subject of ongoing clinical trials.

Simultaneous analysis of liposomal doxorubicin and doxorubicin using capillary electrophoresis and laser induced fluorescence

A method based on a capillary electrophoresis with laser induced fluorescence detection was developed and validated for simultaneous separation of doxorubicin (DOX) and liposomal encapsulated DOX. The separation was accomplished using a fused silica capillary (60 cm in total length, 75 μm I.D.) and potassium phosphate buffer [12.5 mM, pH 7.4] as the running buffer. The effect of sample preparation conditions on maintaining liposomal integrity was also investigated. The limit of detection for DOX was 0.1 μg/ml and the precision and accuracy of CE/LIF method was within the ranges of FDA guidelines. The validated method was successfully used to quantify DOX in human plasma using a direct injection of a 4-fold dilution of spiked liposomal DOX in human plasma.

Formulation and characterization of doxorubicin nanovesicles

Ascorbyl palmitate formed vesicles in combination with cholesterol and a negatively charged lipid (dicetyl phosphate). Niosomes were prepared by the film hydration method, followed by sonication, in which aqueous doxorubicin solution (in phosphate buffer saline pH=7.4) was encapsulated in aqueous regions of lipid layers. Vesicle’s formation was confirmed by either scanning electron microscopy images or observation of unsonicated vesicles by light microscope. The percent efficiencies of doxorubicin encapsulation of the different formulations were determined by fluorescence spectrophotometry. Cholestrol content in niosomes did not exhibit any relation to vesicle size, zeta potential, percent entrapment, or release rate. Differential scanning calorimetric data of pure lipids, vesicle dispersion, and mixture of lipids confirmed the formation of niosomes.

Usefulness of hexamethylenetetramine in combination with chemotherapy using free and pegylated liposomal doxorubicin in vivo, referring to the effect on quiescent cells

SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all intratumor proliferating (P) cells. They received hexamethylenetetramine (HMTA) either once intraperitoneally or continuously subcutaneously together with chemotherapy using intraperitoneally administered free doxorubicin (DXR) or intravenously injected pegylated liposomal doxorubicin (PLD). One hour after the free DXR loading or 24 h after the PLD loading, the response of intratumor quiescent (Q) cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. The response of the total (P + Q) tumor cell population was determined from the tumors not treated with BrdU. Encapsulation of DXR into pegylated liposomes significantly enhanced cytotoxicity, especially in Q cells. HMTA, especially when administered continuously, efficiently increased the sensitivity to DXR, particularly in Q cells. The increase in sensitivity on the continuous rather than single administration of HMTA was a little clearer in the total cell population than in Q cells. DXR's encapsulation into pegylated liposomes and combination with HMTA, particularly when administered continuously, apparently reduced the difference in sensitivity to free DXR between the total and Q cell populations. In terms of the tumor cell-killing effect as a whole, including Q cells, the encapsulation of DXR into pegylated liposomes and combination with HMTA, particularly through continuous administration, are very promising, taking into account that HMTA has been used clinically.

Self-assembled oligopeptide nanostructures for co-delivery of drug and gene with synergistic therapeutic effect

In this study, oligopeptide amphiphile containing three blocks of amino acids, Ac-(AF)6-H5-K15-NH2 (FA32), were synthesized and evaluated as carriers for co-delivery of drug and gene. Doxorubicin (DOX), luciferase reporter gene, and p53 gene were used as a model drug and genes. The peptide amphiphile self-assembled into cationic core–shell nanostructures (i.e. micelles), with a CMC value of around 0.042mg/mL, estimated by fluorescent spectroscopy technique. FA32 nanostructures had an average size of 102±19nm, and a zeta potential of 22.8±0.2mV. These nanostructures had a high capacity for DOX encapsulation, with a DOX loading level of up to 22%. In addition, DOX release from the micelles was sustained without obvious initial burst. DOX-loaded micelles were effectively taken up by HepG2 cells, with an IC50 of 1.8mg/L for DOX-loaded FA32, which was higher than that of free DOX (0.25mg/L). In addition, FA32 micelles condensed DNA efficiently to form small complexes with net positive charge on the surface. In vitro gene transfection studies showed that FA32 induced comparable gene expression level to polyethylenimine. Co-delivery of drug and gene using FA32 micelles was demonstrated via confocal imaging, luciferase expression in the presence of DOX, and synergy in cytotoxic effect between p53 gene and DOX. It was shown that through simultaneous delivery of both p53 gene and DOX using FA32 micelles, an increase in p53 mRNA expression level as well as end point cytotoxicity towards HepG2 cells was achieved. FA32 micelles, therefore, have a great potential in delivering hydrophobic anticancer drug and gene simultaneously for improved cancer therapy.

Doxorubicin pharmacokinetics following two different liposomal formulations (CAELYX™ vs MYOCET™) in advanced breast cancer patients.

Abstract Abstract #2128 Background: Liposomal encapsulation of Doxorubicin (DOX) was designed to minimize healthy tissue distribution by altering pharmacokinetics (PK) thus reducing cardiotoxicity while preserving antitumour efficacy. Study objective was to compare the PK behaviour of the two main liposomal formulations of DOX, i.e. pegylated (Caelyx, C) versus non pegylated (Myocet, M), in a single center study including 17 MBC patients, all Anthracyclin pretreated with additional risk faktors.
 Methods: Cohort 1 consisted of 10pts (mean age 62, 49-77) treated by C as 1HR infusion of 50mg/m²/4 wks. Plasma samples were collected at timepoints min 0, 30, 60, 120, 180 and d1, 7, 14, 28. In cohort 2, 7 pts (mean age 58, 51 – 70) received M 75mg/m² 1H Infusion / 3wks, plasma levels determined at min 0, 30, 60, 90, 120, 180 and d1, 2, 3. Total amount of DOX was determined following the 1st infusion respectively, analytic procedure included solid phase extraction quantification by HPLC and PK analysis by Win Nonlin Pro. Doxorubicinol was detected after M only.
 Results: in Cohort1 after C, mean Cmax of 6.8 µg/ml surprisingly occured at a mean tmax of 2.3 d, log conc time curve showed linear decrease of DOX conc suggesting continuous release into the tissue (mean conc d 7 3.3, d 14 1.0, d 21 0.3, d 28 0.09 µg/ml) . No metabolites were found, AUC (does not represent reliable drug exposure and bioavailability of DOX) expressed as µg/ml.d was 54.6. In contrast AUClast of M was calculated as 35µg ml.H and mean Cmax by 7.5 µg/ml at infusion end. DOXol was measured in all M treated patients at a slower metabolisation rate and late appearance compared to conventional DOX. t/2 el of C was 6 times longer than that of M (HR 90 vs 15). Differences in plasma disposition and PK of M vs C are depicted in table 1 and compared to pooled data of conventional DOX derived from former studies (50 mg/m² 30min inf, n=40). Cltot of conv. DOX was 10 fold higher than M and 500 fold higher than C, Vss about 12 times that of M and 70 times that of C. These PK differences underlines a different toxicity profile between PEGL (C, PPE syndrome) and NPEGL (M, myelotox, nausea), both reducing cardiac tox by avoiding peak plasma levels due to prolonged circulation time.
 
 Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2128.

Doxorubicin-Loaded Polymeric Micelles Based on Amphiphilic Polyphosphazenes with Poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) and Ethyl Glycinate as Side Groups: Synthesis, Preparation and In Vitro Evaluation

To construct novel doxorubicin-loaded polymeric micelles based on polyphosphazenes containing N-isopropylacrylamide copolymers and evaluate their various properties as well as in vitro anticancer effect. These amphiphilic graft polyphosphazenes PNDGP were synthesized via thermal ring-opening polymerization and subsequent two-step substitution reaction of hydrophilic and hydrophobic side groups. Micellization behavior in an aqueous phase was confirmed by fluorescence technique, DLS and TEM. Doxorubicin (DOX) was physically loaded into micelles by dialysis or O/W emulsion method. CLSM and MTT test were applied to observe intracellular drug distribution and determine cytotoxicity of drug-loaded micelles on Hela and HepG2 cells lines, respectively. A series of PNDGPs with controlled substitution ratios were obtained. Poly(NIPAm-co-DMAA) can act as hydrophilic segments in micellular system since its LCST was over 37 degrees C when PNIPAm was copolymerized with DMAA. The CMC value was decreased with the increase of Glyet content. In addition, more hydrophobic group content introduced into the polymer would facilitate DOX encapsulation into the micelle. DOX-loaded micelle could achieve comparative cytotoxicity as free drug via endocytosis and succedent drug release into cytoplasm of cancer cells. The results suggest that these polymers might be used as potential carriers of hydrophobic anti-tumor drug for cancer therapy.

Liposome-Encapsulated Doxorubicin Is Highly Active in Patients with B- as Well as T/NK-Cell Lymphomas and Cardiac Comorbidity or Older Age

Conventional anthracyclines may have severe dose-limiting side effects in patients with pre-existing cardiac comorbidity, particularly if they are elderly. In routine practice, this will lead to dose reductions or preclude the use of anthracyclines. This may result in lower remission and shorter lymphoma-free or overall survival rates. Non-pegylated liposome-encapsulated doxorubicin (Myocet®) in combination with Rituximab, cyclophosphamide, vincristine and prednisone (R-COMP) has been shown to be effective with a low cardiotoxicity profile in patients with diffuse large B-cell lymphoma (DLBCL) (Rigacci et al., Hematol Oncol. 2007; 25:198–203). We have treated 37 lymphoma patients with pre-existing cardiac disorders or elderly patients with reduced general condition who were considered ineligible or high risk for anthracycline containing therapy. Thirty seven patients with various histologies were included: 20 DLBCL, 9 T-/NK cell lymphomas, 3 follicular lymphomas, 2 post-transplant lymphoproliferative disease (PTLD), 2 chronic lymphocytic leukemia (CLL) and 1 multiple myeloma (MM). Reasons for the use of liposome-encapsulated doxorubicin were: pre-existing cardiac disorders (N=19) including myocardial infarction, cardiomyopathy, reduced left ventricular ejection fraction LVEF (N=6), elevated B-type natriuretic peptide (BNP) levels (N=10, median 680 pg/ml), aortic valve problems, or heart transplantation; older age (N=12; median age 78 years); previous therapy with anthracyclines (N=3); other comorbity or reduced general condition (N=4). Twenty-nine patients (78%) were previously untreated. The following combination regimens were given: R-COMP (N=25), COMP (N=11), and BMD (N=1). The median number of Myocet® containing cycles administered was 5. For various reasons, liposome encapsulated doxorubicin was reduced to a dose of less than 50mg/m2 in 19 patients. High remission rates were observed for this poor risk population: Complete remission rates were 70% (14/20) for DLBCL and 57% (4/7 evaluable patients) for T-/NK cell lymphomas. 31 patients (83%) are still alive at 2–9 months after the end of therapy. Five patients died from progressive disease, one of unknown reasons. Thirteen patients with DLBCL are in continuous CR 3 months after the end of therapy (range 1–8 months). No major cardiac or gastrointestinal toxicity was observed. Of note, paravasation of liposome-encapsulated doxorubicin occurred in 2 patients, but resulted in mild inflammation only without tissue damage. Hematologic toxicity was comparable to that of conventional anthracycline containing regimens: CTC Grade 3 or 4 toxicity for leukocytopenia/neutropenia occurred in 22 patients (59%). Nineteen patients (51%) received G-CSF for primary or secondary prophylaxis in at least one cycle of treatment. We conclude that liposome-encapsulated doxorubicine is highly active in combination chemotherapy for B-cell lymphomas with low cardiac toxicity in patients with pre-existing cardiac disorders or older age. Moreover, we herewith report for the first time efficacy in T/NK-cell lymphomas in this high-risk population. A randomized study evaluating cardiac effects of substitution of conventional doxorubicin by liposome-encapsulated doxorubicin in combination chemotherapy (R-CHOP vs. R-COMP) is currently being conducted by the Austrian Cooperative Group on Cancer Drug Therapy (AGMT).

Interest of liposomal doxorubicin as a radiosensitizer in malignant glioma xenografts

Malignant glioma patients have a life expectancy reduced to about 15 months despite aggressive surgery, radiotherapy (RT), and chemotherapy. Doxorubicin has shown a marked cytotoxic effect against malignant glioma cells in vitro. The brain exposure to this drug is, however, hindered by the blood-brain barrier. Encapsulation of doxorubicin in liposomal carriers has been shown to reduce toxicities and to improve brain tumors exposure to doxorubicin. In this study, we evaluated the radiosensitizing properties of a nonpegylated liposomal doxorubicin (Myocet, MYO) on two subcutaneous (U87 and TCG4) and one intracranial (U87) malignant glioma models xenografted on nude mice. Doxorubicin biodistribution was assessed by a high-performance liquid chromatography method. Antitumor efficacy was investigated by tumor volume measurements and mice survival determination. We showed that (i) encapsulation of doxorubicin ensured a preferential deposition of doxorubicin in tumoral tissue in comparison with free doxorubicin; (ii) doxorubicin accumulated in both subcutaneous and intracranial tumors during repeated injections of MYO and this accumulation was linked to the potentiation of RT efficacy on two subcutaneous models; (iii) MYO was unable to improve the antitumoral efficacy of RT on an intracranial glioma model. Finally, this study emphasizes the importance of performing preclinical studies on models closer as possible of human tumors and localization to be more predictive of therapeutic effects observed in humans.

Pharmacokinetics of intra-arterial applied liposomal encapsulated doxorubicin in liver metastases or hepatocellular carcinoma: A phase I study

13523 Background: Doxorubicin has been one of the most widely used anti-neoplastic agents as a single-agent systemic therapy for HCC. Doxorubicin is also used in locoregional therapy approaches. Caelyx (Doxil) is a doxorubicin formulation of polyethylene glycol-coated liposomes. For this new form of doxorubicin, a superior pharmacokinetic profile over that of free doxorubicin is postulated. Blood flow reduction by micro embolization with degradable starch microspheres (DSM; e.g. Spherex, EmboCept) is effective in HCC and liver metastasis. Purpose: Evaluation of the pharmacokinetics (PK) of the encapsulated as well as the released fraction of doxorubicin in addition to the determination of the maximum tolerated dose of intra-arterially applied liposomal encapsulated doxorubicin (Caelyx) in HCC and liver metastases in combination with DSM. Methods: All patients with histologically proven HCC or liver metastases had shown progressive disease when previously treated with systemic chemotherapy. Six dose escalation steps from 15 mg/m2 to 100 mg/m2 were performed with two or three patients in each dose group. Each patient received one single application of Caelyx/DSM. The distribution of Caelyx depended on the tumor size in the left and right liver lobe. For the quantification of Caelyx, doxorubicin, and doxorubicinol, a validated RP-HPLC method with fluorometric detection (excitation wavelength: 470 nm, emission wavelength: 550 nm) was used. Results: PK was calculated for 13 out of 14 treated patients. The mean (terminal) half life for Caelyx was calculated as 3 days, mean residence time about 5 days, the Vss 1.5 l, clearance was determined to be less than 1 ml/min. The increase in peak plasma concentration as well as AUC was linear between 15 and 100 mg/m2 Caelyx. The MTD was reached at a single dose of 100 mg/m2 due to WHO- grade 3 myelosuppression in two patients without further SAE. Conclusions: These first results indicate that hepatic intra arterial chemotherapy with Caelyx in combination with DSM is well tolerated and achieved encouraging responses in patients with liver metastases or HCC. The main PK parameter for Caelyx in combination with DSM did not significantly differ from those obtained after systemic intravenous infusion. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Essex Pharma GmbH, PharmaCept GmbH

Development of novel polymeric micellar drug conjugates and nano-containers with hydrolyzable core structure for doxorubicin delivery

Novel micelle-forming poly(ethylene oxide)- block-poly(ε-caprolactone) (PEO- b-PCL) block copolymers bearing doxorubicin (DOX) side groups (PEO- b-P(CL-DOX)) on the PCL block were synthesized. Prepared block copolymers were characterized, assembled to polymeric micellar drug conjugates and assessed for the level of DOX release at pH 7.4 and pH 5.0 using a dialysis membrane to separate released and conjugated drug. The possibility for the degradation of PCL backbone for PEO- b-P(CL-DOX) micelles was investigated using gel permeation chromatography. Micelle-forming DOX conjugate did not show any signs of DOX release at 37 °C within 72 h of incubation at both pHs, but revealed signs of poly(ester) core degradation at pH 5.0. In further studies, PEO- b-PCL micelles bearing benzyl, carboxyl or DOX groups in the core were also used as micellar nano-containers for the physical encapsulation of DOX, where maximum level of drug-loading and control over the rate of DOX release was achieved by polymeric micelles containing benzyl groups in their core, i.e., PEO- b-poly(α-benzylcarboxylate-ε-caprolactone) (PEO- b-PBCL) micelles. The in vitro cytotoxicity of chemically conjugated DOX as part of PEO- b-P(CL-DOX) and physically encapsulated DOX in PEO- b-PBCL against B16F10 murine melanoma cells was assessed and compared to that of free DOX. Consistent with the results of in vitro release study, cytotoxicity of micellar PEO- b-P(CL-DOX) conjugate (IC 50 of 3.65 μg/mL) was lower than that of free and physically encapsulated DOX in PEO- b-PBCL (IC 50 of 0.09 and 3.07 μg/mL, respectively) after 24 h of incubation. After 48 h of incubation, the cytotoxicity of conjugated DOX (IC 50 of 0.50 μg/mL) was still lower than the cytotoxicity of free DOX (IC 50 of 0.03 μg/mL), but surpassed that of physically encapsulated DOX in PEO- b-PBCL (IC 50 of 1.54 μg/mL). The results point to a potential for PEO- b-P(CL-DOX) and PEO- b-PBCL as novel polymeric micellar drug conjugates and nano-containers bearing hydrolyzable cores for DOX delivery.