Dox Concentration Research Articles

Targeted delivery of doxorubicin using stealth liposomes modified with transferrin

Site-specific delivery of drugs and therapeutics can significantly reduce drug toxicity and increase the therapeutic effect. Transferrin (Tf) is one suitable ligand to be conjugated to drug delivery systems to achieve site-specific targeting, due to its specific binding to transferrin receptors (TfR), highly expressed on the surfaces of tumor cells. Stealth liposomes are effective vehicles for drugs, genes and vaccines and can be easily modified with proteins, antibodies, and other appropriate ligands, resulting in attractive formulations for targeted drug delivery. In this study, we prepared doxorubicin-loaded stealth liposomes (Tf-SL-DOX) by film dispersion followed by ammonium sulphate gradient method, then conjugated Tf to the liposome surface by an amide bound between DSPE–PEG 2000–COOH and Tf. The results of the intracellular uptake study indicated that Tf-modified SL was able to enhance the intracellular uptake of the entrapped DOX by HepG2 cells compared to SL-DOX. We studied tissue distribution and therapeutic effects of Free DOX, SL-DOX and Tf-SL-DOX in tumor-bearing mice and pharmacokinetics in rats. The pharmacokinetic behavior of Tf-SL-DOX in the plasma was closed to SL-DOX. Administration of Tf-SL-DOX to tumor-bearing mice could be used to deliver DOX effectively to the targeted site, significantly increasing DOX concentration in tumor and decreasing DOX concentration in heart and kidney. In summary, our study indicated that the Tf-coupled PEG liposomes (Tf-SL) could be as the targeted carriers to facilitate the delivery of the encapsulated anticancer drugs into tumor cells by receptor-mediated way.

Protective Effect of Memantine Against Doxorubicin Toxicity in Primary Neuronal Cell Cultures: Influence a Development Stage

One of the serious unwanted effects of the anthracycline anticancer drug doxorubicin (Dox, adriamycin) is its neurotoxicity, which can be evoked by the activation of extracellular (FAS/CD95/Apo-1) pathway of apoptosis in cells. Since memantine, a clinically used N-methyl-D: -aspartic acid (NMDA) receptor antagonist, shows antiapoptotic action in several models of neuronal cell damage, in this study we evaluated the effect of memantine on the cell death induced by Dox in primary neuronal cell cultures. First, we investigated the effect of different concentrations of Dox (0.1-5 microM) on mouse neocortical, hippocampal, striatal, and cerebellar neurons on 7- and 12-day in vitro (DIV). The 7 DIV neuronal cell cultures were more prone to Dox-induced cell death than 12 DIV cultures. The cerebellar neurons were the most resistant to Dox-induced apoptosis in comparison to neuronal cell cultures derived from the forebrain. Memantine (0.1-2 microM) attenuated the Dox-evoked lactate dehydrogenase release in 7 DIV neuronal cell cultures with no significant effect on 12 DIV cultures. The ameliorating effect of memantine on Dox-mediated cell death was also confirmed by an increase in cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. There was no effect of memantine on Dox-induced caspase-8 and -3 activity and Dox-evoked decrease in mitochondrial potential, although attenuation in the number of cells with apoptotic DNA fragmentation was observed. We also showed that the antiapoptotic effect of memantine in our model was NMDA receptor-independent, since two other antagonists of this receptor, MK-801 and AP-5, did not attenuate Dox-induced cell death. Furthermore, memantine did not influence the Dox-evoked increase in cytoplasmic Ca2+ level. The obtained data suggest developmental regulation of both, the Dox-mediated neurotoxicity and efficacy of memantine in alleviating the Dox-induced cell damage in neuronal cell cultures. Moreover, this neuroprotective effect of memantine seems not to be dependent on caspase-3 activity and on the antagonistic action on NMDA receptor.

Facile biosynthesis, separation and conjugation of gold nanoparticles to doxorubicin

Particle shape and size determine the physicochemical and optoelectronic properties ofnanoscale materials, including optical absorption, fluorescence, and electric andmagnetic moments. It is thus desirable to be able to synthesize and separatevarious particle shapes and sizes. Biosynthesis using microorganisms has emergedas a more ecologically friendly, simpler, and more reproducible alternative tochemical synthesis of metal and semiconductor nanoparticles, allowing the generationof rare forms such as triangles. Here we show that the plant pathogenic fungusHelminthosporum solani, when incubated with an aqueous solution of chloroaurate ions,produces a diverse mixture of extracellular gold nanocrystals in the size range from2 to 70 nm. A plurality are polydisperse spheres, but a significant number arehomogeneously sized rods, triangles, pentagons, pyramids, and stars. The particles can beseparated according to their size and shape by using a sucrose density gradient in atabletop microcentrifuge, a novel and facile approach to nanocrystal purification.Conjugation to biomolecules can be performed without further processing, asillustrated with the smallest fraction of particles which were conjugated to theanti-cancer drug doxorubicin (Dox) and taken up readily into HEK293 cells. Thecytotoxicity of the conjugates was comparable to that of an equivalent concentration ofDox.

Prevention of MDR development in leukemia cells by micelle-forming polymeric surfactant

Doxorubicin (Dox) incorporated in nanosized polymeric micelles, SP1049C, has shown promise as monotherapy in patients with advanced esophageal carcinoma. The formulation contains amphiphilic block copolymers, Pluronics, that exhibit the unique ability to chemosensitize multidrug resistant (MDR) tumors by inhibiting P-glycoprotein (Pgp) drug efflux system and enhancing pro-apoptotic signaling in cancer cells. This work evaluates whether a representative block copolymer, Pluronic P85 (P85) can also prevent development of Dox-induced MDR in leukemia cells. For in vitro studies murine lymphocytic leukemia cells (P388) were exposed to increasing concentrations of Dox with/without P85. For in vivo studies, BDF1 mice bearing P388 ascite were treated with Dox or Dox/P85. The selected P388 cell sublines and ascitic tumor-derived cells were characterized for Pgp expression and functional activity (RT-PCR, Western Blot, rhodamine 123 accumulation) as well as Dox resistance (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). The global gene expression was determined by oligonucleotide gene microarrays. We demonstrated that P85 prevented development of MDR1 phenotype in leukemia cells in vitro and in vivo as determined by Pgp expression and functional assays of the selected cells. Cells selected with Dox in the presence of P85 in vitro and in vivo exhibited some increases in IC 50 values compared to parental cells, but these values were much less than IC 50 in respective cells selected with the drug alone. In addition to mdr1, P85 abolished alterations of genes implicated in apoptosis, drug metabolism, stress response, molecular transport and tumorigenesis. In conclusion, Pluronic formulation can prevent development of MDR in leukemia cells in vitro and in vivo.

Improved cytotoxicity of doxorubicin by enhancing its nuclear delivery mediated viananosized micelles

For antitumor drugs with an intracellular action site in the nucleus, effectiveinternalization of the drugs into cancer cells and accumulation in the nucleusshould be the determinant step for high antitumor activity. We synthesized a novelchitosan derivative by grafting stearic acid onto chitosan. The derivative can formself-aggregated micelles with about 50 nm size in the aqueous medium, and then can load apoorly soluble antitumor drug (doxorubicin, DOX) with high entrapment efficiencyand drug loading. DOX release from the micelles was retarded significantly asa result of the encapsulation of the micelles. DOX concentration in nuclei wasincreased significantly via the transport of the micelles. Consequently, cytotoxicityof DOX loaded micelles was improved sharply due to the accumulation of thedrug in its intracellular action site. The present micelles are a promising carriercandidate for effective therapy of antitumor drugs with the action site in thenucleus.

Morphological alterations induced by doxorubicin on H9c2 myoblasts: nuclear, mitochondrial, and cytoskeletal targets

Doxorubicin (Dox) is a very potent antineoplastic agent used against several types of cancer, despite a cumulative cardiomyopathy that reduces the therapeutic index for treatment. H9c2 myoblast cells have been used as an in vitro model to study biochemical alterations induced by Dox treatment on cardiomyocyte cells. Despite the extensive work already published, few data are available regarding morphological alterations of H9c2 cells during Dox treatment. The purpose of the present work was to evaluate Dox-induced morphological alterations in H9c2 myoblasts, focusing especially on the nuclei, mitochondria, and structural fibrous proteins. Treatment of H9c2 cell with low concentrations of Dox causes alterations in fibrous structural proteins including the nuclear lamina and sarcomeric cardiac myosin, as well as mitochondrial depolarization and fragmentation, membrane blebbing with cell shape changes, and phosphatidylserine externalization. For higher Dox concentrations, more profound alterations are evident, including nuclear swelling with disruption of nuclear membrane structure, mitochondrial swelling, and extensive cytoplasm vacuolization. The results obtained indicate that Dox causes morphological alterations in mitochondrial, nuclear, and fibrous protein structures in H9c2 cells, which are dependent on the drug concentration. Data obtained with the present study allow for a better characterization of the effects of Dox on H9c2 myoblasts, used as a model to study Dox-induced cardiotoxicity. The results obtained also provide new and previously unknown targets that can contribute to understand the mechanisms involved in the cardiotoxicity of Dox.

Monitoring incorporation, transformation and subcellular distribution of N- l-leucyl-doxorubicin in uterine sarcoma cells using capillary electrophoretic techniques

Previous reports have demonstrated that N- l-leucyl-doxorubicin (LeuDox) is less toxic than its parent drug, Dox, but the underlying causes of this reduced toxicity have yet to be fully elucidated. In this study, the incorporation of LeuDox into (i) the MES-SA human uterine sarcoma cell line and (ii) its Dox resistant counterpart, MES-SA/Dx5 cell line and the subsequent transformation of LeuDox into Dox and its subcellular distribution, were investigated by micellar electrokinetic chromatography with laser-induced fluorescence detection (MEKC-LIF). In both cell lines the cellular uptakes of Dox and LeuDox were similar at equimolar doses, while the percent transformation of LeuDox into Dox in MES-SA/Dx5 cells was about twice as great as its transformation in MES-SA cells, which is beneficial for reaching Dox cytotoxic levels in this resistant cell line. When both cells lines were treated with IC 35 concentrations of either Dox and LeuDox, the intracellular Dox amounts were 6-fold higher in the resistant cell line than in the sensitive cell line, suggesting that other cellular processes play a role in the cytotoxicity of Dox in the resistant cell line. The amounts and ratios of Dox and LeuDox in four subcellular fractions of LeuDox-treated MES-SA/Dx5 cells were also investigated. The highest Dox/LeuDox ratio (i.e. 2.92) was found in the nuclear fraction, followed by the ratio in the low density organelle fraction (i.e. 1.92) that contains lysosomes, organelles in which lysosomal hydrolytic enzymes, capthesins, transform LeuDox into Dox.

Combined effects of doxorubicin and STI571 on growth, differentiation and apoptosis of CML cell line K562.

STI571 (imatinib mesylate; Gleevec) is an inhibitor that targets the tyrosine kinase activity of Bcr-Abl present in chronic myelogenous leukemia (CML) cells. Some preclinical studies have demonstrated that the combination of STI571 with chemotherapeutic drugs results in enhanced toxicity in Bcr-Abl-positive leukemias. We investigated the potential benefit of using STI571 to down-regulate Bcr-Abl activity for the enhancement of doxorubicin anti-proliferative action in K562 cell line derived from blast crisis of CML. At low concentrations of both drugs (40 nM doxorubicin combined with STI571 in the range of 100-150 nM), the antiproliferative effects were mainly due to cellular differentiation as assessed by benzidine staining for hemoglobin synthesis level and real-time PCR for gamma-globin expression. Higher concentrations of STI571 used in combinations with doxorubicin caused mainly apoptosis as shown by DNA degradation and nuclear fragmentation visualized by fluorescence microscopy after DAPI staining, changes in cell morphology observed after Giemza-May Grünwald staining and cellular membrane organization estimated by flow cytometry after Annexin V staining. As compared with either drug alone, cotreatment with STI571 and DOX induced stronger cellular responses. A low concentration of STI571 in combination with a low concentration of DOX might be tested as an alternative approach to increasing the efficacy of chemotherapy against CML.

Regulated expression of active biotinylated G-protein coupled receptors in mammalian cells

We have developed a mammalian expression system suitable for the production of enzymatically biotinylated integral membrane proteins. The key feature of this system is the doxycycline (dox)-regulated co-expression of a secreted variant of Escherichia coli biotin ligase (BirA) and a target protein with a 13-residue biotin acceptor peptide (BioTag) appended to its extracellular domain. Here we describe the expression and functional analysis of three G-protein coupled receptors (GPCRs): protease-activated receptors (PARs) 1 and 2, and the platelet ADP receptor, P2Y12. Clonal Chinese hamster ovary (CHO) Tet-On cell lines that express biotinylated GPCRs were rapidly isolated by fluorescence-activated cell sorting following streptavidin–FITC staining, thereby circumventing the need for manual colony picking. Analysis by Western blotting with streptavidin–HRP following endoglycosidase treatment revealed that all three GPCRs undergo N-linked glycosylation. The expression of biotinylated GPCRs on the cell surface was regulated by the concentration of dox in the medium, reaching a maximum at approximately 1μg/mL dox. Similarly, the extent of GPCR biotinylation was dependent on biotin concentration, with maximum and complete biotinylation achieved upon supplementation with 50μM biotin. Biotinylated PAR1 and PAR2 were readily and specifically cleaved on the surface of intact cells by their cognate proteases, and were capable of transducing extracellular stimuli, resulting in the downstream phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, P2Y12 mediated agonist-induced ERK phosphorylation only when it was expressed at low levels on the cell surface, highlighting the utility of regulated expression for the production of functionally active GPCRs in mammalian cells.

Doxorubicin-induced cardiotoxicity: direct correlation of cardiac fibroblast and H9c2 cell survival and aconitase activity with heat shock protein 27

The use of doxorubicin (Dox) and its derivatives as chemotherapeutic drugs to treat patients with cancer causes dilated cardiomyopathy and congestive heart failure due to Dox-induced cardiotoxicity. In this work, using heat shock factor-1 wild-type (HSF-1(+/+)) and HSF-1 knockout (HSF-1(-/-)) mouse fibroblasts and embryonic rat heart-derived cardiac H9c2 cells, we show that the magnitude of protection from Dox-induced toxicity directly correlates with the level of the heat shock protein 27 (HSP27). Western blot analysis of normal and heat-shocked cells showed the maximum expression of HSP27 in heat-shocked cardiac H9c2 cells and no HSP27 in HSF-1(-/-) cells (normal or heat-shocked). Correspondingly, the cell viability, measured [with (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay] after treatment with various concentrations of Dox, was the highest in heat-shocked H9c2 cells and the lowest in HSF-1(-/-) cells. Depleting HSP27 in cardiac H9c2 cells by small interfering (si)RNA also reduced the viability against Dox, confirming that HSP27 does protect cardiac cells against the Dox-induced toxicity. The cells that have lower HSP27 levels such as HSF-1(-/-), were found to be more susceptible for aconitase inactivation. Based on these results we propose a novel mechanism that HSP27 plays an important role in protecting aconitase from Dox-generated O(2)*(-), by increasing SOD activity. Such a protection of aconitase by HSP27 eliminates the catalytic recycling of aconitase released Fe(II) and its deleterious effects in cardiac cells.

Phase I dose escalation and PK study of thermally sensitive liposomes containing doxorubicin given during radiofrequency ablation (RFA) in patients with non-resectable primary and metastatic liver cancer

15010 Purpose: This phase I dose escalation and pharmacokinetic (PK) study in patients with non-resectable primary or metastatic hepatic tumors undergoing radiofrequency ablation (RFA) uses a 30-minute IV infusion of ThermoDox (TDox) starting 15 minutes prior to RFA treatment. TDox liposomes are engineered to release doxorubicin (Dox) locally at temperatures greater than 39.5 °C. High local concentrations of Dox could allow for increased drug concentration targeted at the tumor margins in an effort to achieve improved local recurrence and tumor control near these RFA-induced thermal lesions. The phase I study goals are to determine the maximum tolerated dose and dose-limiting toxicity of TDox. Patients and Methods: Patients (pts) must be eligible for RFA for primary (HCC) or metastatic liver cancer (MLC). Main inclusion criteria are = 4 lesions and = 7 cm in greatest diameter. Dose escalation is: cohorts of 3–6 pts treated with a single dose of 20, 30, 40, 50, 60 or 70 mg/m2. RFA is administered via percutaneous or surgical approach. RFA treatment without TDox can be repeated for recurrent hepatic (distant or local) tumors. Patients requiring systemic chemotherapy following RFA are removed from the study. MRI, PET and contrast enhanced CT (CE-CT) scans are done pre-, one and three months post-treatment (q3 months thereafter for patients on trial). CE-CT scans are also performed immediately following RFAs. Patients are assessed for safety, PK, and lesion diameters on CT. RFA+TDox lesion diameters will be compared to patients treated by RFA alone (control) at the same institution. Results: A total of 22 pts have been treated as of January 2007 submission date (3, 6, 6, 6, 1 patients at 20, 30, 40, 50, and 60 mg/m2, respectively). This population includes 8 pts with HCC and 14 pts with MLC. Grade 3/4 toxicity (reversible neutropenia) has been observed to be dose dependent. 1 patient at 50 mg/m2 has met DLT criteria. Conclusions: TDox has been safely administered in combination with percutaneous or surgical RFA procedures in 22 patients with liver tumors. There has been limited, manageable toxicity thus far. Enrollment continues as the MTD and DLT have yet to be defined. [Table: see text]

The anti-hepatitis drug DDB chemosensitizes multidrug resistant cancer cells in vitro and in vivo by inhibiting P-gp and enhancing apoptosis

DDB (dimethyl-4,4'-dimethoxy-5,6,5'6'-dimethylene dioxybiphenyl-2,2'-dicarboxylate) is a synthetic hepatoprotectant which has been widely used to treat chronic viral hepatitis B patients in China for more than 20 years. In this study, we evaluated DDB as a multidrug resistance (MDR) chemosensitizing agent. A panel of sensitive and resistant cancer cell lines were treated with various concentration of DDB, and the effect on chemosensitivity and accumulation of anticancer drugs; promotion of apoptosis and P-glycoprotein (P-gp) expression were determined by MTT (Dimethyl thiazolyl-2,5-diphenyltetrazolium bromide) assay, fluorospectrometry and flow cytometry respectively. Drug resistance reversal activity of DDB was also examined in BALB/c nude mice bearing both acquired MDR human nasopharyngeal carcinoma KBv200 and parental KB xenografts. The effect of DDB on the pharmacokinetics of Dox and hematological toxicity induced by Dox was measured in ICR and C(57)/BL mice, respectively. DDB at nontoxic concentrations of 12.5, 25 and 50 microM partly reversed the resistance to vincristine, doxorubicin, paclitaxel in acquired MDR breast carcinoma MCF-7/Adr cells, KBv200 and intrinsic MDR human hepatocarcinoma Bel(7402) cells, whereas no chemosensitizing effect of DDB was observed in sensitive KB and MCF-7 cells. DDB increased the intracellular accumulation of doxorubicin and inhibited surface P-gp expression in MCF-7/Adr cells. Furthermore, it was found that DDB promoted doxorubicin-induced apoptosis of Bel(7402) cells through enhanced caspase-3 activation. Co-administration of DDB at 300 and 500 mg/kg orally to nude mice increased the antitumor activity of vincristine to KBv200 xenografts without a significant increase in toxicity. In contrast, Co-administration of DDB did not inhibit the growth of KB xenografts. DDB also markedly reduced the decrease of leukocytes in doxorubicin-treated C(57)/BL mice. Co-administration of DDB increased Dox concentration in ICR mice bearing S180 sarcoma, but no pharmacokinetical interaction with Dox was observed. These results indicate that DDB has MDR reversal activity by inhibiting P-gp and when used in combination with anti-cancer drugs, it could potentially be used as a clinical treatment for P-gp-mediated MDR cancers.

Cytotoxicity of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles in rat glioma cell lines using different assays

The cytotoxicity of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles (Dox-PBCA-NP) was investigated in the rat glioma cell lines GS-9L, F-98 and RG-2. MTT and LDH assays were used as cytotoxic assays. In general, the cytotoxicity of nanoparticle-bound doxorubicin (Dox) was enhanced compared to the free drug in solution. However, responses of the cell lines towards the drug effects were different. In the case of free Dox in solution, this difference correlated with different intracellular concentrations of Dox, which in turn, depended on the level of P-glycoprotein (P-gp) expression in these cell lines. Accordingly, the 9L gliosarcoma (GS-9L) cells, which appeared to be most resistant towards Dox, were characterized by the highest P-gp expression.Additionally, the influence of surfactants on the cytotoxic effect was investigated at different Dox concentrations. It was shown that the presence of polysorbate 80 (Tween® 80) in the nanoparticle formulation significantly enhanced the cytotoxicity, whereas poloxamer 188 (Pluronic® F68) and poloxamine 908 (Tetronic® 908) had a negligible influence.

Electroporation Enhances Radiation and Doxorubicin-Induced Toxicity in Solid Tumor In Vivo

Treatment of cancer patients is subject to limitations in radiotherapy and chemotherapy. This necessitates development of new protocols, and the present work reports on the effects of a combination of local electroporation with ionizing radiation and/or anticancer drug doxorubicin hydrochloride (DOX) on subcutaneous solid tumor murine fibrosarcoma. Localized treatment of fibrosarcoma tumor, grown in right hind leg of Swiss mice, has been carried out using DOX (0.6 mg/kg body weight), radiation (Co 60 gamma-rays, dose rate 0.37 Gy/min) and electroporation (1 kV/cm, 200 micros, 8 pulses per burst, 10 bursts) individually or in combinations. Measurements of the tumor growth kinetics after treatment with combinations have revealed significant growth delay. The treatment groups, (i) radiation and electroporation, (ii) DOX and electroporation, and (iii) radiation, DOX and electroporation, have yielded tumor growth delays (TGDs) of 1.22, 1.5, and 1.73 days, respectively, compared to control with the tumor volumes being 53%, 57%, and 49% that of control on the final day of observation. These results suggest that the antitumor effects of a moderate dose of gamma radiation and low concentration of DOX can be significantly enhanced by combination with electroporation.

Comparison of gene expression in cell culture to that in the intact animal: relevance to drugs and environmental toxicants. Focus on “Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes”

to study the effects of drugs and other environmental toxicants on the liver, many believe that the use of hepatocyte-derived cultures offers advantages over studies in the liver of intact animals. Cell cultures can be manipulated more easily, and they are far less expensive to maintain than a

Alteration of Genomic Responses to Doxorubicin and Prevention of MDR in Breast Cancer Cells by a Polymer Excipient: Pluronic P85

Polymer therapeutics has emerged as a new clinical option for the treatment of human diseases. However, little is known about pharmacogenetic responses to drugs formulated with polymers. In this study, we demonstrate that a formulation containing the block copolymer Pluronic P85 and antineoplastic drug doxorubicin (Dox) prevents the development of multidrug resistance in the human breast carcinoma cell line, MCF7. Specifically, MCF7 cells cultured in the presence of Pluronic were unable to stably grow in concentrations of Dox that exceeded 10 ng of Dox/mL of culture medium. In sharp contrast, MCF7 cells cultured in the absence of the block copolymer resulted in the selection and stable growth of cells that tolerated a 1000 times higher concentration of the drug (10 000 ng of Dox/mL of culture medium). Detailed characterization of the isolated sublines demonstrated that those cells selected in the polymer-drug formulation did not show amplification of the MDR1 gene, likely resulting in their high sensitivity to the drug. Conversely, cells selected with Dox alone showed an elevated level in the expression of the MDR1 gene along with a corresponding increase in the expression level of the drug efflux transporter, Pgp, and likely contributing to the high resistance of the cells to Dox. Global analysis of the expression profiles of 20K genes by DNA microarray revealed that the use of Pluronic in combination with Dox drastically changed the direction and magnitude of the genetic response of the tumor cells to Dox and may potentially enhance therapeutic outcomes. Overall, this study reinforces the need for a thorough assessment of pharmacogenomic effects of polymer therapeutics.

Investigation of the influence of modulation of P-glycoprotein by a multiple dosing regimen of tamoxifen on the pharmacokinetics and toxicodynamics of doxorubicin

The in vivo effect of modulators of P-glycoprotein (Pgp) on organ accumulation of substrates of Pgp has not been fully investigated. We investigated the influence of a Pgp modulator (tamoxifen, TAM) on the pharmacokinetics and toxicodynamics of a Pgp substrate (doxorubicin, DOX) in rats. TAM was administered daily for 11 days before the administration of DOX in male Sprague-Dawley rats, with all doses being clinically relevant. The experimental design of the project consisted of two different protocols. One was to investigate the effect of DOX on the time course of Pgp-ATPase activity, sarcoplasmic reticulum Ca(2+) -ATPase (SERCA) activity, and DOX concentration in the heart, liver, and kidneys of TAM-pretreated animals; the other protocol was to study the effect of TAM pretreatment on the disposition of DOX in the body by investigating its time course in plasma, urine and bile. The simultaneous curve fitting of plasma data with urine and bile data with the help of the related pharmacokinetic equations provided the calculated parameters and constants. The first-order rate constants between the central and the myocardial compartments (k(1H) and k(H1)) were decreased in the TAM-treated group. The treatment also significantly reduced the k(1H)/k(H1) ratio in comparison to that of the control group. The first-order biliary elimination rate constant (k(b)) was significantly decreased (29%) in the TAM-treated group. The reduction was estimated in comparison with that of the control group. This reduction could be attributed to the inhibitory effect of TAM on Pgp located on biliary canicular membranes. The initial reduction of Pgp activity in TAM-treated group was at 60% of the basal level. The activity declined and reached a plateau at 20% of the basal activity after 6 h and remained at that level for 24 h. The area under the curves of Pgp-ATPase activity time (AUC(Activity 0-24)) following DOX administration in TAM-treated group was significantly lower than that of the control group, indicating an overall inhibitory effect of TAM on Pgp-ATPase activity under the protocol of this study. The area under the curves of the SERCA activity-time curve following DOX administration in TAM-treated group demonstrated a 15% reduction in AUC(Activity 0-24) in comparison with that of the control group, an indication of increased toxicity. The amount of myocardial Pgp in the 24-h period following DOX administration was comparable to the control group and showed no significant deviation from the basal levels of the protein. The effect of TAM on DOX accumulation in the myocardial tissue and the increase in cardiotoxicity can be related to the net inhibitory effect of TAM on the efflux activity of Pgp in the heart. The results of the present study supported the hypothesis of the project that multiple regimen pretreatment with Pgp modulator TAM increases the DOX accumulation in the heart and promotes DOX-induced cardiotoxicity.

143. Regulated, High Capacity Adenoviral Vectors Mediated Long-Term Gene Expression in the Brain Even in the Presence of a Peripheral Immune Response to Adenovirus

For gene therapy of chronic neurological disorders, vector systems should allow long-term transduction of brain cells in the complete absence of undesirable long-term side-effects. We have engineered a novel regulatable system consisting of a tetracycline-dependent reverse-transactivator, rtTA2S-M2, in combination with Tet-repressor, tTSKid, under the control of either the major immediate early human cytomegalovirus (hCMV) promoter or murine (mCMV) promoter, encoded within high capacity, helper-dependent adenovirus vectors (HC-Adv). We assessed inducibility, leakiness and time course of expression in-vitro and in-vivo within the central nervous system (CNS). We also assessed cell-type specific expression within neurons and glial cells, and the efficiency of the regulatable vectors in the presence of a systemic immune response to adenovirus. HC-Adv vectors, i.e., STK120mBM [pSTK120m (TRE-|[beta]|-Gal-pA)-(mCMV pr.-rtTA2S-M2-IRES-tTSKid-pA)-Kanamycin] and STK120hBM [pSTK120.1 (TRE-|[beta]|-Gal-pA)-(hCMV pr.-rtTA2S-M2-IRES-tTSKid-pA)-Kanamycin] were injected into the striatum of rat brains at 1|[times]|107 blue forming units. Twenty-four hours prior to injection, the rats were given drinking water with 2 mg/ml doxycycline (Dox) and 1% sucrose, or 1% sucrose only. At the appropriate time points, rats were perfused and brains were post-fixed in 4% PFA for 3 days and vibratome sectioned for immunohistochemistry. Assessment of transgene expression levels in vivo revealed higher potency of the mCMV driven TetOn switch when compared to the hCMV driven switch in the presence of Dox. Both mCMV and hCMV encoded regulatable switches produced negligible gene expression levels in the absence of Dox. Our results demonstrate that the rtTA2S-M2 transactivator in conjunction with IRES and tTSKid transcriptional silencer displays strong and stringent induction kinetics with negligible basal activity in the uninduced state; in vivo data show stringent regulation kinetics of |[beta]|-Galactosidase expression with co-localization of |[beta]|-Galactosidase within MAP-2 (neurons) and GFAP (astrocytes) immunoreactive cells in the striatum; expression of |[beta]|-Galactosidase was dependent on the concentration of Dox and the time after HC-Adv administration. Regulated, long-term transduction in the CNS was attained even in the presence of a systemic immune response to adenovirus as could be encountered in human patients, making HC-Adv vectors very attractive for CNS gene therapy.

PHARMACOKINETICS AND BIODISTRIBUTION STUDIES OF DOXORUBICIN LOADED POLY(BUTYL CYANOACRYLATE) NANOPARTICLES SYNTHESIZED BY TWO DIFFERENT TECHNIQUES

The aim of the study is to determine and compare the pharmacokinetics and tissue distribution of Doxorubicin (Dox) delivered as solution or through nanoparticles after intravenous (i.v.) and intraperitoneal (i.p.) injection. Doxorubicin loaded poly(butyl cyanoacrylate) nanoparticles were synthesized by dispersion polymerization (DP) and emulsion polymerization (EP) techniques. The drug loaded DP and EP nanoparticles were administered by i.v. or i.p. routes and the respective pharmacokinetics and tissue distribution were determined. Both types of nanoparticles significantly enhanced the elimination half-life (T(1/2)), mean residence time (MRT) AUC(0-8), AUC(0-infinity) and AUMC(0-8) of Dox in blood after i.v. injection. Dox delivered through DP nanoparticles rapidly disappeared from blood and distributed to the organs of reticuloendothelial system (RES). But, the clearance of Dox delivered through EP nanoparticles from blood was slower than this of the DP nanoparticles and Dox solution. After i.p. injection, the Dox loaded into DP nanoparticles quickly appeared in blood and undergone rapid distribution to the organs of RES, while the Dox loaded into EP nanoparticles absorbed slowly into blood and remained in the circulation for longer time. The absorption into blood of Dox delivered through DP and EP nanoparticles after i.p. injection was relatively rapid and higher than Dox solution. The T(1/2), MRT, AUC(0-8), AUC(0-infinity) and AUMC(0-8) of Dox in blood were significantly higher and the clearance (Cl) was lower than for the Dox solution after i.p. injection. The tissue concentrations of Dox delivered through nanoparticles after i.p. injection were significantly lower than after i.v. injection. The bioavailability (F) of Dox was greatly enhanced by DP (approximately 1.9 fold) and EP nanoparticles (approximately 2.12 fold) compared to Dox solution after i.p. injection. EP nanoparticles significantly enhanced the bioavailability, MRT, T(1/2), AUC(0-8), AUC(0-infinity) and AUMC(0-8) of Dox than DP nanoparticles. This signifies the advantage of EP nanoparticles in increasing the elimination half-life of Dox both after i.v. and i.p. injection and enhanced bioavailability after i.p. injection, which is expected to improve the therapeutic efficacy of Dox and reduce the Dox-associated systemic toxicity. Importantly, both DP and EP nanoparticles greatly reduced the distribution of Dox to heart both after i.v. and i.p. injection, suggesting their potential in reducing Dox-associated cardiotoxicity.

Enhanced Tumour Uptake of Doxorubicin Loaded Poly(butyl cyanoacrylate) Nanoparticles in Mice Bearing Dalton's Lymphoma Tumour

The objective of this study is to enhance the delivery of Doxorubicin hydrochloride to Dalton's lymphoma solid tumour through poly(butyl cyanoacrylate) (PBC) nanoparticles. Doxorubicin loaded PBC (DPBC) nanoparticles were prepared by emulsion polymerization and characterized by particle size analysis, zeta potential and scanning electron microscopy. Doxorubicin HCl (Dox) and DPBC nanoparticles were radiolabeled with 99mTc by reduction method using stannous chloride and optimized the labeling parameters to obtain high labeling efficiency. The in vitro stability of 99mTc-labeled complexes was determined by DTPA and cysteine challenge test. The labeled complexes showed very low transchelation and high in vitro and serum stability. 99mTc labeled complexes of Dox and DPBC nanoparticles were administered subcutaneously below the Dalton's lymphoma tumour and biodistribution was studied. The distribution of DPBC nanoparticles to the blood, heart and organs of RES such as liver, lung and spleen was biphasic with a rapid initial distribution, followed by a significant decrease later at 6 h post-injection. The distribution of Dox to tissues was very low initially and increased significantly at 6 h post-injection indicating its accumulation at the injection site for a longer time. The concentration of DPBC nanoparticles was also found high in tissues at 6 h post-injection indicating their accumulation at the subcutaneous site and consequent disposition to tissues with time. A significantly high tumour uptake of DPBC nanoparticles (∼13 fold higher at 48 h post-injection) (P <0.001) was found compared to free Dox. The tumour concentrations of both Dox and DPBC nanoparticles increased with time indicating their slow penetration from the injection site into tumour. The concentration of DPBC nanoparticles in the femur bone in the tumour region was also significantly higher (P <0.001) than free Dox and increased with time. The study signifies the advantage of delivering Dox to Dalton's lymphoma through PBC nanoparticles by facilitating enhanced tumour uptake and prolonged tumour retention, which are expected to lead to greater therapeutic effect in the form of tumour regression.