Intracellular Drug Levels Research Articles

A Nanosystem of Amphiphilic Oligopeptide-Drug Conjugate Actualizing Both αvβ3 Targeting and Reduction-Triggered Release for Maytansinoid.

To design a prodrug-based self-assembling nanosystem with both ligand targeting and stimuli-responsive features, and elucidate the superiority of each targeting strategy and the synergistic effect between them, we synthesized four small molecule amphiphilic peptide-drug conjugates (APDCs) using maytansinoid (DM1) as a cytotoxic agent, cRGDfK as a homing peptide, and disulfide (SS) or thioether (SMCC) as linker. Owing to their amphiphilicity, the APDCs could self-assemble into nanoparticles (APDC@NPs) which were evaluated in vitro in three different cell lines and in vivo in tumor-bearing C57BL/6 mice. The RSSD@NPs showed the strongest interaction with αvβ3 integrin, highest cell uptake and intracellular free drug level, and best antitumor efficacy in vitro and in vivo, while it shared the same goodness with other test nanosystems in terms of high drug loading, EPR effect and free of potentially toxic polymers. Especially, the in vivo efficacy of RSSD@NPs was 2 fold of free DM1 which is too cytotoxic to be a drug, while the active targeted APDC@NPs demonstrated acceptable system, tissue and blood compatibility. In αvβ3-positive cells or tumors, the RGD targeting contributed much more than disulfide in anticancer effect. The maximum synergism of the two strategies reached to 22 fold in vitro and 3 fold in vivo. Generally, the active targeting, prodrug and nanosystem could significantly decrease the toxicity of free DM1 and improve its therapy outcome via combining active targeting, prodrug and nanopreparation, especially the dual targeting strategies and their synergism.

Biomarkers and biometric measures of adherence to use of ARV-based vaginal rings.

IntroductionPoor adherence to product use has been observed in recent trials of antiretroviral (ARV)-based oral and vaginal gel HIV prevention products, resulting in an inability to determine product efficacy. The delivery of microbicides through vaginal rings is widely perceived as a way to achieve better adherence but vaginal rings do not eliminate the adherence challenges exhibited in clinical trials. Improved objective measures of adherence are needed as new ARV-based vaginal ring products enter the clinical trial stage.MethodsTo identify technologies that have potential future application for vaginal ring adherence measurement, a comprehensive literature search was conducted that covered a number of biomedical and public health databases, including PubMed, Embase, POPLINE and the Web of Science. Published patents and patent applications were also searched. Technical experts were also consulted to gather more information and help evaluate identified technologies. Approaches were evaluated as to feasibility of development and clinical trial implementation, cost and technical strength.ResultsNumerous approaches were identified through our landscape analysis and classified as either point measures or cumulative measures of vaginal ring adherence. Point measurements are those that give a measure of adherence at a particular point in time. Cumulative measures attempt to measure ring adherence over a period of time.DiscussionApproaches that require modifications to an existing ring product are at a significant disadvantage, as this will likely introduce additional regulatory barriers to the development process and increase manufacturing costs. From the point of view of clinical trial implementation, desirable attributes would be high acceptance by trial participants, and little or no additional time or training requirements on the part of participants or clinic staff. We have identified four promising approaches as being high priority for further development based on the following measurements: intracellular drug levels, drug levels in hair, the accumulation of a vaginal analyte that diffuses into the ring, and the depletion of an intrinsic ring constituent.ConclusionsWhile some approaches show significant promise over others, it is recommended that a strategy of using complementary biometric and behavioural approaches be adopted to best understand participants’ adherence to ARV-based ring products in clinical trials.

Differential Mechanisms of Tenofovir and Tenofovir Disoproxil Fumarate Cellular Transport and Implications for Topical Preexposure Prophylaxis.

Intravaginal rings releasing tenofovir (TFV) or its prodrug, tenofovir disoproxil fumarate (TDF), are being evaluated for HIV and herpes simplex virus (HSV) prevention. The current studies were designed to determine the mechanisms of drug accumulation in human vaginal and immune cells. The exposure of vaginal epithelial or T cells to equimolar concentrations of radiolabeled TDF resulted in over 10-fold higher intracellular drug levels than exposure to TFV. Permeability studies demonstrated that TDF, but not TFV, entered cells by passive diffusion. TDF uptake was energy independent but its accumulation followed nonlinear kinetics, and excess unlabeled TDF inhibited radiolabeled TDF uptake in competition studies. The carboxylesterase inhibitor bis-nitrophenyl phosphate reduced TDF uptake, suggesting saturability of intracellular carboxylesterases. In contrast, although TFV uptake was energy dependent, no competition between unlabeled and radiolabeled TFV was observed, and the previously identified transporters, organic anion transporters (OATs) 1 and 3, were not expressed in human vaginal or T cells. The intracellular accumulation of TFV was reduced by the addition of endocytosis inhibitors, and this resulted in the loss of TFV antiviral activity. Kinetics of drug transport and metabolism were monitored by quantifying the parent drugs and their metabolites by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Results were consistent with the identified mechanisms of transport, and the exposure of vaginal epithelial cells to equimolar concentrations of TDF compared to TFV resulted in ∼40-fold higher levels of the active metabolite, tenofovir diphosphate. Together, these findings indicate that substantially lower concentrations of TDF than TFV are needed to protect cells from HIV and HSV-2.

Inhibition of cisplatin-resistance by RNA interference targeting metallothionein using reducible oligo-peptoplex

Effective intracellular level of a platinum anti-cancer drug, cisplatin, following repeated injections can be decreased either by the active efflux via ATP pump or by interactions with glutathione and metallothionein. Cisplatin in cytoplasm preferably binds to cysteine-rich proteins such as glutathione and metallothionein (MT). Detoxification of cisplatin by intracellular thiol-containing proteins has been considered to be major hurdles to overcome. The short hairpin RNA targeting MT (shMT) was tested to down-regulate MT and recover cisplatin resistance. A reducible polymer, poly(oligo-d-arginine) (rPOA), formed stable complex with shMT and demonstrated superior transfection efficiency. Efficient transfection of shMT/rPOA oligo-peptoplexes was found to significantly inhibit MT over-expression, resulting in 45% decrease of cell viability compared to the cisplatin alone group. This decrease was mediated by the synergistic effect of shMT/rPOA oligo-peptoplex and cisplatin. Co-administration of shMT/rPOA oligo-peptoplex and cisplatin in in vivo tumor model showed noticeable tumor-suppressing effect by inducing reversal of cisplatin resistance following effective intracellular delivery of shMT by rPOA. Combination therapy through co-administration of shMT/rPOA oligo-peptoplex and cisplatin was found to effectively reverse cisplatin resistance by RNA interference and consequently improve anti-cancer activity of cisplatin.

Update on Antifungal Drug Resistance.

Invasive fungal infections remain a major source of global morbidity and mortality, especially among patients with underlying immune suppression. Successful patient management requires antifungal therapy. Yet, treatment choices are restricted due to limited classes of antifungal agents and the emergence of antifungal drug resistance. In some settings, the evolution of multidrug-resistant strains insensitive to several classes of antifungal agents is a major concern. The resistance mechanisms responsible for acquired resistance are well characterized and include changes in drug target affinity and abundance, and reduction in the intracellular level of drug by biofilms and efflux pumps. The development of high-level and multidrug resistance occurs through a stepwise evolution of diverse mechanisms. The genetic factors that influence these mechanisms are emerging and they form a complex symphony of cellular interactions that enable the cell to adapt and/or overcome drug-induced stress. Drivers of resistance involve a complex blend of host and microbial factors. Understanding these mechanisms will facilitate development of better diagnostics and therapeutic strategies to overcome and prevent antifungal resistance.

Inhibition of mdr1 by G-quadruplex oligonucleotides and reversal of paclitaxel resistance in human ovarian cancer cells.

The expression of ATP-dependent efflux pump P-glycoprotein (P-gp) in cancer cells generally results in multidrug resistance (MDR) to chemotherapeutic drugs, which is the main cause of chemotherapy failure in cancer treatment. The intracellular drug levels could be increased by some MDR reversal agents that inhibited the drug efflux activity of P-gp. The synthesized DNA nucleic acids of G-quadruplex represent a novel and unique class of anti-cancer agents. While there was no report on the roles of DNA G-quadruplex oligonucleotides (GQ-ODNs) in the MDR reversal, the present study was performed to investigate the ability of synthesized GQ-ODNs to reverse P-gp-mediated MDR and its mechanism in paclitaxel (PTX)-resistant SKOV3 (SKOV3/PTX) cells and their sensitive cell lines SKOV3. The ability of GQ-ODNs to reverse drug resistance was evaluated by MTS assay. The results showed that GQ-ODNs can reverse PTX resistance effectively. The potential of GQ-ODNs as reversal agents was evaluated with the nude mice tumor xenograft model and showed that the co-administration of the GQ-ODNs and PTX had better effects and was also more evident than treatment with only PTX. The P-gp expression was assessed by the Western blot; it showed that SKOV3/PTX cells showed highly expressed P-gp protein, while their sensitive cells scarcely showed P-gp. The presence of GQ-ODNs efficiently decreased the P-gp expression, showing that GQ-ODNs could reverse P-gp-mediated MDR by decreasing the expression of P-gp. This study indicated that GQ-ODNs could effectively reverse P-gp-mediated PTX resistance by inhibiting the expression of P-gp and by the co-administration of GQ-ODNs and PTX that could increase the apoptosis of SKOV3/PTX cells. Thus, the synthesized GQ-ODNs may be a potential inhibitor to overcome drug resistance.

Anti-HIV Drug-Combination Nanoparticles Enhance Plasma Drug Exposure Duration as Well as Triple-Drug Combination Levels in Cells Within Lymph Nodes and Blood in Primates

HIV patients on combination oral drug therapy experience insufficient drug levels in lymph nodes, which is linked to viral persistence. Following success in enhancing lymph node drug levels and extending plasma residence time of indinavir formulated in lipid nanoparticles, we developed multidrug anti-HIV lipid nanoparticles (anti-HIV LNPs) containing lopinavir (LPV), ritonavir (RTV), and tenofovir (PMPA). These anti-HIV LNPs were prepared, characterized, scaled up, and evaluated in primates with a focus on plasma time course and intracellular drug exposure in blood and lymph nodes. Four macaques were subcutaneously administered anti-HIV LNPs and free drug suspension in a crossover study. The time course of the plasma drug concentration as well as intracellular drug concentrations in blood and inguinal lymph nodes were analyzed to compare the effects of LNP formulation. Anti-HIV LNPs incorporated LPV and RTV with high efficiency and entrapped a reproducible fraction of hydrophilic PMPA. In primates, anti-HIV LNPs produced over 50-fold higher intracellular concentrations of LPV and RTV in lymph nodes compared to free drug. Plasma and intracellular drug levels in blood were enhanced and sustained up to 7 days, beyond that achievable by their free drug counterpart. Thus, multiple antiretroviral agents can be simultaneously incorporated into anti-HIV lipid nanoparticles to enhance intracellular drug concentrations in blood and lymph nodes, where viral replication persists. As these anti-HIV lipid nanoparticles also prolonged plasma drug exposure, they hold promise as a long-acting dosage form for HIV patients in addressing residual virus in cells and tissue.

Long-acting three-drug combination anti-HIV nanoparticles enhance drug exposure in primate plasma and cells within lymph nodes and blood.

Insufficient HIV drug levels in lymph nodes have been linked to viral persistence. To overcome lymphatic drug insufficiency, we developed and evaluated in primates a lipid-drug nanoparticle containing lopinavir, ritonavir, and tenofovir. These nanoparticles produced over 50-fold higher intracellular lopinavir, ritonavir and tenofovir concentrations in lymph nodes compared to free drug. Plasma and intracellular drug levels in blood were enhanced and sustained for 7 days after a single subcutaneous dose, exceeding that achievable with current oral therapy.

Mechanisms of Antifungal Drug Resistance.

Antifungal therapy is a central component of patient management for acute and chronic mycoses. Yet, treatment choices are restricted because of the sparse number of antifungal drug classes. Clinical management of fungal diseases is further compromised by the emergence of antifungal drug resistance, which eliminates available drug classes as treatment options. Once considered a rare occurrence, antifungal drug resistance is on the rise in many high-risk medical centers. Most concerning is the evolution of multidrug- resistant organisms refractory to several different classes of antifungal agents, especially among common Candida species. The mechanisms responsible are mostly shared by both resistant strains displaying inherently reduced susceptibility and those acquiring resistance during therapy. The molecular mechanisms include altered drug affinity and target abundance, reduced intracellular drug levels caused by efflux pumps, and formation of biofilms. New insights into genetic factors regulating these mechanisms, as well as cellular factors important for stress adaptation, provide a foundation to better understand the emergence of antifungal drug resistance.

The novel bis-benzylisoquinoline PY35 reverses P-glycoprotein-mediated multidrug resistance.

Multidrug resistance (MDR) to chemotherapeutic drugs is the main cause of chemotherapy failure in cancer treatment, and it generally results from expression of ATP-dependent efflux pump P-glycoprotein (P-gp). MDR reversal agents typically act by inhibiting the drug efflux activity of P-gp, thereby increasing intracellular drug levels. PY35 is a novel 5-substituted tetrandrine (Tet) derivative (CNApplication No.201210238709.6). The present study was performed to investigate the ability of PY35 to reverse P-gp-mediated MDR and its mechanism in resistant K562/Adriamycin(ADM), MCF-7/ADM cells and their sensitive cell lines K562 and MCF-7. The ability of PY35 to reverse drug resistance was evaluated by MTT assay. The results showed that PY35 can reverse MDR more effectively than the drug prototype‑Tet. The P-gp function was assessed by the Rhodamine 123(Rho-123; a P-gp substrate) uptake assay with flow cytometry(FCM) and laser scanning confocal microscopes(LSCM); it showed that the MDR cells pumped Rho-123 out the cells, while their sensitive cells scarcely showed efflux. The presence of PY35 efficiently decreased the efflux of the Rho-123, showing that PY35 can reverse P-gp-mediated MDR by increasing the intracellular concentration of Rho-123. The intracellular accumulation of ADM was analyzed by FCM and showed that the coadministration of PY35 and ADM had clearer accumulation than the treatment of Tet and ADM, and was also more evident than treatment with only ADM. The effect of PY35 on the expression of P-gp was assessed by western blotting. The results indicated that PY35 does not inhibit the expression level of the P-gp. This study indicated that PY35 can effectively reverse P-gp-mediated MDR, not by inhibiting the expression of P-gp, but by the coadministration of PY35 and ADM that could increase the intracellular accumulation of drugs. Thus, PY35 may be a potential inhibitor to overcome drug resistance.

Development and anti‐tumor activities of claudin‐4‐specific monoclonal antibodies (1062.8)

Claudin (CLDN) is a key component of the epithelial tight junction. Expression of the various CLDNs differs among tissues and is deregulated in malignant tumors. For example, CLDN4 is frequently overexpressed in pancreatic, breast, and ovarian cancers and therefore may be an attractive target for anticancer drugs. However, the low antigenicity of CLDN4 has hampered the development of antibody‐based CLDN4‐targeted cancer therapy. We successfully generated monoclonal antibodies (mAbs) to CLDN4 and evaluated their anti‐tumor activity. Briefly, we immunized rats with plasmids containing the CLDN4 coding sequence. We fused the resulting B cells with myeloma cells and screened 8 hybridoma clones that produced anti‐CLDN4 mAbs. All 8 mAbs bound to CLDN4‐expressing cells but not to those expressing CLDN1, 2, 5, 6, 7or 9. All 8 mAbs showed CLDN4 binding‐dependent activation of the Fcγ receptor. A clone with high affinity to CLDN4 suppressed tumor growth in vitro and in vivo. Interestingly, treatment of cancer cells with the clone of CLDN4‐specific mAb and an anti‐cancer drug (5‐flurouracil or cisplatin) had a synergistic cytotoxic effect. Specifically, mAb treatment increased intracellular drug levels. Our findings indicate that CLDN4 mAbs are promising tools for CLDN4‐targeted cancer therapy.Grant Funding Source: supported by the Ministry of Health, Labor, and Welfare of Japan

A metabolomics investigation into the effects of HIV protease inhibitors on HPV16 E6 expressing cervical carcinoma cells

Recently, it has been reported that anti-viral drugs, such as indinavir and lopinavir (originally targeted for HIV), also inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells. In order to understand more about the mode-of-action(s) of these drugs the metabolome of HPV16 E6 expressing cervical carcinoma cell lines was investigated using mass spectrometry (MS)-based metabolic profiling. The metabolite profiling of C33A parent and E6-transfected cells exposed to these two anti-viral drugs was performed by ultra performance liquid chromatography (UPLC)-MS and gas chromatography (GC)-time of flight (TOF)-MS. Using a combination of univariate and multivariate analyses, these metabolic profiles were investigated for analytical and biological reproducibility and to discover key metabolite differences elicited during anti-viral drug challenge. This approach revealed both distinct and common effects of these two drugs on the metabolome of two different cell lines. Finally, intracellular drug levels were quantified, which suggested in the case of lopinavir that increased activity of membrane transporters may contribute to the drug sensitivity of HPV infected cells.

Colon Cancer Therapy: Recent Developments in Nanomedicine to Improve the Efficacy of Conventional Chemotherapeutic Drugs

The number of patients with colorectal cancer, the third most frequently diagnosed malignancy in the world, has increased markedly over the past 20 years and will continue to increase in the future. Despite recent advances in chemotherapy, currently used anticancer molecules are unable to improve the prognosis of advanced or recurrent colorectal cancer, which remains incurable. The transport of classical drugs by nanoparticles has shown great promise in terms of improving drug distribution and bioavailability, increasing tissue half-life and concentrating anticancer molecules in the tumor mass, providing optimal drug delivery to tumor tissue, and minimizing drug toxicity, including those effects associated with pharmaceutical excipients. In addition, colon cancer targeting may be improved by incorporating ligands for tumor-specific surface receptors. Similarly, nanoparticles may interact with key drug-resistance molecules to prevent a reduction in intracellular drug levels drug. Recently published data have provided convincing pre-clinical evidence regarding the potential of active-targeted nanotherapeutics in colon cancer therapy, although, unfortunately, only a few of these therapies have been translated into early-phase clinical trials. As nanotechnology promises to be a new strategy for improving the prognosis of colon cancer patients, it would be very useful to analyze recent progress in this field of research. This review discusses the current status of nanoparticle-mediated cancer-drug delivery, the challenges restricting its application, and the potential implications of its use in colon cancer therapy.

Kinase Inhibitor Therapy in CML: It's What's Inside That Counts

Kinase Inhibitor Therapy in CML: It's What's Inside That Counts

Threshold Levels of ABL Tyrosine Kinase Inhibitors Retained in Chronic Myeloid Leukemia Cells Determine Their Commitment to Apoptosis

The imatinib paradigm in chronic myelogenous leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKI). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCR-ABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKI-induced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular fluorescence-activated cell sorting and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies. Importantly, significant intracellular TKI stores were detected following drug washout, levels of which tracked with onset of apoptosis and incomplete return of BCR-ABL signaling, particularly pSTAT5, to baseline. Among TKIs tested, ponatinib showed the most robust capacity for apoptotic commitment showing sustained suppression of BCR-ABL signaling even at low intracellular levels following extensive washout, consistent with high-affinity binding and slow dissociation from ABL kinase. Together, our findings suggest commitment of CML cells to apoptosis requires protracted incomplete restoration of BCR-ABL signaling mediated by intracellular retention of TKIs above a quantifiable threshold. These studies refine our understanding of apoptotic commitment in CML cells and highlight parameters important to design of therapeutic kinase inhibitors for CML and other malignancies.

Inhibition of Drug Efflux Pumps in Staphylococcus Aureus : Current Status of Potentiating Existing Antibiotics

The emergence of multidrug-resistant Staphylococcus aureus coupled with a declining output of new antibiotic treatment options from the pharmaceutical industry is a growing worldwide healthcare problem. Multidrug efflux pumps are known to play a role in antibiotic and biocide resistance in S. aureus. These membrane transporters are capable of extruding drugs and other structurally unrelated compounds, hence decreasing intracellular concentration and increasing survival. Coadministration of efflux pump inhibitors (EPIs) with antibiotics that are pump substrates could increase intracellular drug levels, thus bringing renewed efficacy to existing antistaphylococcal agents. Numerous EPIs have been identified or synthesized over the past two decades; these include existing pharmacologic drugs, naturally occurring compounds, and synthetic derivatives thereof. This review describes the current progress in EPI development for use against S. aureus.

Efficient induction of apoptosis in HeLa cells by a novel cationic porphycene photosensitizer

In the present study we analyze the photobiological properties of 2,7,12-tris(α-pyridinio-p-tolyl)–17-(p-(methoxymethyl)phenyl) porphycene (Py3MeO-TBPo) in Hela cells, in order to assess its potential as a new photosensitizer for photodynamic therapy of cultured tumor cells. Using 0.5 μM Py3MeO-TBPo, flow cytometry studies demonstrated an increase of intracellular drug levels related to the incubation time, reaching a maximum at 18 h. LysoTracker® Green (LTG) and MitoTracker® Green (MTG) probes were used to identify the subcellular localization. Upon exposure to ultraviolet excitation, red porphycene fluorescence was detected as red granules in the cytoplasm that colocalized with LTG. No significant toxic effects were detected for Py3MeO-TBPo in the dark at concentrations below 1 μM. In contrast, Py3MeO-TBPo combined with red-light irradiation induced concentration- and fluence-dependent HeLa cells inactivation. Besides, all photodynamic protocols assayed induced a clear effect of cell detachment inhibition after trypsin treatment. Both apoptotic and necrotic cell death mechanisms can occur in HeLa cells depending on the experimental protocol. After 18 h incubation with 0.5 μM Py3MeO-TBPo and subsequent red light irradiation (3.6 J/cm2), a high number of cells die by apoptosis, as evaluated by morphological alterations, immunofluorescent relocalization of Bax from cytosol to mitochondria, and TUNEL assay. Likewise, immunofluorescence techniques showed that cytochrome c is released from mitochondria into cytosol in cells undergoing apoptosis, which occurs immediately after relocation of Bax in mitochondria. The highest amount of apoptosis appeared 24 h after treatment (70%) and this cell death occurred without cell detachment to the substrate. In contrast, with 0.75 μM Py3MeO-TBPo and 3.6 J/cm2 irradiation, morphological changes showed a preferential necrotic cell death.Singlet oxygen was identified as the cytotoxic agent involved in cell photoinactivation. Moreover, cell cultures pre-exposed to the singlet oxygen scavenger sodium azide showed pronounced protection against the loss of viability induced by Py3MeO-TBPo and light.Different changes in distribution and organization of cytoskeletal elements (microtubules and actin microfilaments) as well as the protein vinculin, after apoptotic and necrotic photodynamic treatments have been analyzed. Neither of these two cell death mechanisms (apoptosis or necrosis) induced cell detachment.In summary, Py3MeO-TBPo appears to meet the requirements for further scrutiny as a very good photosensitizer for photodynamic therapy: it is water soluble, has a high absorption in the red spectral region (where light penetration in tissue is higher), and is able to induce effective high apoptotic rate (70%) related to the more widely studied photosensitizers.

Chemoembolization for Colorectal Liver Metastases after FOLFOX Failure

Colon carcinoma remains the third most common cancer in the United States among men and women, and accounts for 9% of all cancer deaths (1). The majority (50%–60%) of patients with colon cancer will eventually develop liver metastases, and 80%–90% of these individuals will present with unresectable disease. Liver metastases remain the most common cause of mortality in these patients, and they are therefore the focus of many current therapies. The oncologic rationale for transarterial chemoembolization of colorectal metastases shares similarities with transarterial chemoembolization performed for hepatocellular carcinoma and other chemoembolic-responsive tumor pathologic proceses. A high first-pass effect of cytotoxic agents, augmented by prolonged intracellular drug levels from embolic effects on tumor vasculature, produces a high rate of local response. However, in the United States, a relative paucity of data to support chemoembolization for colorectal metastases has limited broader use. Limited prospective data of transarterial chemoembolization for colon metastases have led some third-party payors to decline reimbursement, citing that the therapy is still considered investigational or experimental. The most recent treatment guidelines of the National Comprehensive Cancer Network (2) consider arterially directed therapies such as chemoembolization a ‘‘category 3’’ recommendation (ie, major disagreement that the intervention is appropriate) based on insufficient data and variations in techniques among institutions. Several retrospective studies performed in the 1990s in the United States suggested that transarterial chemoembolization extends survival time in patients with colorectal metastases without significant toxicity. These include the Puget Sound Oncology Group (3) and Northwestern University studies (4). The patients in these studies were not naive to systemic chemotherapy, and response rates of 63%–70% were observed, with median survival times of 14–29 months. These studies added to previous encouraging results during a time when median survival for patients with metastatic colon carcinoma to the liver was 8–12 months with conventional 5-fluorouracil (5-FU)/ leucovorin chemotherapy (5,6). Since that time, the paradigm of chemotherapy for these patients has significantly changed. The response rates to first-line chemotherapy with 5-FU/leucovorin/oxaliplatin (FOLFOX) and 5-FU/leucovorin/irinotecan (FOLFIRI) have increased to 50%–65%, compared with 30% for 5-FU/leucovorin (2). In addition, incremental benefits of the addition of biologic therapies such as bevacizumab, cetuximab, and panitumumab to cytotoxic regimens have extended survival times even further. The number of treatment algorithms supported by randomized trials has increased treatment options for patients who show disease progression with firstor second-line therapies or develop drug-related toxicities (7–11). Nevertheless, there remains an important subset of patients who show disease progression or recurrence after sequential systemic therapies, and transarterial therapies have the potential to further improve survival outcomes while maintaining quality of life. More recent experience with transarterial chemoembolization from the author’s institution (12), which achieved a median survival of 27 months, indicates that salvage therapy for metastatic colon carcinoma continues to hold promise. Other transarterial therapies have emerged in the past decade that are gaining broader acceptance for colon metastases, particularly yttrium-90 radioembolization. Although radioembolization was initially used as salvage therapy for patients following sequential chemotherapy (13,14), several prospective randomized trials are under way to evaluate the incremental benefit of radioembolization administered concurrently with firstor second-line chemotherapy. Technologic improvements in transarterial chemoembolization have also emerged through the use of drug-eluting beads. These agents may enable transarterial chemoembolization to become better tolerated by patients and provide more controlled drug delivery to tumor cells while using cytotoxic agents with higher response rates (eg, The author has not identified a conflict of interest. From the Section of Interventional Radiology, Department of Radiology, University of Pennsylvania School of Medicine, Penn Presbyterian Medical Center, 39th and Market Sts., Philadelphia, PA 19104. Received October 15, 2012; accepted October 16, 2012. Address correspondence to T.W.I.C.; Email: timothy.clark@uphs.upenn.edu

Cellular Pharmacology and Potency of HIV-1 Nucleoside Analogs in Primary Human Macrophages

Understanding the cellular pharmacology of antiretroviral agents in macrophages and subsequent correlation with antiviral potency provides a sentinel foundation for definition of the dynamics between antiretroviral agents and viral reservoirs across multiple cell types, with the goal of eradication of HIV-1 from these cells. Various clinically relevant nucleoside antiviral agents, and the integrase inhibitor raltegravir, were selected for this study. The intracellular concentrations of the active metabolites of the nucleoside analogs were found to be 5- to 140-fold lower in macrophages than in lymphocytes, and their antiviral potency was significantly lower in macrophages constitutively activated with macrophage colony-stimulating factor (M-CSF) during acute infection than in resting macrophages (EC(50), 0.4 to 9.42 μM versus 0.03 to 0.4 μM, respectively). Although tenofovir-treated cells displayed significantly lower intracellular drug levels than cells treated with its prodrug, tenofovir disoproxil fumarate, the levels of tenofovir-diphosphate for tenofovir-treated cells were similar in lymphocytes and macrophages. Raltegravir also displayed significantly lower intracellular concentrations in macrophages than in lymphocytes, independent of the activation state, but had similar potencies in resting and activated macrophages. These data underscore the importance of delivering adequate levels of drug to macrophages to reduce and eradicate HIV-1 infection.

Early Molecular Response to Imatinib in CP-CML Patients: The Significance of Early Dose Intensity and OCT-1 Activity in Responders and Efficacy of Dose Escalation and Switch to Nilotinib in Non-Responders

Abstract Abstract 693 Background: Accurate and early response prediction in CML patients treated with imatinib (IM) will facilitate timely therapeutic intervention in those patients predicted to respond poorly. The response of CP-CML patients to imatinib can be predicted by the BCR-ABL level at 3 months (early molecular response – EMR) with a level of ≥ 10% (IS) (EMR failure) associated with inferior outcomes. Response can also be predicted by OCT-1 activity (OA); a measure of active transport of IM into target leukemic cells. In the ALLG TIDEL II study patients were treated with 600mg of IM upfront, and dose escalate (800mg) if IM blood levels are <1000ng/ml at day 21. Patients were also dose escalated or switched to nilotinib for failure to achieve, or loss of, specified time dependent molecular milestones, including EMR failure. The aims of this study were to (1) assess the predictive value of EMR (2) assess the impact of factors which likely contribute to EMR such as dose intensity and OA, and (3) assess factors that modify the risk profile within the cohort who achieve EMR. Results: 210 patients were enrolled to TIDEL II. For this analysis 200 patients were analysed based on the availability of complete OA, day 21 blood levels and 3 month BCR-ABL% data. EMR was associated with a significantly higher rate of major molecular response (MMR) compared to that of patients with EMR failure (n=25/200) at both 12 (70% vs 20% p<0.001) and 24 months (82% vs 24% p<0.001), and a significantly higher event free survival (EFS)^ (83% vs 60%, p=0.013). Similarly, patients with high OA achieved MMR at a higher rate than those with low OA (12 mo 73% vs 53% p=0.003 and 24 mo 82% vs 66% p<0.001), and had significantly higher EFS (89% vs 71% p=0.011). IM drug levels at day 21 were significantly lower in patients with EMR failure (median 1050 vs 1620ng/ml: p=0.004). Levels <1000ng/ml were associated with a significantly higher rate of EMR failure (26% compared with 9% if levels >1000ng/ml p<0.001). Importantly, the negative impact of low IM levels was confined to patients with low OA. EMR failure was no different in patients with high OA regardless of drug level (17% vs 9% p>0.05), whereas 35% of patients with low OA and IM levels <1000 ng/ml had EMR failure compared to 11% in patients with low OA and satisfactory drug levels. (p=0.008). This suggests that IM dose and the intracellular level of drug achieved (OA) are key determinants of the 3 month response. 22/25 patients with EMR failure remained on study at 6 months, 21 of whom were either dose increased or switched to nilotinib. Four of 8 patients with EMR failure and high OA achieved 1% BCR-ABL (IS) by 6 months, compared to 1/14 (7%) of patients with low OA and EMR failure. This suggests that early intervention in patients with EMR failure may not be effective in patients with low OA. Interestingly, the risk of events for patients with EMR failure, compared to those who achieve EMR but have low OA was equally high (EFS; 60% v 74% p=0.280). Importantly, 5/6 (83%) patients who transformed had low OA however 3/5 had achieved EMR. Similarly, the risk of mutation development was significantly associated with low OA, and not predicted by EMR (Table 1). While no patient with EMR failure achieved MR4.5 by 24 months, there was a significant difference in the proportion of patients achieving MR4.5, based on OA, within the cohort of patients who achieve EMR (p=0.003) (Table 1). Conclusion: Despite early intervention to increase dose intensity in cases of low drug levels or EMR failure (dose escalation and/or switch to nilotinib), OA, drug levels, and EMR all remain strong predictors of outcome. Importantly the achievement of EMR does not represent a safe haven for patients with low OA. These data suggest that optimising outcomes in high-risk CML cases may not be achievable even with early proactive intervention. Hence, the use of OA and other potential biomarkers at diagnosis may better identify patients with high-risk CML in whom therapy, other than currently available TKIs, may be required. Disclosures: White: Novartis Oncology: Honoraria, Research Funding; BMS: Research Funding; CSL: Research Funding. Yeung:Novartis Pharmaceuticals: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Grigg:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Membership on an entity's Board of Directors or advisory committees; CSL: Research Funding.