Synthesis and activity of a novel Autotaxin inhibitor-Icodextrin conjugate

© Copyright 2018 American Chemical Society. Autotaxin is an extracellular phospholipase D that catalyses the hydrolysis of lysophosphatidyl choline (LPC) to generate the bioactive lipid lysophosphatidic acid (LPA). Autotaxin has been implicated in many pathological processes relevant to cancer. Intraperitoneal administration of an autotaxin inhibitor may benefit patients with ovarian cancer, however low molecular mass compounds are known to be rapidly cleared from the peritoneal cavity. Icodextrin is a polymer that is already in clinical use because it is slowly eliminated from the peritoneal cavity. Herein we report conjugation of the autotaxin inhibitor HA-155 to icodextrin. The conjugate inhibits autotaxin activity (IC50 = 0.86 ± 0.13 μg mL-1) and reduces cell migration. Conjugation of the inhibitor increased its solubility, decreased its membrane permeability and improved its intraperitoneal retention in mice. These observations demonstrate the first application of icodextrin as a covalently-bonded drug delivery platform with potential use in the treatment of ovarian cancer

imulations showing long-term tumor xenograft response to combination therapy.

<p>Following the experimental protocol in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081582#pone.0081582-Witham1" target="_blank">[13]</a>, the administration of a weekly dose of 30 mg/kg carboplatin together with a daily dose of 100 mg/kg ABT-737 is simulated, and simulations allowed to run until tumor cell numbers averaged over 7 days (the period of carboplatin administration) achieve a steady-state. <b>A</b>, Predicted steady states of average tumor cell numbers as a function of carboplatin infusion time . As is increased from 0 hours (corresponding to a bolus dose) to 120 hours, the average tumor cell number steady-state decreases rapidly to (see inset), and then increases, with minimal survival of tumor cells predicted for . <b>B</b>, Predicted values of , the length of time therapy must be administered to achieve minimal residual disease (defined as cell remaining) as is varied between 1 and 25 hours. A minimum value of days is predicted for weekly carboplatin infusions lasting 8 hours. <b>C</b>, Minimum values of are predicted to decrease to 215 days as , the arrested cell sensitivity to intracellular Bax is increased. In all cases, a carboplatin infusion time of 8 hours minimized the cure time.</p

Model schematic and fits to experimental data.

<p><b>A</b>, Model schematic. Ovarian cancer cells, proliferate and undergo apoptosis at a rate dependent on intracellular Bax concentration. Administration of carboplatin induces DNA damage, leading to cell cycle arrest. Arrested cells, subsequently undergo apoptosis at a rate proportional to the amount of DNA damage sustained at the time of arrest and on their intracellular Bax concentration, and may also recover to the proliferating population. The rates at which and undergo cell death are elevated on application of ABT-737, which leads to increased levels of intracellular Bax. <b>B</b>, Intracellular reaction diagram of the heterodimerization reaction between Bcl-xL and Bax molecules, and the inhibition of Bcl-xL by intracellular ABT-737 . <b>C, D</b>, Fit to time-course tumor xenograft growth inhibition data taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081582#pone.0081582-Witham1" target="_blank">[13]</a>. Briefly, ovarian cancer (IGROV-1 cell) xengrafts were established in mice and treatment started 19 days post-transplantation. Levels of tumor growth inhibition were recorded periodically. Experimental data is represented by black squares and red triangles, while solid curves show best fits. Values represent mean and standard deviation (from experimental data). <b>C</b>, Weekly treatment with vehicle (black squares and curve) or daily treatment with 100 mg/kg of ABT-737 (red triangles and curve). <b>D</b>, Weekly treatment with 30 mg/kg carboplatin (black squares and curve) or a combination of carboplatin and ABT-737 (red triangles and curve).</p

Optimizing carboplatin and ABT-737 doses when given in combination.

<p><b>A</b>, Predicted Combination Index (CI) values computed for a desired tumor growth inhibition level of 67%, for various combinations of carboplatin and ABT-737. Following the experimental protocol in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081582#pone.0081582-Witham1" target="_blank">[13]</a>, treatment is initiated at 19 days post-transplantation and continued for 4 weeks. Simulations indicate that a combination of 17.8 mg/kg carboplatin given weekly combined with a daily dose of 86.5 mg/kg ABT-737 minimizes the CI and hence maximizes the synergy between the two drugs (dashed lines). <b>B</b>, Predicted tumor growth dynamics corresponding to the optimal dose combination found above. Plots show tumor cell numbers averaged over 7 days (the period of carboplatin administration) versus time. <b>C</b>, Predicted time-courses of tumor growth inhibition levels as compared to the control (no treatment) case, corresponding to the optimal dose combination found above.</p

Exploiting the Synergy between Carboplatin and ABT-737 in the Treatment of Ovarian Carcinomas

<div><p>Platinum drug-resistance in ovarian cancers mediated by anti-apoptotic proteins such as Bcl-xL is a major factor contributing to the chemotherapeutic resistance of recurrent disease. Consequently, concurrent inhibition of Bcl-xL in combination with chemotherapy may improve treatment outcomes for patients. Here, we develop a mathematical model to investigate the potential of combination therapy with ABT-737, a small molecule inhibitor of Bcl-xL, and carboplatin, a platinum-based drug, on a simulated tumor xenograft. The model is calibrated against <i>in vivo</i> experimental data, wherein xenografts established in mice were treated with ABT-737 and/or carboplatin on a fixed periodic schedule. The validated model is used to predict the minimum drug load that will achieve a predetermined level of tumor growth inhibition, thereby maximizing the synergy between the two drugs. Our simulations suggest that the infusion-duration of each carboplatin dose is a critical parameter, with an 8-hour infusion of carboplatin given weekly combined with a daily bolus dose of ABT-737 predicted to minimize residual disease. The potential of combination therapy to prevent or delay the onset of carboplatin-resistance is also investigated. When resistance is acquired as a result of aberrant DNA-damage repair in cells treated with carboplatin, drug delivery schedules that induce tumor remission with even low doses of combination therapy can be identified. Intrinsic resistance due to pre-existing cohorts of resistant cells precludes tumor regression, but dosing strategies that extend disease-free survival periods can still be identified. These results highlight the potential of our model to accelerate the development of novel therapeutics such as BH3 mimetics.</p></div

Intrinsic resistance to carboplatin therapy.

<p>A small fraction (1 in 60,000) of carboplatin-resistant cells is assumed to be present prior to treatment initiation. The treatment of a late-stage tumor is simulated. Plots show 7 day-averages of carboplatin-sensitive (blue curve), carboplatin-resistant (red curve) and total (dashed black curve) tumor cell numbers versus time, as treatment strategy is varied. In all cases, therapy is administered for a period of 1 year. Increasing the weekly bolus dose of carboplatin administered as a single agent from <b>A</b>, 30 mg/kg, <b>B</b>, 600 mg/kg, to <b>C</b>, 1300 mg/kg, cannot prevent the onset of carboplatin-resistance, with overall tumor size returning to pre-treatment levels eventually. <b>D, E, F</b>, In fact, a combination therapy of 30 mg/kg carboplatin delivered weekly as a bolus (<b>D</b>) or via 8-hour infusion (<b>E, F</b>) together ABT-737 delivered daily at a dose of 100 mg/kg (<b>D, E</b>) or 500 mg/kg (<b>F</b>) is also unable to prevent the emergence of resistance. However, combination therapy is predicted to result in partial tumor growth control, with a steady-state tumor size reaching 65.2% of pre-treatment levels for a combination of 30 mg/kg carboplatin and 100 mg/kg ABT-737 (<b>D, E</b>). Increasing ABT-737 dosage to 500 mg/kg is predicted to induce extended periods of disease-free survival, as evidenced by a dip in the total tumor size graph (<b>F</b>).</p

The emergence of acquired resistance to carboplatin therapy.

<p>DNA-mismatch repair in arrested cells recovering to the proliferating population is assumed to be the cause of resistance. The treatment of a late-stage tumor is simulated. Plots show 7 day-averages of carboplatin-sensitive (blue curve), carboplatin-resistant (red curve) and total (dashed black curve) tumor cell numbers versus time, as treatment strategy is varied. In all cases, therapy is administered for a period of 1 year. Increasing the weekly bolus dose of carboplatin administered as a single agent from <b>A</b>, 30 mg/kg, <b>B</b>, 300 mg/kg, to <b>C</b>, 800 mg/kg, cannot prevent the emergence of carboplatin-resistance. <b>D</b>, In fact, a weekly dose of 1300 mg/kg carboplatin is required to induce tumor regression. <b>E</b>, A combination of 30 mg/kg carboplatin delivered weekly as a bolus together with 100 mg/kg ABT-737 delivered daily is predicted to prevent the emergence of resistance and lead to tumor growth control at 6.5% of pre-treatment levels. <b>F</b>, The same combination dose, with carboplatin delivered via a 8-hour infusion is predicted to induce tumor regression within 150 days.</p

The BH3 Mimetic Obatoclax Accumulates in Lysosomes and Causes Their Alkalinization

<div><p>Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive preclinical and clinical evaluation as a cancer therapeutic. Despite this, it is clear that obatoclax has additional pharmacological effects that contribute to its cytotoxic activity. It has been claimed that obatoclax, either alone or in combination with other molecularly targeted therapeutics, induces an autophagic form of cell death. In addition, obatoclax has been shown to inhibit lysosomal function, but the mechanism of this has not been elucidated. We have evaluated the mechanism of action of obatoclax in eight ovarian cancer cell lines. Consistent with its function as a BH3 mimetic, obatoclax induced apoptosis in three cell lines. However, in the remaining cell lines another form of cell death was evident because caspase activation and PARP cleavage were not observed. Obatoclax also failed to show synergy with carboplatin and paclitaxel, chemotherapeutic agents which we have previously shown to be synergistic with authentic Bcl-2 family antagonists. Obatoclax induced a profound accumulation of LC-3 but knockdown of Atg-5 or beclin had only minor effects on the activity of obatoclax in cell growth assays suggesting that the inhibition of lysosomal function rather than stimulation of autophagy may play a more prominent role in these cells. To evaluate how obatoclax inhibits lysosomal function, confocal microscopy studies were conducted which demonstrated that obatoclax, which contains two basic pyrrole groups, accumulates in lysosomes. Studies using pH sensitive dyes demonstrated that obatoclax induced lysosomal alkalinization. Furthermore, obatoclax was synergistic in cell growth/survival assays with bafilomycin and chloroquine, two other drugs which cause lysosomal alkalinization. These studies explain, for the first time, how obatoclax inhibits lysosomal function and suggest that lysosomal alkalinization contributes to the cytotoxic activity of obatoclax.</p></div

Combinations of obatoclax with carboplatin or paclitaxel.

<p><b>A</b>. Cells were treated with obatoclax and either carboplatin or paclitaxel, combined at the ratio of the IC₅₀s determined in cell growth/survival assays with the individual drugs. After 72 hours, the surviving cell number was estimated by staining with SRB and the combination index calculated as described in the methods. The results are expressed as the combination index at fraction affected = 0.5 (mean ± S.D., n = 3–10) and were significantly different from 1.0 where indicated (*, <i>P</i> < 0.05, t-test using Welch’s correction). <b>B</b>. Scheduled combinations of obatoclax and carboplatin. Cells were exposed to: a) obatoclax for 48 hours then carboplatin for 48 hours; b) carboplatin for 48 hours, then obatoclax for 48 hours; c) carboplatin and obatoclax for 48 hours then culture medium for a further 48 hours; d) culture medium for 48 hours, then carboplatin and obatoclax for 48 hours. In each case, the cells were treated with 18 different concentrations of carboplatin and obatoclax combined at the ratio of their IC₅₀s determined in experiments with the individual drugs. Combination indices were determined at fraction affected = 0.5 mean ± S.D., n = 3–4) and were significantly different from 1.0 where indicated (*, <i>P</i> < 0.05, t-test using Welch’s correction).</p

Obatoclax induces cleavage of PARP and accumulation of LC-3.

<p>Cells were treated with obatoclax for 48 hours at the indicated multiple of the IC₅₀ determined in cell proliferation assays (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150696#pone.0150696.t001" target="_blank">Table 1</a>). Lysates were prepared and PARP cleavage and accumulation of LC-3 was determined by immunoblotting. LC3-I and LC3-II were almost impossible to resolve in in most cell lines due to the very robust increase in LC3, although LC3-I (arrowed) and LC3-II could be distinguished in Ovcar-3 cells and obatoclax induced accumulation of LC3-II. The results shown are representative of 2–4 experiments per cell line.</p