PDF file, 785KB, High dose TPI-287 inhibited proliferation and increased acetylated tubulin in primary tumors while low dose TPI-287 had no effect on brain metastasis prevention.

Supplementary Tables 1-2 from OSI-930: A Novel Selective Inhibitor of Kit and Kinase Insert Domain Receptor Tyrosine Kinases with Antitumor Activity in Mouse Xenograft Models

Supplementary Figures 1-3 from OSI-930: A Novel Selective Inhibitor of Kit and Kinase Insert Domain Receptor Tyrosine Kinases with Antitumor Activity in Mouse Xenograft Models

PDF file, 74KB, Pharmacokinetics for TPI-287 in rodents.

Abstract Purpose: This study aimed to: (1) evaluate the antitumor efficacy of a controlled release cabazitaxel cellulose nanoparticle (CBZed-Nano) in an orthotopic bone model of docetaxel-resistant LNCaP C4-2B prostate cancer, and (2) confirm the efficacy of dose preparation by scalable instrumentation (NanoAssemblr), in a subcutaneous PC3 prostate xenograft model. Methods: mPEG-OH and cabazitaxel (CBZ) were coupled to a carboxymethylcellulose acetate polymer to yield CBZed-Nano, according to established coupling and purification methods (1). Particles were prepared by nanoprecipitation of polymer into saline by either a manual vortex technique (1) or a NanoAssemblr™ (NA) microfluidic-based instrument. Particles were purified by dialysis and sterile filtration (1). Particle size was measured by a DLS Malvern Zetasizer, morphology was screened by TEM, and residual substances were measured by LC. Male NOD-SCID mice were inoculated via intrafemoral injection with docetaxel-resistant LNCaP C42B prostate cancer. One day after inoculation, mice were treated with vortex prep CBZed-Nano (55 mg/kg) or CBZ (2 mg/kg) on a q1,5,9d schedule. Survival curves were generated (n = 10 mice per group). To directly compare preparation technique, nude mice bearing s.c. PC3 prostate cancer xenografts were treated at Bolder BioPath (Boulder, US) with CBZed-Nano (NA and vortex prep, 85 to 142 mg/kg) and CBZ (15-25 mg/kg) on a q1,5,9d schedule. Results: The CBZed-Nano polymer conjugate contained 36 wt% CBZ and 11 wt% PEG, with residual free CBZ < 0.01 wt% of CBZ content. Particles prepared by vortex and NA were 94 and 70 nm respectively (by DLS). By TEM analysis, NA particles exhibit a more uniform spherical morphology. Mice bearing docetaxel resistant LNCaP C42B tumors exhibited a partial response to CBZ treatment, with a median survival of 61 days, compared to 34 days for vehicle controls. In contrast, mice treated with CBZed-Nano exhibited a significantly improved response, with 70% durable cure. Efficacy was observed regardless of CBZed-Nano preparation technique. Mice treated with CBZ exhibited a 38-63% durable cure in the 60, 75, and 100% MTD groups. In comparison, mice treated with CBZed-Nano exhibited a 92-100% durable cure, confirming both the superior activity of CBZed-Nano relative to free CBZ, and confirming antitumor activity of the scalable NanoAssemblr production. Conclusion: The CBZed-Nano preparation technique is scalable, with manual and NanoAssemblr™ produced dose being equally efficacious in a PC3 prostate cancer model. Significant efficacy (70% durable cure) in a docetaxel-resistant prostate cancer of the bone model was also observed. The data suggests that CBZed-Nano is more tolerable than CBZ, and that it may overcome taxane-resistant solid tumor indications. (1) Bioconjugate Chem 22 2011, 2474-2486 (2) Biomaterials 33 2012, 3931-3941 Citation Format: Joseph Bteich, Bryan Hoang, Elijus Undzys, Mohammed Mohammed, Ai Lin Su, Kevin Ou, David Emerson, Kenneth Sokoll, Shyh-Dar Li, Mohit Trikha, Henry Lowman, Mark J. Ernsting. A production-scalable cabazitaxel cellulose nanoparticle exhibits significant efficacy in a bone model of docetaxel-resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1342.

Abstract Brain metastases of breast and other cancers remain resistant to chemotherapeutic regimens that are effective systemically, in part due to the blood–brain barrier. We report that TPI-287, a new microtubule-stabilizing agent, displays in vitro cytotoxic activity similar to taxanes and epothilones. Unlike the taxanes, TPI-287 is permeable through the blood–brain barrier. Brain-to-plasma ratios of TPI-287 after a single injection typically exceeded one and were as high as 63.8 in the rat and 14.1 in the mouse. A brain-tropic derivative of the MDA-MB-231 triple-negative breast cancer cell line, 231-BR, was used to test whether TPI-287 may be efficacious at preventing or treating brain metastases. TPI-287 had growth inhibitory effects comparable with paclitaxel when 231-BR tumor cells were injected into the mammary fat pad. Brain metastatic colonization was determined by intracardiac injection of 231-BR cells, with treatment beginning on day 3 to 4 postinjection, culminating in a histologic count of brain metastases in brains necropsied days 25 to 28 postinjection. In this assay, paclitaxel, ixabepilone, and nab paclitaxel did not have significant inhibitory activity. TPI-287 was ineffective in the same assay using a 6 mg/kg every week schedule; however an 18 mg/kg dose delivered on days 3, 7, and 11 significantly reduced the outgrowth of brain metastases (55% reduction, P = 0.028) and reduced proliferation in brain metastases (16% reduction, P = 0.008). When TPI-287 treatment was delayed until days 18, 22, and 26 postinjection, efficacy was reduced (17% reduction, not significant). These data suggest that TPI-287 may have efficacy when administered early in the course of the disease. Mol Cancer Ther; 11(9); 1959–67. ©2012 AACR.

1441 The treatment of glioblastoma with chemotherapeutic agents is hampered by the inability of most drugs to sufficiently penetrate the blood brain barrier and deliver adequate drug exposure. Taxanes have been an important class of agents in cancer treatment, but have contributed little to therapeutic advances in glioblastoma treatment. TPI 287 is a highly lipophylic third generation taxane which avoids the GP170 drug efflux pump, is orally bioavailable, and penetrates the blood brain barrier and accumulates in the brain of rats and mice. After a single 20mg/kg iv dose, TPI 287 brain/plasma ratio increases to 63.79 at 96h post dose, with 261 nM drug concentration in the brain of mice.The mean residence time for TPI 287 exposure was 42.5h and 17.6h for brain and plasma, respectively. In cell culture the U251 glioblastoma tumor cell line was shown to be very sensitive to TPI 287 with a 6 nM IC50 in standard MTS cytotoxicity assay. In addition, subcutaneous tumors were also sensitive to a single Q4Dx3 cycle of treatment by iv. and po. drug administration. In the U251 intracranial glioblastoma antitumor efficacy study TPI 287 as single agent or in combination with temozolomide increased survival of treated mice p

A192 Tumor resistance involves several factors including the extrusion of chemotherapeutic agents by constitutively expressed or upregulated ABC transporter pumps such as MDR1 (P-gp 170 kDa) which is capable of exporting a variety of hydrophobic compounds before they reach their drug targets. This pump is also found in the cells of the intestine, liver, kidney, and blood-brain barrier, sites which affect the ability of the drug to reach the tumor. TPI 287 is a taxane derivative with previously shown enhanced oral bioavailability and penetrance of the blood-brain barrier in rodents. Because of the molecular complexity of the P-gp pump, we explored several different assays to show that TPI 287 behaves differently from paclitaxel and ortataxel. In a 72 hr proliferation assay, mdr1(-) tumor cells from several different tissues (breast, colon, brain, lung, ovary) exhibit paclitaxel IC50s below 0.1nM and TPI 287 IC50s in the 0.5-10nM range. However, in mdr1(+) tumor cells (as determined by Western blot), the paclitaxel IC50 is 50-200 times higher than that for TPI 287. Further, TPI 287 blocks the efflux of Rhodamine 123 (an MDR1 substrate) from mdr1(+) cells, as does ortataxel, whereas paclitaxel and docetaxel do not. This fluorescence-based assay with the SK-N-FI(mdr1+) and MV522/mdr1+ cell lines generates a 30-40 fold higher Blocking Factor (BF) for TPI 287 versus docetaxel or paclitaxel. TPI 287 and ortataxel generate similar high BFs for R123 efflux in MV522/mdr1+ cells. In contrast, TPI 287 does not block efflux of an MRP1 substrate from an mrp1+ cell type any more than docetaxel. In a luciferase assay, with cell-free P-gp membranes, which measures the ATPase activity of the P-gp pump, TPI 287 behaves differently from paclitaxel and ortataxel. TPI 287 causes a 2-3 fold higher consumption of ATP than paclitaxel at any given concentration, and ortataxel stimulates a very low level of ATPase activity. When tested against the high verapamil stimulation of the P-gp ATPase, Cyclosporin-A and ortataxel cause a dose dependent decrease in the level of ATPase activity, but paclitaxel and TPI 287 have little effect. In addition, Caco-2 and MDCKII/mdr1+ permeability analyses show that paclitaxel is a good substrate of the P-gp pump in the basolateral to apical direction (5-fold greater activity than in the apical to basal direction in MDCKII/mdr1+), but TPI 287 shows no such polarization, suggesting that it is not a substrate of the active P-gp transport system in these cells. In xenograft studies of MDR1 tumors in nude mice, TPI 287 has shown efficacy where paclitaxel has none, suggesting avoidance or modulation of the tumor cell MDR1 pump. And in a pancreatic tumor xenograft, oral administration of TPI 287 had very similar efficacy to IV administration, suggesting avoidance of the MDR1 pump in brush border cells of the intestine. These data demonstrate that TPI 287 is distinct from other taxanes and functions in mdr1+ tumor cells by avoidance of the P-gp efflux mechanism and may act as a blocking agent in some circumstances in order to reduce drug efflux. This profile further supports the development of TPI 287 as an oral anticancer agent.

493 The new taxane TPI 287 has demonstrated the ability to avoid the MDR1 efflux pump in transport assays, differential cell cytotoxicity assays and in xenografts. Further characterization of TPI 287 was performed in rat pharmacokinetic studies comparing iv to po administration. Sprague Dawley rats weighing 225 to 300 g were fitted with a jugular vein catheter and dosed with either 9 mg/kg TPI 287 po or 6 mg/kg TPI 287 iv. Two different formulations of TPI 287 were dosed po, Group 1: a suspension in 1% carboxymethyl cellulose, 0.5% Tween 80 in water, and Group 2: a soluble formulation of 5% ethanol, 5% cremophor in D5W, (po; iv). Bioanalytical sample analysis was performed by a validated LC/MS/MS method, and analysis of data was performed with WinNonlin version 4.0. The total exposure to TPI 287 was assessed by estimating AUClast, AUCinf and AUCinf/Dose. The percent of AUCinf extrapolated beyond Clast ranged from 3.2% to 14.7% for all individual profiles following po dose administration and 8.7% to 22.9% following iv administration. These indicators confirmed that the dynamic range of the bioanalytical assay and scheduled blood collection were sufficient to assess and measure exposure. Exposure appeared to be appreciably higher (nearly twice) in females than in males after either oral or iv dosing. There were no significant sex differences with respect to the oral bioavailability. The oral bioavailability of TPI 287 based on AUClast was 26.9% and 27.3% in females and males, respectively, in Group 1, and 36.5% and 38.9% in females and males, respectively, in Group 2. However, test article absorption was approximately 10% greater when formulated in Group 2. The mean elimination half-life (HL) was slightly greater in females than in males, and tended to be longer following oral administration when the test article was formulated in the Group 2. Oral antitumor activity of TPI 287 was demonstrated in DU145 prostate tumor xenografts in nude mice where a dose response effect on tumor growth was seen when dosed on a Q2Dx4 schedule of 10, 12, 14 mg/kg, generating a % TGI of 40%, 51%, and 64%, respectively. Finally, TPI 287 was compared to paclitaxel in cell cytotoxicity assays with three ovarian tumor cell lines. The wild type A2780 ovarian cell line, PTX10 cell line with the tubulin mutation [phenylalanine (TTT) → valine (GTT ) at nucleotide 810] and the PTX22 cell line with mutation [alanine (GCA) → threonine (ACA) at nucleotide 1092]. The results demonstrated a 4-30 fold increase in potency of TPI 287 relative to paclitaxel in the mutant cell line and similar potency in the wild type cell line. In conclusion, these data indicate that TPI 287 has greater in vitro activity against tumor cells with mutant tubulin compared to paclitaxel, and TPI 287 has sufficient oral activity when dosed alone whereas paclitaxel requires co-administration with an MDR1 inhibitor to achieve efficacious exposure.

A series of [2-imidazol-1-yl-2-(6-alkoxy-naphthalen-2-yl)-1-methyl-ethyl]-dimethyl-amines were designed and synthesized as CYP26 inhibitors, serving as retinoic acid metabolic blocking agents (RAMBA’s).

677 The full potential and optimal utilization of targeted kinase inhibitors in combination with standard chemotherapeutic regimens has yet to be realized. OSI-930 potently inhibited the closely-related kinases Kit, KDR, and PDGFRβ in cell-based assays (IC 50 values . A FOLFOX-like regimen (5-FU/ oxaliplatin) was selected for combination studies with OSI-930 in two human CRC models, SW48 and COLO 205. 5-FU was administered at 20 mg/kg IV Day 1-5, oxaliplatin at 10 mg/kg IP on Day 1, and vehicle or OSI-930 200 mg/kg PO administered as maintenance therapy Day 8-21. Treatment with maintenance OSI-930 resulted in enhanced tumor growth inhibition (TGI) and growth delay (GD) compared to vehicle maintenance in both tumor models (SW48: TGI=84.4% compared to 68.9%, GD=6.8 days over vehicle; COLO 205: TGI=70.8% compared to 28.4%, GD=12.4 days over vehicle). In a separate COLO 205 study, OSI-930 administered at 100 mg/kg PO either concomitantly (Day 1-14) or as maintenance therapy (Day 8-21) improved tumor growth delay by 2-3 fold and increased TGI by 15-20% compared to vehicle maintenance. Both dosing schedules were well-tolerated with body weight loss in both groups

To obtain further information concerning the structure of Fc IgG-binding sites on human peripheral blood lymphocytes, enriched T-cells were surface-radioiodinated and treated with nonionic detergent, and the soluble supernatant was submitted to affinity chromatography selecting for components binding complexed IgG. Analysis of eluted material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and two-dimensional electrophoresis demonstrated the major proteins to be of M, 56,000, PI 4.8-5.1 and M, 60,000, PI 5.0-5.6, and these were radiolabeled, indicating an origin in part from the Tcell membrane. While the M. 56,000 band gave positive reactions upon transblotting with antisera to Gc protein, the identity of the M, 60,000 protein remains unknown. Three other components were detected, although with less consistency; M, 78,000, 42,000, and 20,000-25,000, respectively. Immunocytochemical experiments showed that <5% freshly isolated native T-cells were positive with antiserum to human Gc, but, after IgG antibody-coated erythrocyte rosetting, the number of positive cells increased to 15-25%, in close agreement with the percentage of IgG antibody-coated erythrocyte rosette-positive T-cells. These findings therefore indicate that, in addition to interaction with components of M, 60,000, 42,000, and 20,00025,000, complexed IgG binding to Fc, of human peripheral blood T-cells also becomes spatially associated with Gc protein.

The use of liposomal drug delivery systems to improve the therapeutic index of pharmaceutical agents is exemplified by camptothecin-based drugs. This highly active class of anticancer agents possesses a unique mechanism of action with some inherent shortcomings, which might have been solved by liposomal formulation. Recent studies have revealed the protective action, increased tumor delivery and prolonged plasma exposure of these liposomal formulated drugs. These advances in pharmaceutical development could increase the levels of activity of these agents, as well as increase their clinical utility as new emerging anticancer therapies.

Tumor establishment and metastasis are dependent on extracellular matrix proteolysis, tumor cell migration, and angiogenesis. Urokinase plasminogen activator (uPA) and its receptor are essential mediators of these processes. The purpose of this study was to investigate the effect of a recombinant human uPAR antagonist on growth, establishment, and metastasis of tumors derived from human cancer cell lines. A noncatalytic recombinant protein, consisting of amino acids 1-137 of human uPA and the CH2 and CH3 regions of mouse IgG1 (uPA-IgG), was expressed, purified, and shown to bind specifically to human uPAR and to saturate the surface of human tumor cells which express uPAR. Daily i.p. administration of uPA-IgG to nude mice extended latencies of unstaged tumors derived from Lox melanoma and SW48 colon carcinoma cells by 7.7 and 5.5 days, respectively. uPA-IgG treatment did not affect the growth of Lox or KB tumors staged to 200 mg before antagonist treatment commenced. The effect of uPA-IgG on the establishment of micrometastases was assessed in SCID mice. KB head/neck tumor cells were injected in the tail vein and allowed to seed for 48 h before initiation of daily i.p. injections of uPA-IgG for 24 days. The number of lung colonies ranged between 5 and 30% of vehicle-treated mice in two separate experiments. Furthermore, a single 800 μg dose of uPA-IgG administered 1 h prior to tail vein injection of KB cells reduced lung colony formation to just 3.5% of vehicle-treated SCID mice. These data demonstrate that antagonism of uPAR arrested metastasis and inhibited the establishment of primary tumors and micrometastases. Thus, small molecule uPAR antagonists may serve as useful adjuvant agents in combination with existing cancer chemotherapy. © Rapid Science 1998

Four potent, synthetic inhibitors of matrix metalloproteinases (MMPs) were assessed as inhibitors of tumor growth and spontaneous metastasis to the lung. Mat Ly Lu rat prostate tumor, LOX human melanoma and M27 murine Lewis lung tumor were implanted subcutaneously (s.c.) in mice and allowed to grow for 3–12 days. The lungs of the tumor-bearing mice were then removed and implanted s.c. into untreated mice, and the outgrowth of secondary tumors from the implanted lungs measured. The incidence and rate of outgrowth of secondary tumors increased with the length of primary tumor growth, validating these measurements as indices of spontaneous metastasis to the lung. Compounds were tested by sc. implantation of minipumps which delivered compound throughout the period of primary tumor growth and spontaneous metastasis to the lung at steady-state drug concentrations orders of magnitude greater than the concentrations needed to either inhibit collagenase, gelatinase or stromelysin in vitro. Inhibitor treatment slowed the growth of primary s.c. Mat Ly Lu and LOX tumors by 40–60% but had no significant effect on the growth of primary M27 tumors. Surprisingly, inhibitor treatment had no significant effect on the ability of the lung to generate secondary tumors when reimplanted s.c. in untreated mice. Because of the possible importance of cathepsins B, H and L in tumor growth and metastasis, the irreversible inhibitor E-64 was also infused by s.c. minipump. E-64 had no effect on the growth or spontanous metastasis of Mat Ly Lu or M27 tumors.

Antibody reactivity to melanocyte-derived cells was investigated in patients with alopecia areata or totalis by use of Western blot analysis of detergent-solubilized membrane antigens of a human melanoma cell line, M14. Reactivity was detected in the sera of 9 of 27 alopecia areata or totalis patients, 8 of 13 vitiligo patients, and 6 of 24 normal control subjects. Significant differences between patient and control sera were found in the number and distribution of antibody specificities detected. In vitiligo sera, there was an increased prevalence of reactivity to a melanoma antigen of 52,000 mol wt. In contrast, the predominant specificities in alopecia areata sera were for antigens of 74,500 and 70,800 mol wt, and the majority of positive sera were from patients with total hair loss. These findings suggest that autoreactivity to pigmented cells occurs in certain patients with alopecia areata or totalis.