In vitro and in vivo antitumor activity of methotrexate conjugated to human serum albumin in human cancer cells

To avoid systemic toxicity of the cytotoxic drug methotrexate (MTX) and to improve tumor selectivity, MTX was bound to human serum albumin (HSA) as a drug carrier. To understand more about the mechanism of action of MTX conjugated to HSA (MTX-HSA), the uptake of MTX-HSA into the cell was determined as well as the effect of MTX-HSA on thymidylate synthase (TS), cell cycle distribution, and cell proliferation. Different uptake kinetics were observed for [(3)H]MTX and [(3)H]MTX-HSA. However, similar uptake kinetics were measured for (125)I-HSA and (125)I-MTX-HSA (2.1 and 1.8 pmol/10(7) cells/h when cells were treated with 10 micro M (125)I-HSA and (125)I-MTX-HSA, respectively), suggesting that MTX-HSA enters the cells by albumin-mediated endocytosis. We observed no effect of MTX-HSA on TS when folate receptor-expressing KB cells were treated for 4 h (IC(50), >50 micro M). However, 24 h after incubation, MTX-HSA inhibited TS with an IC(50) of 6.9 micro M. In addition, we found that MTX-HSA had a delayed effect on the cell cycle compared with MTX and that this effect could be inhibited with the lysosomal inhibitor methylamine, suggesting that MTX-HSA activity is dependent on lysosomal processes. The proliferation of different wild-type and MTX-resistant tumor cell lines was inhibited at IC(50) concentrations between 2 and 78 micro M, respectively. MTX-HSA accumulates in vivo in the tumor tissue. Local concentrations of 18-29 micro M were measured, which are effective antiproliferative concentrations as determined in vitro. We also investigated the antitumor activity of MTX-HSA in vivo in different human tumor xenografts grown s.c. in nude mice. Fourteen tumors from eight different tissues were tested. Nine of 14 tumors (64%) showed a clear response with tumor inhibition, stasis, or regression; 5 of 14 (36%) gave a moderate response with tumor growth delay or no response. In conclusion, MTX-HSA is effectively taken up by the cells via albumin receptor- or folate receptor-mediated endocytosis and time-dependently released as an active compound into the cytosol to exert an inhibiting effect on TS and to induce cell cycle alterations. In vivo, effective concentrations of MTX-HSA were reached in tumor tissue to exhibit antitumor activity

Targeting Transforming Growth Factor beta 2 (TGF-β2) mRNA with ISTH0036 as Novel Therapeutic Intervention in Neovascular Ocular Disease

TITLE: Targeting Transforming Growth Factor beta 2 (TGF-β2) mRNA with ISTH0036 as Novel Therapeutic Intervention in Neovascular Ocular Disease ABSTRACT BODY: Purpose: Although anti-VEGF therapeutics represent a significant step forward in treating neovascular eye diseases, insufficient response to anti-VEGF-treatments represents a significant clinical problem. Therefore, novel therapies with alternative molecular mechanisms of action are desirable. We recently reported potent activity of ISTH0036, a LNAmodified antisense oligonucleotide specifically targeting the sequence of TGF-β2 mRNA, in a murine glaucoma filtration surgery model. The aim of the present studies was to evaluate the therapeutic potential of ISTH0036 in a murine model of laser-induced choroidal neovascularization (CNV). Methods: Effect of ISTH0036 on TGF-β2 mRNA expression and cell migration was explored on murine astrocytes to ensure target engagement and pharmacological effect in these cells. The posterior eye tissue distribution was determined following intravitreal (IVT) injection of DIG-labeled ISTH0036. Subsequently, the effect of the drug was studied in a murine CNV model by performing 3 laser-induced burns on the Bruch’s membrane of the eyes (n= 5-9 mice/group). IVT injections of test items were performed directly after laser treatment. Experimental outcomes included inflammation (CD45 staining, on day 5), presence of CNV pathology (FA and OCT, on days 5-14), angiogenesis (FITC-dextran staining, on day 14), and fibrotic processes (collagen I deposition, on day 28). Results: ISTH0036 showed potent downregulation of target mRNA and inhibition of migration of murine astrocytes in cell based assays. ISTH0036 was detectable in most of the tested posterior eye tissues; including sclera, retina, choroid and ciliary body. In the murine CNV model, ISTH0036 was shown to significantly reduce the process of angiogenesis in a dose-and sequence-dependent manner. Beneficial inhibitory effect of ISTH0036 on the progression of CNV pathology and vascular leakage was demonstrated. In addition, and in contrast to existing anti-VEGF treatments, deposition of collagen was significantly reduced with ISTH0036. Conclusions: Our results confirm that targeting TGF-β2 in CNV may represent an attractive modality for the effective treatment of neovascular ocular diseases such as wet AMD and diabetic retinopathy. Demonstrated potent inhibitory effects on vascular leakage, angiogenesis and fibrosis support further clinical development of ISTH0036 in these relevant indications.status: publishe

Modulation of signaling enhances the efficacy of the combination of satraplatin and erlotinib

The active metabolite (JM118) of the oral platinum analog satraplatin (JM216) was investigated for potential synergism with erlotinib, an epidermal growth factor receptor (EGFR) inhibitor. JM118 sensitivity of 7 cancer cell lines (ovarian: 2008, A2780; colon: Lovo92, WiDr; lung: A549, SW1573; epidermoid: A431), was enhanced most pronounced when JM118 preceded erlotinib, which was associated with increased formation of DNA-platinum adducts. The combination increased G2/M phase accumulation and enhanced apoptosis. JM118 increased the phosphorylation of the cell cycle proteins CDK2 and CHK1 after 24 hr exposure. JM118/erlotinib enhanced Erk and Akt phosphorylation after 2 hr. JM118 significantly decreased the phosphorylation of PTEN, VEGFR, EPHA1, ERBB4, FGF-R, andSTAT3 by 20 (PTEN) to >90% (STAT3). Erlotinib enhanced the effects of JM118, even in cells with mutations in Ras. The mechanism of synergy involved a combination of effects on platinum-DNA adduct formation, cell cycle distribution and signalin

CONSORT flow diagram.

<p>Overview indicates the patient allocation.</p

First-in-human phase I study of ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-β2), in subjects with open-angle glaucoma undergoing glaucoma filtration surgery

<div><p>Purpose</p><p>To evaluate the safety and tolerability of intravitreal ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-β2), in patients with primary open angle glaucoma (POAG) undergoing trabeculectomy (TE; glaucoma filtration surgery).</p><p>Methods</p><p>In this prospective phase I trial glaucoma patients scheduled for TE with mitomycin C (MMC) received a single intravitreal injection of ISTH0036 at the end of surgery in escalating total doses of 6.75 μg, 22.5 μg, 67.5 μg or 225 μg, resulting in calculated intraocular ISTH0036 concentrations in the vitreous humor of approximately 0.3 μM, 1 μM, 3 μM or 10 μM after injection, respectively. Outcomes assessed included: type and frequency of adverse events (AEs), intraocular pressure (IOP), numbers of interventions post trabeculectomy, bleb survival, visual acuity, visual field, electroretinogram (ERG), slit lamp biomicroscopy and optic disc assessment.</p><p>Results</p><p>In total, 12 patients were treated in the 4 dose groups. Main ocular AEs observed were corneal erosion, corneal epithelium defect, or too high or too low IOP, among others. No AE was reported to be related to ISTH0036. All other safety-related analyses did not reveal any toxicities of concern, either. The mean medicated preoperative IOP at decision time-point for surgery was 27.3 mmHg +/- 12.6 mmHg (SD). Mean IOP (±SD) for dose levels 1, 2, 3, and 4 were at Day 43 9.8 mmHg ± 1.0 mmHg, 11.3 mmHg ± 6.7 mmHg, 5.5 mmHg ± 3.0 mmHg and 7.5 mmHg ± 2.3 mmHg SD; and at Day 85 9.7 mmHg ± 3.3 mmHg, 14.2 mmHg ± 6.5 mmHg, 5.8 mmHg ± 1.8 mmHg and 7.8 mmHg ± 0.6 mmHg, respectively. In contrast to IOP values for dose levels 1 and 2, IOP values for dose levels 3 and 4 persistently remained below 10 mmHg throughout the observation period.</p><p>Conclusion</p><p>This first-in-human trial demonstrates that intravitreal injection of ISTH0036 at the end of TE is safe. Regarding IOP control, single-dose ISTH0036 administration of 67.5 μg or 225 μg at the time of TE resulted in IOP values persistently < 10 mmHg over the three month postoperative observation period.</p></div

Bleb filtering and morphology evaluation (Wuerzburg bleb classification Score).

<p>Bleb filtering and morphology evaluation (Wuerzburg bleb classification Score).</p