Phase 1b Study of IGF-Methotrexate Conjugate in the Treatment of High-grade Myelodysplastic Syndromes.

BACKGROUND/AIM: The insulin-like growth factor type 1 receptor (IGF-1R) is overexpressed in myelodysplastic syndrome (MDS) cells, and 765IGF-Methotrexate (IGF-MTX) is a conjugate of methotrexate and a variant of insulin-like growth factor-1 (IGF-1) designed to selectively target cancer cells through binding to IGF-1R. The aim of this study was to determine whether IGF-MTX would be effective to treat MDS. PATIENTS AND METHODS: In this phase I clinical trial, two patients with high grade MDS or oligoblastic acute myeloid leukemia (O-AML) that had failed standard therapy were treated with IGF-MTX. RESULTS: No dose-limiting toxicity was observed. Both patients had stable or improved cell counts and CD34+ myelodysplastic cell counts and exceeded their life expectancy (both alive at 1.9 years despite a life expectancy of less than 6 months). Bone marrow blast counts decreased from 22% to 5% in one patient, and from 17% to 16% in the other. CONCLUSION: In conclusion, IGF-MTX at 0.20 µM equivalents per kg was well tolerated, caused no cytopenia, and produced stable disease and extension of life.

Immune characteristics correlating with HSV-1 immune control and effect of squaric acid dibutyl ester on immune characteristics of subjects with frequent herpes labialis episodes.

INTRODUCTION: Differences in immune characteristics, including immune gene expression by peripheral blood mononuclear cells (PBMCs), correlating with herpes labialis and good or poor immune control of herpes simplex virus type 1 (HSV-1), and how these characteristics change after dosing with squaric acid dibutyl ester (SADBE), were investigated. METHODS: PBMCs were collected from persons positive for IgG against HSV-1 and having frequent, infrequent, or no herpes labialis outbreaks. The PBMCs were tested for proliferation against HSV-1 and a fungal antigen (Candida) and immune gene expression in the presence of HSV-1 and Candida. On day 1 after blood collection the subjects with frequent outbreaks were dosed topically on the arm once with SADBE, and their PBMCs were collected and tested 8 weeks later. RESULTS: Those with good immune control of their HSV-1 infection (fewer outbreaks) differ from those with poorer immune control in these ways: (1) Greater PBMC proliferation in vitro to HSV-1, HSV-1-infected cell extracts, and Candida considered together (P < 0.01). (2) Higher expression of IFNG and five other immune-related genes (P < 0.05 for each) and lower expression of IL5 and two other immune-related genes (P < 0.05 for each) in PBMCs in vitro stimulated with HSV-1 virus. The subjects with frequent outbreaks were treated once with SADBE, and 56 days later the PBMCs of these subjects differed from PBMCs from the same subjects taken on day 1 before treatment in exactly the same ways listed above as differences between those with good and poor immune control of HSV-1, and at the same levels of significance. CONCLUSIONS: Higher IFNG and lower IL5 expression by PBMCs in the presence of HSV-1 correlate with fewer herpes labialis outbreaks, and a single topical dose of SADBE to the arm of subjects with frequent herpes labialis episodes improves immune response to HSV-1.

Novel insulin-like growth factor-methotrexate covalent conjugate inhibits tumor growth in vivo at lower dosage than methotrexate alone.

The insulin-like growth factor receptor is overexpressed on many types of cancer cells and has been implicated in metastasis and resistance to apoptosis. We report here the development of a novel covalent conjugate that contains the antifolate drug methotrexate coupled to an engineered variant of insulin-like growth factor-1 (IGF-1), long-R3-IGF-1, which was designed to target methotrexate to tumor cells that overexpress the membrane IGF-1 receptor. The IGF-methotrexate conjugate was found to contain at least 4 methotrexate molecules per IGF-1 protein. The IGF-methotrexate conjugate bound to MCF7 breast cancer cells with greater than 3.3-fold higher affinity than unconjugated long-R3-IGF-1 in a competition binding assay against radiolabeled wild-type IGF-1. Compared with free methotrexate, the IGF-methotrexate conjugate required slightly higher concentrations to inhibit the in vitro growth of the human prostate cancer cell line LNCaP. In vivo, however, in a mouse xenograft model using LNCaP cells, the IGF-methotrexate conjugate was more effective than free methotrexate even at a 6.25-fold lower molar dosage. Similarly, MCF7 xenografts were inhibited more effectively by the IGF-methotrexate conjugate than free methotrexate, even at a 4-fold lower molar dosage. Our results suggest that the targeting of the IGF receptor on tumor cells and tumor-related tissues with IGF-chemotherapy conjugates may substantially increase the specific drug localization and therapeutic effect in the tumor.

Atrazine chlorohydrolase from Pseudomonas sp. strain ADP is a metalloenzyme.

Atrazine chlorohydrolase (AtzA) from Pseudomonas sp. ADP initiates the metabolism of the herbicide atrazine by catalyzing a hydrolytic dechlorination reaction to produce hydroxyatrazine. Sequence analysis revealed AtzA to be homologous to metalloenzymes within the amidohydrolase protein superfamily. AtzA activity was experimentally shown to depend on an enzyme-bound, divalent transition-metal ion. Loss of activity obtained by incubating AtzA with the chelator 1,10-phenanthroline or oxalic acid was reversible upon addition of Fe(II), Mn(II), or Co(II) salts. Experimental evidence suggests a 1:1 metal to subunit stoichiometry, with the native metal being Fe(II). Our data show that the inhibitory effects of metals such as Zn(II) and Cu(II) are not the result of displacing the active site metal. Taken together, these data indicate that AtzA is a functional metalloenzyme, making this the first report, to our knowledge, of a metal-dependent dechlorinating enzyme that proceeds via a hydrolytic mechanism.