Isolation and {\it in vitro}-immunosuppressive characterization of a novel cyclosporine metabolite

Cyclosporine (CsA) has shown great benefit to organ transplant recipients, as an immunosuppressive drug. To optimize CsA immunosuppressive therapy, pharmacodynamic evaluation of serial patient serum samples after CsA administration, using mixed lymphocyte culture (MLC) assays, revealed in vitro serum immunosuppressive activity of a CsA-like, ether-extractable component, associated with good clinical outcome in vivo. Since the in vitro immunosuppressive CsA metabolites, M-17 and M-1, are erythrocyte-bound, the immunosuppressive activity demonstrated in patient serum suggests that other immunosuppressive metabolites need exist. To test this hypothesis and obtain CsA metabolites for study, ether-extracted bile from tritiated and nonradioactive CsA-treated pigs was processed by novel high performance liquid and thin-layer chromatography (HPLC and HPTLC) techniques. Initial MLC screening of potential metabolites revealed a component, designated M-E, to have immunosuppressive activity. Pig bile-derived M-E was characterized as a CsA metabolite, by radioactive CsA tracer studies, by 56% crossreactivity in CsA radioimmunoassay, and by mass spectrometric (MS) analysis. MS revealed a CsA ring structure, hydroxylated at a site other than at amino acid one. M-E was different than M-1 and M-17, as demonstrated by different retention properties for each metabolite, using HPTLC and a novel rhodamine B/ $\alpha$-cyclodextrin stain, and using HPLC, performed by Sandoz, that revealed M-E to be different than previously characterized metabolites. The immunosuppressive activity of M-E was quantified by determination of mean metabolite potency ratio in human MLC assays, which was found to be 0.79 $\pm$ 0.23 (CsA, 1.0). Similar to parent drug, M-E revealed inter-individual differences in its immunosuppressive activity. M-E demonstrates inhibition of IL-2 production by concanavalin A stimulated C3H mouse spleen cells, similar to CsA, as determined with an IL-2 dependent mouse cytotoxic T-cell line. ^

MOUSE MAMMARY TUMOR VIRUS-RELATED PROTEINS: ISOLATION AND CHARACTERIZATION OF A UNIQUE GLYCOPROTEIN

Mouse mammary tumor virus (MMTV) contained six major proteins, identified as gp55, gp33, p25, pp20, p12, and p10. Immunoprecipitation of cytoplasmic extracts from MMTV-infected, pulse-labeled cells identified three MMTV core-specific precursor proteins, termed Pr78('gag), Pr110('gag), Pr110('gag), and Pr180('gag+). The major intracellular core-specific precursor polyprotein, Pr78('gag), contained antigenic determinants and tryptic peptides characteristic of p25, p12, and p10. Pr110('gag) contained all but one of the leucine-containing tryptic peptides of Pr78('gag), plus several additional peptides. In addition to Pr78('gag) and Pr110('gag), monospecific antisera to virion p12 and p25 also precipitated from pulse-labeled cells a small amount of Pr180('gag+). This large polyprotein contained nearly all of the leucine-containing tryptic peptides of Pr78('gag) and Pr110('gag) plus several additional peptides. By analogy to type-C viral systems, Pr180('gag+) is presumed to represent a gag-pol-specific common precursor which is the major translation product in the synthesis of MMTV RNA-dependent-DNA polymerase. Immunoprecipitation of cytoplasmic extracts from pulse-labeled cells with antisera to gp55 identified two envelope-specific proteins, designated gPr76('env) and gP79('env). The major envelope-specific precursor, gPr76('env), could be labeled with radioactive glucosamine and contained antigenic determinants and tryptic peptides characteristic of gp55 and gp33. A quantitatively minor glycoprotein, gP79('env), contained both fucose and glucosamine and was precipitable from cytoplasmic extracts with monospecific serum to gp55. It is suggested that gP79('env) represents fucosylated gPr76('env) which is transiently synthesized and cleaved rapidly into gp55 and gp33.^ A glycoprotein of 130,00 molecular weight (gP130) was precipitable from the cytoplasm of GR-strain mouse mammary tumor cells by a rabbit antiserum (anti-MMTV) to Gr-strain mouse mammary tumors virus (GR-MMTV). Two dimensional thin layer analysis of ('35)S-methionine-containing peptides revealed that five of nine gp33 peptides and one of seven gp55 peptides were shared by gP130 and gPr76('env). Six of ten p25 peptides and four more core-related peptides were shared by Pr78('gag) and gP130. Protein gP130 also contained several tryptic peptides not found in gPr76('env), or in the core protein precursors Pr78('gag), Pr110('gag), or Pr180('gag+). both gP130 and a second protein, p30, were found in immunoprecipitates of detergent disrupted, isotopically labeled GR-MMTV treated with anti-MMTV serum. Results suggest that antibodies to gP130 in the anti-MMTV serum are capable of recognizing those protein sequences which are not related to viral structural proteins. These gP130-unique peptides are evidently host specific. Polyproteins consisting of juxtaposed host- and virus-related protein tracts have been implicated in the process of cell transformation in other mammalian systems. Therefore, gP130 may be instrinsic to the oncogenic potential of MMTV. ^

A novel treatment planning methodology for high dose 166Ho-DOTMP therapy in patients with multiple myeloma

Bone marrow ablation, i.e., the complete sterilization of the active bone marrow, followed by bone marrow transplantation (BMT) is a comment treatment of hematological malignancies. The use of targeted bone-seeking radiopharmaceuticals to selectively deliver radiation to the adjacent bone marrow cavities while sparing normal tissues is a promising technique. Current radiopharmaceutical treatment planning methods do not properly compensate for the patient-specific variable distribution of radioactive material within the skeleton. To improve the current method of internal dosimetry, novel methods for measuring the radiopharmaceutical distribution within the skeleton were developed. 99mTc-MDP was proven as an adequate surrogate for measuring 166Ho-DOTMP skeletal uptake and biodistribution, allowing these measures to be obtained faster, safer, and with higher spatial resolution. This translates directly into better measurements of the radiation dose distribution within the bone marrow. The resulting bone marrow dose-volume histograms allow prediction of the patient disease response where conventional organ scale dosimetry failed. They indicate that complete remission is only achieved when greater than 90% of the bone marrow receives at least 30 Gy. ^ Comprehensive treatment planning requires combining target and non-target organ dosimetry. Organs in the urinary tract were of special concern. The kidney dose is primarily dependent upon the mean transit time of 166 Ho-DOTMP through the kidney. Deconvolution analysis of renograms predicted a mean transit time of 2.6 minutes for 166Ho-DOTMP. The radiation dose to the urinary bladder wall is dependent upon numerous factors including patient hydration and void schedule. For beta-emitting isotopes such as 166Ho, reduction of the bladder wall dose is best accomplished through good patient hydration and ensuring a partially full bladder at the time of injection. Encouraging the patient to void frequently, or catheterizing the patient without irrigation, will not significantly reduce the bladder wall dose. ^ The results from this work will produce the most advanced treatment planning methodology for bone marrow ablation therapy using radioisotopes currently available. Treatments can be tailored specifically for each patient, including the addition of concomitant total body irradiation for patients with unfavorable dose distributions, to deliver a desired patient disease response, while minimizing the dose or toxicity to non-target organs. ^

Targeted cancer diagnosis with radiolabeled endostatin

Nuclear imaging is used for non-invasive detection, staging and therapeutic monitoring of tumors through the use of radiolabeled probes. Generally, these probes are used for applications in which they provide passive, non-specific information about the target. Therefore, there is a significant need for actively-targeted radioactive probes to provide functional information about the site of interest. This study examined endostatin, an endogenous inhibitor of tumor angiogenesis, which has affinity for tumor vasculature. The major objective of this study was to develop radiolabeled analogues of endostatin through novel chemical and radiochemical syntheses, and to determine their usefulness for tumor imaging using in vitro and in vivo models of vascular, mammary and prostate tumor cells. I hypothesize that this binding will allow for a non-invasive approach to detection of tumor angiogenesis, and such detection can be used for therapeutic monitoring to determine the efficacy of anti-angiogenic therapy. ^ The data showed that endostatin could be successfully conjugated to the bifunctional chelator ethylenedicysteine (EC), and radiolabeled with technetium-99m and gallium-68, providing a unique opportunity to use a single precursor for both nuclear imaging modalities: 99mTc for single photon emission computed tomography and 68Ga for positron emission tomography, respectively. Both radiolabeled analogues showed increased binding as a function of time in human umbilical vein endothelial cells and mammary and prostate tumor cells. Binding could be blocked in a dose-dependent manner by unlabeled endostatin implying the presence of endostatin receptors on both vascular and tumor cells. Animal biodistribution studies demonstrated that both analogues were stable in vivo, showed typical reticuloendothelial and renal excretion and produced favorable absorbed organ doses for application in humans. The imaging data provide evidence that the compounds quantitate tumor volumes with clinically-useful tumor-to-nontumor ratios, and can be used for treatment follow-up to depict changes occurring at the vascular and cellular levels. ^ Two novel endostatin analogues were developed and demonstrated interaction with vascular and tumor cells. Both can be incorporated into existing nuclear imaging platforms allowing for potential wide-spread clinical benefit as well as serving as a diagnostic tool for elucidation of the mechanism of action of endostatin. ^

A comparative analysis of Texas and Louisiana Oil-field NORM Waste Policy

NORM (Naturally Occurring Radioactive Material) Waste Policies for the nation's oil and gas producing states have been in existence since the 1980's, when Louisiana was the first state to develop a NORM regulatory program in 1989. Since that time, expectations for NORM Waste Policies have evolved, as Health, Safety, Environment, and Social responsibility (HSE & SR) grows increasingly important to the public. Therefore, the oil and gas industry's safety and environmental performance record will face challenges in the future, about its best practices for managing the co-production of NORM wastes. ^ Within the United States, NORM is not federally regulated. The U.S. EPA claims it regulates NORM under CERCLA (superfund) and the Clean Water Act. Though, there are no universally applicable regulations for radium-based NORM waste. Therefore, individual states have taken responsibility for developing NORM regulatory programs, because of the potential radiological risk it can pose to man (bone and lung cancer) and his environment. This has led to inconsistencies in NORM Waste Policies as well as a NORM management gap in both state and federal regulatory structures. ^ Fourteen different NORM regulations and guidelines were compared between Louisiana and Texas, the nation's top two petroleum producing states. Louisiana is the country's top crude oil producer when production from its Federal offshore waters are included, and fourth in crude oil production, behind Texas, Alaska, and California when Federal offshore areas are excluded. Louisiana produces more petroleum products than any state but Texas. For these reasons, a comparative analysis between Louisiana and Texas was undertaken to identify differences in their NORM regulations and guidelines for managing, handling and disposing NORM wastes. Moreover, this analysis was undertaken because Texas is the most explored and drilled worldwide and yet appears to lag behind its neighboring state in terms of its NORM Waste Policy and developing an industry standard for handling, managing and disposing NORM. As a result of this analysis, fourteen recommendations were identified.^

Characterization of Jak, STAT, and Src interactions in Head and Neck Squamous Cell Carcinoma

Recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is common; thus, it is essential to improve the effectiveness and reduce toxicity of current treatments. Proteins in the Src/Jak/STAT pathway represent potential therapeutic targets, as this pathway is hyperactive in HNSCC and it has roles in cell migration, metastasis, proliferation, survival, and angiogenesis. During short-term Src inhibition, Janus kinase (Jak) 2, and signal transducer and activator of transcription (STAT) 3 and STAT5 are dephosphorylated and inactivated. Following sustained Src inhibition, STAT5 remains inactive, but Jak2 and STAT3 are reactivated following their early inhibition. To further characterize the mechanism of this novel feedback pathway we performed several experiments to look at the interactions between Src, Jak2, STAT5 and STAT3. We attempted to develop a non-radioactive kinase assay using purified recombinant Jak2 and Src proteins, but found that phospho-tyrosine antibodies were non-specifically binding to purified recombinant proteins. We then performed in vitro kinase assays (IVKAs) using purified recombinant Jak2, Src, STAT3, and STAT5 proteins with and without Src and Jak2 pharmacologic inhibitors. We also examined the interactions of these proteins in intact HNSCC cells. We found that recombinant Jak2, STAT3, and STAT5 are direct substrates of Src and that recombinant Src, STAT3, and STAT5 are direct substrates of Jak2 in the IVKA. To our knowledge, the finding that Src is a Jak substrate is novel and has not been shown before. In intact HNSCC cells we find that STAT3 can be reactivated despite continuous Src inhibition and that STAT5 continues to be inhibited despite Jak2 reactivation. Also, Jak2 inhibition did not affect Src or STAT5 activity but it did cause STAT3 inhibition. We hypothesized that the differences between the intact cells and the IVKA assays were due to a potential need for binding partners in intact HNSCC cells. One potential binding partner that we examined is the epidermal growth factor receptor (EGFR). We found that EGFR activation caused increased activation of Src and STAT5 but not Jak2. Our results demonstrate that although STAT3 and STAT5 are capable of being Src and Jak2 substrates, in intact HNSCC cells Src predominantly regulates STAT5 and Jak2 regulates STAT3. Regulation of STAT5 by Src may involve interactions between Src and EGFR. This knowledge along with future studies will better define the mechanisms of STAT regulation in HNSCC cells and ultimately result in an ideal combination of therapeutic agents for HNSCC.