Showing papers by "Eli Lilly and Company published in 1996"

Identification of an Estrogen Response Element Activated by Metabolites of 17β-Estradiol and Raloxifene

Abstract: 17β-Estradiol modulates gene transcription through the estrogen receptor and the estrogen response element in DNA. The human transforming growth factor-β3 gene was shown to be activated by the estrogen receptor in the presence of estrogen metabolites or estrogen antagonists. Activation was mediated by a polypurine sequence, termed the raloxifene response element, and did not require the DNA binding domain of the estrogen receptor. Interaction of the estrogen receptor with the raloxifene response element appears to require a cellular adapter protein. The observation that individual estrogens modulate multiple DNA response elements may explain the tissue-selective estrogen agonist or antagonist activity of compounds such as raloxifene.

Olanzapine versus placebo: results of a double-blind, fixed-dose olanzapine trial.

Abstract: Olanzapine is a potential new “atypical” antipsychotic agent. This double-blind, acute phase study compared two doses of olanzapine [1 mg/day (Olz1.0); 10 mg/day (Olz10.0)] with placebo in the treatment of 152 patients who met the DSM-III-R criteria for schizophrenia and had a Brief Psychiatric Rating Scale (BPRS)-total score (items scored 0–6) ≥24. In overall symptomatology improvement [BPRS-total score and Positive and Negative Syndrome Scale (PANSS)-total score], Olz10.0 was statistically significantly superior to placebo. In positive symptom improvement (PANSS-positive score, BPRS-positive score), Olz10.0 was statistically significantly superior to placebo. In negative symptom improvement (PANSS-negative score), Olz10.0 was statistically superior to placebo. Olz 1.0 was clinically comparable to placebo in all efficacy comparisons. The only adverse event to show an overall statistically significant incidence difference was anorexia (reported for 10% of placebo-treated and 0% of Olz10.0-treated patients). The Olz10.0-treated patients improved over baseline with respect to parkinsonian and akathisia symptoms, and these changes were comparable with those observed with placebo. There were no dystonias associated with Olz10.0 treatment. At endpoint, the incidence of patients with elevated prolactin values did not differ statistically significantly between placebo-treated and Olz10.0-treated patients. Olanzapine appears to be not only safe and effective, but a promising atypical antipsychotic candidate.

Reversal of P-Glycoprotein-mediated Multidrug Resistance by a Potent Cyclopropyldibenzosuberane Modulator, LY335979

Abstract: Overexpression of P-glycoprotein (Pgp) by tumors results in multidrug resistance (MDR) to structurally unrelated oncolytics. MDR cells may be sensitized to these oncolytics when treated with a Pgp modulator. The present study evaluates LY335979 as a modulator both in vitro and in vivo. LY335979 (0.1 µm) fully restored sensitivity to vinblastine, doxorubicin (Dox), etoposide, and Taxol in CEM/VLB100 cells. Ly335979 modulated Dox cytotoxicity even when LY335979 (0.5 µm) was removed 24 h prior to the cytotoxicity assay. LY335979 blocked [3H]azidopine photoaffinity labeling of the Mr ∼170,000 Pgp in CEM/VLB100 plasma membranes and competitively inhibited equilibrium binding of [3H]vinblastine to Pgp (Ki of ∼0.06 µm). Treatment of mice bearing P388/ADR murine leukemia cells with LY335979 in combination with Dox or etoposide gave a significant increase in life span with no apparent alteration of pharmacokinetics. LY335979 also enhanced the antitumor activity of Taxol in a MDR human non-small cell lung carcinoma nude mouse xenograft model. Thus, LY335979 is an extremely potent, efficacious modulator that apparently lacks pharmacokinetic interactions with coadministered anticancer drugs and is, therefore, an exciting new agent for clinical evaluation for reversal of Pgp-associated MDR.

(S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-d ione (LY333531) and related analogues: isozyme selective inhibitors of protein kinase C beta.

Abstract: Protein kinase C (PKC) is a family of closely related serine and threonine kinases. Overactivation of some PKC isozymes has been postulated to occur in several diseases states, including diabetic complications. Selective inhibition of overactivated PKC isozymes may offer a unique therapeutic approach to disease states such as diabetic retinopathy. A novel series of 14-membered macrocycles containing a N-N'-bridged bisindolylmaleimide moiety is described. A panel of eight cloned human PKC isozymes (alpha, beta I, beta II, gamma, delta, epsilon, sigma, eta) was used to identify the series and optimize the structure and associated activity relationship. The dimethylamine analogue LY333531 (1), (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene++ +-1,3(2H)-dione, inhibits the PKC beta I (IC50 = 4.7 nM) and PKC beta II (IC50 = 5.9 nM) isozymes and was 76- and 61-fold selective for inhibition of PKC beta I and PKC beta II in comparison to PKC alpha, respectively. The additional analogues described in the series are also selective inhibitors of PKC beta. LY333531 (1) exhibits ATP dependent competitive inhibition of PKC beta I and is selective for PKC in comparison to other ATP dependent kinases (protein kinase A, calcium calmodulin, caesin kinase, src tyrosine kinase). The cellular activity of the series was assessed using bovine retinal capillary endothelial cells. Retinal endothelial cell dysfunction has been implicated in the development of diabetic retinopathy. Plasminogen activator activity stimulated by a phorbol ester (4 beta-phorbol 12,13-dibutyrate) in endothelial cells was inhibited by the compounds in the series with ED50 values ranging from 7.5 to 0.21 microM. A comparison of the PKC isozyme and related ATP dependent kinase inhibition profiles is provided for the series and compared to the profile for staurosporine, a nonselective PKC inhibitor. The cellular activity of the series is compared with that of the kinase inhibitor staurosporine.

The biologic effects of C225, a chimeric monoclonal antibody to the EGFR, on human prostate carcinoma

Abstract: For prostate cancer, a correlation exists between overexpression of the epidermal growth factor receptor (EGFR) and poor clinical prognosis. In addition, late-stage metastatic disease is characterized by a change from a paracrine to an autocrine mode of expression for TGF-alpha, the ligand for the EGFR. These observations suggest that activation of the EGFR may be important for the growth of prostatic carcinoma in situ, and blockade of the receptor-ligand interaction may offer a means of therapeutic intervention for this disease. We describe the biologic effects of a chimeric anti-EGFR monoclonal antibody, C225, on several human prostate tumor cell lines in culture and the tumor inhibitory properties of the antibody for the treatment of human prostate carcinoma xenografts in nude mice. In vitro analysis of the EGFR from androgen-responsive and independent prostatic carcinoma cell lines revealed that C225 blocked EGF-induced receptor activation and induced internalization of the receptor. In vivo, a treatment regimen of C225 alone or antibody plus doxorubicin significantly inhibited tumor progression of well-established DU145 and PC-3 xenografts in nude mice. These results suggest that C225 may have utility for the treatment of human prostate carcinoma in a clinical setting.

Recyclable medication dispensing device

Abstract: A multi-use pen-shaped medication dispensing device (20) made of a plastic material that is recyclable after the contents of the medication cartridge (40) have been exhausted. The device (20) is made of a minimal number of parts, which include a housing (22), a dial assembly (34), a generally cylindrical button assembly (32) located within the proximal end (50) of the dial assembly (34), an internally threaded nut (36), and an externally threaded leadscrew (38). The device (20) is arranged so that the dial (34) must be rotated to the zero dose position prior to setting a dose. The device (20) includes a lockout mechanism (52) that prevents the dial (34) from being depressed during dosing. The device (20) further includes a mechanism (157) that limits the maximum dosage that can be dialed up and a mechanism (230, 234) that prevents the user from dialing up a dosage greater than that remaining in the cartridge (40).

Platelet-activating factor acetylhydrolase deficiency. A missense mutation near the active site of an anti-inflammatory phospholipase.

Abstract: Deficiency of plasma platelet-activating factor (PAF) acetylhydrolase is an autosomal recessive syndrome that has been associated with severe asthma in Japanese children. Acquired deficiency has been described in several human diseases usually associated with severe inflammation. PAF acetylhydrolase catalyzes the degradation of PAF and related phospholipids, which have proinflammatory, allergic, and prothrombotic properties. Thus, a deficiency in the degradation of these lipids should increase the susceptibility to inflammatory and allergic disorders. Miwa et al. reported that PAF acetylhydrolase activity is absent in 4% of the Japanese population, which suggests that it could be a common factor in such disorders, but the molecular basis of the defect is unknown. We show that inherited deficiency of PAF acetylhydrolase is the result of a point mutation in exon 9 and that this mutation completely abolishes enzymatic activity. This mutation is the cause of the lack of enzymatic activity as expression in E. coli of a construct harboring the mutation results in an inactive protein. This mutation as a heterozygous trait is present in 27% in the Japanese population. This finding will allow rapid identification of subjects predisposed to severe asthma and other PAF-mediated disorders.

Autoimmunity associated with TGF-β1-deficiency in mice is dependent on MHC class II antigen expression

Abstract: The progressive inflammatory process found in transforming growth factor beta1 (TGF-beta1)-deficient mice is associated with several manifestations of autoimmunity, including circulating antibodies to nuclear antigens, immune complex deposition, and increased expression of both class I and class II major histocompatibility complex (MHC) antigens. The contribution of MHC class II antigens to the genesis of this phenotype has been determined by crossing the TGF-beta1-null [TGF-beta1(-/-)] genotype into the MHC class II-deficient [MHC-II(-/-)] background. Mice homozygous for both the TGF-beta1 null allele and the class II null allele [TGF-beta1(-/-);MHC-II(-/-)] are without evidence of inflammatory infiltrates, circulating autoantibodies, or glomerular immune complex deposits. Instead, these animals exhibit extensive extramedullary hematopoiesis with progressive splenomegaly and adenopathy, surviving only slightly longer than TGF-beta1(-/-);MHC-II(+/+) mice. The role of CD4+ T cells, which are also absent in MHC class II-deficient mice, is directly demonstrated through the administration of anti-CD4 monoclonal antibodies in class II-positive, TGF-beta1(-/-) mice. The observed reduction in inflammation and improved survival emphasize the significance of CD4+ cells in the pathogenesis of the autoimmune process and suggest that the additional absence of class II antigens in TGF-beta1(-/-);MHC-II(-/-) mice may contribute to their extreme myeloid metaplasia. Thus, MHC class II antigens are essential for the expression of autoimmunity in TGF-beta1-deficient mice, and normally may cooperate with TGF-beta1 to regulate hematopoiesis.

NCM460, a normal human colon mucosal epithelial cell line.

Abstract: Dear Editor: Many studies of human intestinal physiology and pathology would be augmented with the development of normal human cell lines (1,2). These include investigations of transport, drug delivery, infectious disease, and other processes. Developing normal human cell lines is particularly important for colon cancer where malignant cell lines (8) have dominated the in vitro studies. This has been true because of the lack of easily propagated normal cell lines, even though it has been wellrecognized from detailed genetic analyses of in situ tissues (10) that genetic and other changes in colon cancer progression are complex anti might hest be sludied with more appropriate models. To thai end, we (9) have recently reported on two normal cell lines which were derived from patients with colon cancer. Although they were verified by the pathologist as histologically normal areas of mucosa taken from the wide margins of surgical resection and are of epithelial origin, they display a subset of potentially premalignant features when grown in vitro. Thus, the continued development of additional normal colon mucosa (NCM) cell lines is still of great value. To that end, the NCM460 cell line was initiated and propagated by methods detailed elsewhere (4-7). The patient donor for the source t issue was a 68-year-old Hispanic male, who underwent a partial gastrectomy for removal of a poorly differentiated gastric tu-

Identification of the human cytochromes P450 responsible for the in vitro formation of the major oxidative metabolites of the antipsychotic agent olanzapine.

Abstract: The formation kinetics of 2-hydroxymethyl olanzapine (2-OH olanzapine), 4'-N-oxide olanzapine (N-O olanzapine) and 4'-N-desmethyl olanzapine (NdM olanzapine) were analyzed in vitro. Biphasic kinetics were observed for formation of 2-OH and NdM olanzapine. The high-affinity enzyme responsible for 2-OH olanzapine formation by two human liver samples exhibited an intrinsic clearance (CLint) of 0.2 microliter/min/mg. NdM olanzapine formation by two human liver samples exhibited a CLint of 1.0 microliter/min/mg for the high affinity enzyme. The formation of N-O olanzapine was linear up to 300 microM olanzapine, yielding a CLint of 0.32 to 1.70 microliters/min/mg. The formation of 7-hydroxy olanzapine (7-OH olanzapine) exhibited an apparent Km of 24.2 microM. The rates of 2-OH olanzapine formation correlated with CYP2D6 levels and activity, and it was formed to the greatest extent by cDNA-expressed CYP2D6. N-O olanzapine formation correlated with human liver flavin-containing monooxygenase (FMO3) levels and activity. NdM olanzapine and 7-OH olanzapine formation correlated with CYP1A2 catalytic activities and they were formed to the greatest extent by expressed CYP1A2. These results suggest that CYP1A2 catalyzes NdM olanzapine and 7-OH olanzapine formation, CYP2D6 catalyzes 2-OH olanzapine formation and FMO3 catalyzes N-O olanzapine formation.

Multi-cartridge medication injection device

Abstract: A multi-cartridge dispenser for delivering two liquid medications through a single needle. The dispenser includes independent dual channel metering mechanism, dual channel drive mechanisms, and dual channel lock and pullback mechanisms. The wing must be in its "up" position for metering to take place. The wing reciprocatingly moves a driver between a pre-injection position and a post-injection position to reciprocatingly advance the leadscrews upon an injection stroke. The dosage indicator automatically rotates to its initial zero position upon the injection stroke. The lock and pullback mechanism automatically prevents rotation of the leadscrew upon metering and injection. It also locks out the cartridge retainers so that the retainers can be removed from the housing only while the wing is in its "down" position. The pullback sleeve unloads a pullback key during a cartridge change in order to enable the leadscrew to be spun freely back to its home position.

Reductive Alkylation of Glycopeptide Antibiotics: Synthesis and Antibacterial Activity

Abstract: Reductive alkylation of the A82846 family of glycopeptide antibiotics has the potential of producing seven products. N-Alkylation of the disaccharide amino function can be accomplished selectively, and offers the greatest increase in antibacterial activity. Products resulting from N-alkylation of LY264826 (A82846B) provide the most potent derivatives as compared to other members of this class of antibiotics. Two of these derivatives, LY307599 and LY333328 are approximately 500 times more active than vancomycin against vancomycin-resistant enterococci.

Anti-obesity proteins

Abstract: The present invention provides anti-obesity proteins, which when administered to a patient regulate fat tissue. Accordingly, such agents allow patients to overcome their obesity handicap and live normal lives with much reduced risk for type II diabetes, cardiovascular disease and cancer.

Rac GTPase interacts specifically with phosphatidylinositol 3-kinase

Abstract: The Rac GTP-binding proteins are members of the Rho family and regulate growth factor-stimulated actin assembly in a variety of cells. The formation of phosphorylated inositol lipids has been implicated in control of the processes initiating and regulating such actin polymerization. Associations of Rho family GTP-binding proteins with enzymes involved in lipid metabolism have been described. Here we demonstrate a direct and specific interaction of Rac proteins with phosphatidylinositol (PI) 3-kinase. This interaction is dependent upon Rac being in a GTP-bound state and requires an intact Rac effector domain. In contrast, direct binding of RhoA to PI 3-kinase could not be detected. Rac-GTP also bound to PI 3-kinase in Swiss 3T3 fibroblast and human neutrophil lysates, and increased PI 3-kinase activity became associated with Rac-GTP in platelet-derived growth factor-stimulated cells. Interaction of Rac-GTP with PI 3-kinase in vitro stimulated the activity of the enzyme by 2-9-fold. A specific interaction of active Rac with PI 3-kinase might be important in regulation of the actin cytoskeleton.

Report of the American Diabetes Association's Task Force on Standardization of the Insulin Assay

Abstract: Recent large-scale epidemiological studies demonstrate that blood concentrations of immunoreactive insulin predict the development of NIDDM and IDDM and are associated with the risk of several degenerative diseases, such as coronary and peripheral vessel atherosclerosis, hypertension, and dyslipidemia. The reliability of these measurements is dependent on a biological assay that has not been well standardized between laboratories. Recognizing this, the American Diabetes Association organized a task force to assess comparability of blood insulin measurements between laboratories and to suggest techniques to improve comparability. The task force found that identical serum and plasma samples measured in different laboratories produced widely disparate values that were unacceptable for population comparisons. Use of a single reference standard did little to improve comparability. Assay characteristics such as linearity, recovery, accuracy, and cross-reactivity to proinsulin and its primary conversion intermediates varied among the laboratories, and they did not readily explain differences in the measurements made from assay to assay. Use of the same assay kit in different laboratories did not always ensure comparable measurements. Linear regression of assay results from one laboratory to an arbitrarily chosen reference assay greatly improved comparability and demonstrated the potential value in comparing each assay to a reference method. The task force report defines acceptable assay characteristics and proposes a three-step process of insulin assay proficiency and comparability. A central reference assay and ongoing sample exchange will be needed to allow reliable comparisons of insulin measurements made in different laboratories. Rigorous quality control and continuous quality improvement are needed to maintain reliability of the insulin measurement.

Effects of oxidized low-density lipoprotein on vascular contraction and relaxation: clinical and pharmacological implications in atherosclerosis.

Abstract: Oxidized LDL exerts profound effects on the vasomotor response of isolated blood vessels to various stimuli that closely mimic the vascular dysfunction associated with hypercholesterolemia and atherosclerosis in humans. The beneficial effect of lipid-lowering therapy in normalizing vascular function and greatly decreasing the frequency of clinical events associated with atherosclerosis, combined with the ability of antioxidants to alleviate vasomotor disturbances in hypercholesterolemia and slow the progression of atherosclerosis, strongly support a causative role of oxidized LDL in mediating vascular dysfunction in vivo and contributing to the clinical sequalae of coronary artery disease. Further research to understand more fully the mechanisms of oxidized LDL formation and actions in vivo may reveal novel strategies to inhibit these events, and may prove beneficial in the therapeutic management of atherosclerotic disease.

The human drug metabolizing cytochromes P450

Abstract: The superfamily of heme-thiolate proteins known as the cytochromes P450 is responsible for the oxidative metabolism of the majority of drugs. Thus, the phenotypes of individuals with respect to their levels of catalytically active cytochromes P450 determines to a large part the substantial interindividual variation observed in the metabolic clearance of drugs. Over the past 10 years 15 different human cytochromes P450 involved in drug metabolism have been isolated and characterized to varying degrees. This brief review discusses the characterization of these cytochromes P450 and how this knowledge has been used by the pharmaceutical industry to aid in the development of new drugs.

Expression cloning of a human brain neuropeptide Y Y2 receptor.

Abstract: The 36-amino acid peptide, neuropeptide Y (NPY), is a member of a peptide family that includes the endocrine peptides, peptide YY (PYY), and pancreatic polypeptide (PP). NPY receptors have been broadly subdivided into postsynaptic Y1 receptors and presynaptic Y2 receptors based on the preference of Pro34-substituted analogues for the Y1 receptors and carboxyl-terminal fragments for the Y2. A Y1 receptor has been cloned, and this receptor appears to mediate several effects of NPY, including vasoconstriction and anxiolysis in animal models. We report the cloning of a human brain Y2 receptor from a human brain library. Pools of clones were transiently expressed in COS-1 cells, and 125I-PYY binding pools were identified by autoradiography. After a single positive pool was detected in the original screening, a single clone was isolated by four rounds of sequential enrichment. The clone encoded a 381-amino acid protein of the heptahelix (seven TM) type. Amino acid identity of this receptor with the Y1 receptor was 31% overall with 40% identity in the TM regions. Comparison with the human PP1 receptor indicated 33% overall amino acid identity with 42% identity in the TM regions. Pharmacologically, the receptor exhibited high affinity for NPY, PYY, and carboxyl-terminal fragments of NPY and PYY. In addition, Pro34-substituted analogues had very low affinity. With the use of Northern blot analysis, high levels of Y2 mRNA were detected in a variety of brain regions with little expression in peripheral tissues. Thus, the receptor protein has the pharmacological properties and distribution of the human Y2 receptor.

Effect of ractopamine on growth performance, carcass composition, and cutting yields of pigs slaughtered at 107 and 125 kilograms

Abstract: At approximately 68 kg live weight, crossbred barrows and gilts (n = 144) were allocated to be fed to one of two weight end points (107 kg and 125 kg). Pigs from each weight group were treated with Ractopamine (RAC) (0, 10, or 20 ppm; n = 24/ treatment) for the last 40 kg of gain. Feed consump- tion and weight gain were measured. Pigs were slaughtered and carcass measurements made at 24 h postmortem. Carcasses were fabricated into wholesale, trimmed wholesale, and boneless wholesale cuts for cutting yields. Hams were separated into muscle, fat, and bone. The RAC improved growth characteristics and carcass characteristics. Pigs fed RAC had in- creased ( P < .01) average daily gain and improved ( P < .01) feed:gain ratio over controls in each weight group. Carcasses from pigs treated with RAC had larger ( P < .01) longissimus muscle area and reduced ( P < .01) fat at the 10th rib. Cuts from 125-kg pigs were generally heavier than those from 107-kg pigs. The RAC increased ( P < .05) the boneless cut weights of both weight groups. Percentage of dissected lean from the hams of RAC-treated pigs was ( P < .05) higher than that of controls. Few consistent differences were observed between the 10 and 20 ppm of RAC treatments. Results from this study indicate that RAC had positive effects on the growth charac- teristics, carcass characteristics, and carcass cutting yields of pigs representative of the broad spectrum of market weights.