Showing papers by "Eli Lilly and Company published in 1991"

Action of 2',2'-difluorodeoxycytidine on DNA synthesis.

Abstract: The action of the new deoxycytidine analogue 2′,2′-difluorodeoxycytidine (dFdC) on DNA synthesis was investigated in whole cells and in vitro assay systems with purified DNA polymerases. DNA synthesis in human lymphoblastoid CEM cells was inhibited by dFdC in a concentration-dependent manner that could not be reversed by exogenous deoxynucleosides. The analogue was incorporated into cellular DNA; most of the incorporated dFdC 5′-monophosphate (dFdCMP) residues were in internucleotide linkage. In vitro DNA primer extension assays demonstrated that dFdC 5′-triphosphate (dFdCTP) competed with deoxycytidine triphosphate for incorporation into the C sites of the growing DNA strand. The ratios of the apparent K m values for the incorporation of dFdCTP and dCTP into a C site of M13mp19 DNA were 21.8 and 22.9 for DNA polymerases α and e, respectively. The apparent K l values of dFdCTP were 11.2 µm for DNA polymerase α and 14.4 µm for polymerase e. After dFdCMP incorporation, the primer was extended by one deoxynucleotide before a major pause in the polymerization process was observed. This was in contrast to the action of arabinosylcytosine 5′-triphosphate, which caused both DNA polymerases α and e to pause at the site of incorporation. The 3′ → 5′ exonuclease activity of DNA polymerase e was essentially unable to excise nucleotides from DNA containing dFdCMP at either the 3′-end or at an internal position, whereas arabinosylcytosine monophosphate was removed from the 3′-terminus at 37% the rate for deoxynucleotides. The cytotoxic activity of dFdC was strongly correlated with the amount of dFdCMP incorporated into cellular DNA. Our results demonstrate qualitative and quantitative differences in the molecular actions of dFdC and arabinosylcytosine on DNA metabolism, but are consistent with an important role for such incorporation in the toxicity of dFdC.

Multiple dose injection pen

Abstract: The present invention relates to a hypodermic syringe having the same general appearance as a pen which is specifically adapted to provide for multiple measured injections of materials such as insulin or human growth hormone.

Fluoxetine and suicide: a meta-analysis of controlled trials of treatment for depression.

Abstract: OBJECTIVE--A comprehensive meta-analysis of clinical trial data was performed to assess the possible association of fluoxetine and suicidality (suicidal acts and ideation). DESIGN--Retrospective analysis of pooled data from 17 double blind clinical trials in patients with major depressive disorder comparing fluoxetine (n = 1765) with a tricyclic antidepressant (n = 731) or placebo (n = 569), or both. MAIN OUTCOME MEASURES--Multiple data sources were searched to identify patients with suicidal acts. Suicidal ideation was assessed with item 3 of the Hamilton depression rating scale, which systematically rates suicidality. Emergence of substantial suicidal ideation was defined as a change in the rating of this item from 0 or 1 at baseline to 3 or 4 during double blind treatment; worsening was defined as any increase from baseline; improvement was defined as a decrease from baseline at the last visit during the treatment. RESULTS--Suicidal acts did not differ significantly in comparisons of fluoxetine with placebo (0.2% v 0.2%, p = 0.494, Mantel-Haenszel adjusted incidence difference) and with tricyclic antidepressants (0.7% v 0.4%, p = 0.419). The pooled incidence of suicidal acts was 0.3% for fluoxetine, 0.2% for placebo, and 0.4% for tricyclic antidepressants, and fluoxetine did not differ significantly from either placebo (p = 0.533, Pearson's chi 2) or tricyclic antidepressants (p = 0.789). Suicidal ideation emerged marginally significantly less often with fluoxetine than with placebo (0.9% v 2.6%, p = 0.094) and numerically less often than with tricyclic antidepressants (1.7% v 3.6%, p = 0.102). The pooled incidence of emergence of substantial suicidal ideation was 1.2% for fluoxetine, 2.6% for placebo, and 3.6% for tricyclic antidepressants. The incidence was significantly lower with fluoxetine than with placebo (p = 0.042) and tricyclic antidepressants (p = 0.001). Any degree of worsening of suicidal ideation was similar with fluoxetine and placebo (15.4% v 17.9%, p = 0.196) and with fluoxetine and tricyclic antidepressants (15.6% v 16.3%, p = 0.793). The pooled incidence of worsening of suicidal ideation was 15.3% for fluoxetine, 17.9% for placebo, and 16.3% for tricyclic antidepressants. The incidence did not differ significantly with fluoxetine and placebo (p = 0.141) or tricyclic antidepressants (p = 0.542). Suicidal ideation improved significantly more with fluoxetine than with placebo (72.0% v 54.8%, p less than 0.001) and was similar to the improvement with tricyclic antidepressants (72.5% v 69.8%, p = 0.294). The pooled incidence of improvement of suicidal ideation was 72.2% for fluoxetine, 54.8% for placebo, and 69.8% for tricyclic antidepressants. The incidence with fluoxetine was significantly greater than with placebo (p less than 0.001) and did not differ from that with tricyclic antidepressants (p = 0.296). CONCLUSIONS--Data from these trials do not show that fluoxetine is associated with an increased risk of suicidal acts or emergence of substantial suicidal thoughts among depressed patients.

The Effects of Formulation Variables on the Stability of Freeze-Dried Human Growth Hormone

Abstract: Formulation often has a dramatic effect on degradation of proteins during the freeze-drying process as well as impacting on the "shelf-life" stability of the freeze-dried product. This research presents the results of a formulation optimization study of the "in-process" and shelf-life stability of freeze-dried human growth hormone (hGH). Chemical decomposition via methionine oxidation and deamidation of asparagine residues as well as irreversible aggregation were characterized by HPLC assay methodology. In-process degradation and stability of low moisture freeze-dried solids were studied at 25 and 40 degrees C in a nominal nitrogen headspace (approximately 0.5% O2). Formulation variables included pH, level of salts, and the nature of the lyoprotectant. Studies of the effect of shear on aggregation in solutions indicated that shear comparable to that experienced during filtration does not induce aggregation. Irreversible changes in hGH during the freeze-drying process were minimal, but chemical decomposition via methionine oxidation and asparagine deamidation and aggregation did occur on storage of the freeze-dried solid. Decomposition via methionine oxidation was significant. A combination of mannitol and glycine, where the glycine remains amorphous, provided the greatest protection against decomposition and aggregation. It is postulated that an excipient system that remains at least partially amorphous is necessary for stabilization. However, the observation that dextran 40 formulations showed poor stability toward aggregation demonstrates that an amorphous excipient system is not a sufficient condition for stability. Stability of the solid was optimal when produced from solutions in the pH range, 7-7.5, with severe aggregation being observed at high pH. The level of sodium phosphate buffer affected stability of the solid, although this relationship was complex. Freeze-drying in the presence of NaCl produced severe aggregation and precipitation during the freeze-drying process as well as acceleration of oxidation and/or deamidation.

An electroenzymatic glucose sensor fabricated on a flexible substrate

Abstract: A glucose-oxidase-based electroenzymatic glucose sensor has been developed using thin-/thick-film processing techniques. We believe that this processing scheme will overcome a major impediment to the successful movement of the devices from the research laboratory to the marketplace by removing the difficulty of fabricating large numbers of reproducible and economical sensors. Several hundred sensors have been fabricated and tested in vitro. The sensors respond linearly to glucose, are stable for 72 h, are oxygen independent and have a 90 s response time. The sensors have also responded appropriately during preliminary glucose tolerance tests performed in rabbits.

Thin film electrical component

Abstract: A biocompatible thin film electrical component is configured for use in a human body or other ionic liquid environment. A polyimide substrate is bonded to a glass carrier plate sized for handling by automatic equipment and a multiple-layer metal conductor is deposited on the substrate and patterned to define an electrical circuit or biosensor. The polyimide and the glass establish a bond therebetween that withstands handling yet is broken using biocompatible releasing agents and techniques. The polyimide substrate and glass carrier plate preferably have similar thermal expansion properties to reduce the likelihood of fracture and delamination problems during release of the substrate from the carrier plate. An insulation layer covers the metal conductor and, in one embodiment, is made of a polyimide having a cure temperature lower than the temperature at which interdiffusion occurs in the metal layers in the conductor.

Ion Trap Mass Spectrometry

Abstract: This paper reports on the quadrupole ion trap which is a mass spectrometer whose essential components can be held in one hand. But is has a mass range of about 10{sup 5} daltons per charge, provides molecular weight and structural information on biopolymers, and has the greatest sensitivity of all mass spectrometers. These features, however, has become available only within the past few years. They stem from an almost neglected 1958 invention, one in which interest was maintained by only a few research groups, notably those of John Todd at the University of Kent in England and Ray March at Trent University in Canada. Development of a new scanning method by George Stafford and his coworkers of Finnigan Corp. provided the impetus that led Finnigan to introduce a commercial ion trap in 1983. Since then, the device has been transformed from a simple gas chromatography detector to a high-performance mass spectrometer.

Structure of recombinant human rheumatoid arthritic synovial fluid phospholipase A2 at 2.2 Å resolution

Abstract: Phospholipases A2 (PLA2s) may be grouped into distinct families of proteins that catalyse the hydrolysis of the 2-acyl bond of phospholipids and perform a variety of biological functions. The best characterized are the small (relative molecular mass approximately 14,000) calcium-dependent, secretory enzymes of diverse origin, such as pancreatic and venom PLA2s. The structures and functions of several PLA2s are known. Recently, high-resolution crystal structures of complexes of secretory PLA2s with phosphonate phospholipid analogues have provided information about the detailed stereochemistry of transition-state binding, confirming the proposed catalytic mechanism of esterolysis. By contrast, studies on mammalian nonpancreatic secretory PLA2s (s-PLA2s) have only recently begun; s-PLA2s are scarce in normal cells and tissues but large amounts are found in association with local and systemic inflammatory processes and tissue injury in animals and man. Such s-PLAs have been purified from rabbit and rat inflammatory exudate, from synovial fluid from patients with rheumatoid arthritis and from human platelets. Cloning and sequencing shows that the primary structure of the human s-PLA2 has about 37% homology with that of bovine pancreatic PLA2 and 44% homology with that of Crotalus atrox PLA2. The human s-PLA2 is an unusually basic protein, yet contains most of the highly conserved amino-acid residues and sequences characteristic of the PLA2s sequenced so far. Here we report the refined, three-dimensional crystal structure at 2.2 A resolution of recombinant human rheumatoid arthritic synovial fluid PLA2. This may aid the development of potent and specific inhibitors of this enzyme using structure-based design.

Daptomycin disrupts membrane potential in growing Staphylococcus aureus.

Abstract: Daptomycin (LY146032) caused a calcium-dependent dissipation of the membrane potential (delta psi) in Staphylococcus aureus without noticeably affecting the chemical gradient (delta pH) across the membrane. The effect of daptomycin on membrane energization may account for many of the inhibitory effects on macromolecular biosyntheses and membrane function reported for this antibiotic. Our evidence indicates that the bactericidal activity of daptomycin is dependent on an available delta psi.

Fluoxetine, a selective inhibitor of serotonin uptake

Abstract: In summary, fluoxetine is a highly selective serotonin uptake inhibitor in vitro and in vivo. The conformation of fluoxetine, which resembles that of sertraline and other serotonin uptake inhibitors, appears to be a key feature that enables its high affinity and selective interaction with the serotonin transporter. The para-trifluoromethyl substituent, however, is also a pivotal structural element. The molecular pharmacology of fluoxetine has been well-defined, and its in vivo pharmacological effects appear to be mediated almost exclusively by serotonin uptake inhibition. Its selectivity for the serotonin transporter, lack of affinity for neurotransmitter receptors, and retention of selectivity following metabolism to norfluoxetine make fluoxetine a useful tool to explore pharmacologically induced increases in serotonin neurotransmission. Fluoxetine has found a variety of therapeutic application. Its use in treating depression has been most extensively studied, but controlled clinical studies also suggest the drug may have a role in treating obesity and bulimia. Moreover, a variety of other psychiatric disorders may be treatable with this drug. Regardless of the outcome of these clinical trials, it is apparent that fluoxetine has found a useful niche in therapy, and can be used as a probe to determine the role of serotonin in modulating human pathophysiologies.

Separation of DNA restriction fragments by capillary electrophoresis using coated fused silica capillaries.

Abstract: The abilities of several different capillary electrophoresis techniques to separate DNA restriction fragments up to 23,000 bp were investigated. Methods employing electroosmotic flow in an untreated silica capillary were found to provide, at best, only partial resolution of the 23 fragments in a 1-kbp DNA ladder. By coating the inner walls of a silica capillary with poly(acrylamide) and filling these capillaries with buffers containing methylcellulose as a sleving medium, all fragments in the 1-kbp DNA ladder were separated. In addition, this technique facilitated the separation of the very large fragments in a lambda DNA-HindIII digest. Optimum resolution was obtained at low separation potentials using buffers containing at least 0.5% methylcellulose. The performance of this technique, i.e., resolution and quantitation, make capillary electrophoresis a powerful complement to slab gel electrophoresis and may make it a preferred alternative to both agarose gel electrophoresis and HPLC for applications such as the confirmation of plasmid integrity.

Antibacterial activities and modes of action of vancomycin and related glycopeptides.

Abstract: Vancomycin (21) is produced by Amycolatopsis orientalis (18) (previously designated Nocardia orientalis and Streptomyces orientalis). The antibiotic has been marketed for the past 30 years and continues to be marketed, as the hydrochloride salt, to treat deep-seated gram-positive bacterial infections. It is the drug of choice for infections caused by Staphylococcus aureus, especially those caused by strains resistant to methicillin. Vancomycin is bactericidal to most gram-positive organisms, but gram-negative organisms are resistant. Vancomycin is not absorbed from the gastrointestinal tract, and the antibiotic is used to treat enterocolitis, especially that caused by Clostridium difficile

Reproducible electrodeposition of biomolecules for the fabrication of miniature electroenzymatic biosensors

Abstract: A technique has been developed for reproducibly depositing an enzyme or other biomolecule onto the surface of a conductor. Specifically, glucose oxidase has been electrodeposited onto the surface of an electrode previously electroplated with platinum black. This deposition takes place without the assistance of an electropolymerizable matrix material such as polypyrrole. The deposition process does not require a time-consuming alignment step, characteristic of some deposition techniques utilizing photoimageable matrix materials; yet it results in the enzyme being precisely deposited only on the working electrode. The thickness of the resulting enzyme layer can be varied from Angstroms to microns by varying the deposition parameters. With proper fixturing, potentially hundreds of sensors can be simultaneously coated with enzyme.

Role of Adrenal Cortical Activation in the Immunosuppressive Effects of Chronic Morphine Treatment

Abstract: Implantation of a 75-mg morphine pellet in sham-adrenalectomized male C3H/HeN mice resulted in significant elevations of serum corticosterone levels within 6 h Corticosterone levels remained elevated (3- to 4-fold) for 72 h and had returned to normal by 120 h postimplantation Within 48 h of pellet implantation, morphine-pelleted mice exhibited marked reductions in spleen (35%) and thymus weight (56%) relative to values in placebo-pelleted controls In addition, adrenal hypertrophy was observed in the morphine-pelleted shams (50% increase in adrenal weight relative to placebo) The magnitude of splenic and thymic atrophy was reduced by about 50% in adrenalectomized morphine-pelleted mice (17% and 22% reductions, respectively) compared to that in adrenalectomized mice implanted with placebo pellets Lymphocyte proliferative responses to the T-cell mitogen Concanavalin-A and the B-cell mitogen bacterial lipopolysaccharide were also significantly reduced in the morphine-pelleted sham mice Morphine-induced

Effects of body weight restriction on the development and progression of spontaneous osteoarthritis in guinea pigs.

Abstract: Hartley albino guinea pigs develop spontaneous osteoarthritis (OA) of the knee joint. A study was done to determine the importance of body weight in the pathogenesis of this disease. Two groups of 20 male guinea pigs each were maintained on the same diets. The control group was allowed ad libitum feed consumption and the other group was restricted to 30–35 gm of feed per day. Ten animals from each group were killed at 9 months of age to evaluate histologic features of the knee joints. The severity of the OA lesions was reduced by 40%, in conjunction with a 28% decrease in body weight, in the diet-restricted group. The remaining animals were killed at 18 months of age. Those in the diet-restricted group had a 56% reduction in severity of lesions, with a 29% decrease in body weight. These results indicate that body mass in guinea pigs, as in humans, is an important predisposing factor for the development of spontaneous OA of the knee.