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Soluble targets represent a special challenge when employing ligand binding assays to support pharmacokinetic analysis of monoclonal therapeutics. Target-engaged antibody is not available for binding in immunoassays employing anti-idiotype-specific antibodies or target for capture. We investigated several formats of total antibody assays that show reduced interference of soluble targets: direct target capture, indirect target capture and acid dissociation. While indirect target capture worked well for a regular affinity antibody against DKK1, a high affinity antibody against PCSK9 required an additional acid dissociation step. The choice of a suitable format was antibody and target dependent. Our results offer several choices to approach immunoassay development for soluble targets.

N1-guanyl-1,7-diaminoheptane (GC7) is a potent inhibitor of deoxyhypusine synthase (DHS), the enzyme that catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF-5A). Since eIF-5A is the only known cellular substrate for DHS and GC7 has been reported to block the proliferation of CHO cells, it has been suggested that DHS may be a novel target for anti-cancer therapy. In the present study we investigated the antiproliferative effect of GC7 on several tumorigenic cell lines under various growth conditions. We found that this compound inhibits the proliferation of H9 cells in suspension culture and the growth of HeLa cells and v-src-transformed NIH3T3 cells under both anchorage-dependent and anchorage-independent conditions. Moreover, studies with NIH3T3 cells and v-src-transformed NIH3T3 cells show that GC7 inhibits the growth of both cell lines in monolayer culture with similar potency and could not reverse the transformed phenotype. In addition, the v-src-transformed cells grown under both anchorage-dependent and anchorage-independent conditions showed similar sensitivity toward GC7. These data indicate that GC7 acts as a general antiproliferative agent and does not appear to preferentially target tumorigenic cell types. Cell cycle analysis show that GC7 reduces the CHO-K1 cell population in the G1-phase of the cell cycle by 42% and increases the number of cells in the S-phase by 44%. This cell cycle distribution profile strikingly resembles the distribution of cells treated with puromycin. This result supports the hypothesis that the synthesis of a subset of proteins important for the S-phase progression of CHO-K1 cells might be dependent upon hypusinated eIF-5A. Thus the antiproliferative effect of GC7 appears to be related to its interference with the progression of cell cycle, which also provides a possible explanation for the lack of selectivity of GC7 between nontransformed and transformed cell types tested in this study.

The avian skeletal alpha-actin gene was used as a template for construction of a myogenic expression vector that was utilized to direct expression of a human IGF-I cDNA in cultured muscle cells and in striated muscle of transgenic mice. The proximal promoter region, together with the first intron and 1.8 kilobases of 3'-noncoding flanking sequence of the avian skeletal alpha-actin gene directed high level expression of human insulin-like growth factor I (IGF-I) in stably transfected C2C12 myoblasts and transgenic mice. Expression of the actin/IGF-I hybrid gene in C2C12 muscle cells increased levels of myogenic basic helix-loop-helix factor and contractile protein mRNAs and enhanced myotube formation. Expression of the actin/IGF-I hybrid gene in mice elevated IGF-I concentrations in skeletal muscle 47-fold resulting in myofiber hypertrophy. IGF-I concentrations in serum and body weight were not increased by transgene expression, suggesting that the effects of transgene expression were localized. These results indicate that sustained overexpression of IGF-I in skeletal muscle elicits myofiber hypertrophy and provides the basis for manipulation of muscle physiology utilizing skeletal alpha-actin-based vectors.

Treatment of Fura-2 loaded HEL cells, a human megakaryocyte-like cell line, with P2-purinoceptor nucleotide ligands (ADP, ATP, UTP, 2-methylthio-ATP) evoked a rise in cytosolic calcium. Homologous- and cross-desensitization studies using sequential addition of nucleotides showed that the ADP-induced calcium response in HEL cells was mediated mainly by purinoceptor(s) for which ADP but not ATP was an agonist. There were also minor contributions from purinoceptors for which ATP and ADP are both agonists (probably P2U and P2Y). ATP inhibited the ADP-induced calcium response in HEL cells. This inhibition was overcome by raising the ADP concentration, thus indicating that ATP was an antagonist for the HEL cell ADP receptor. AMP was also an antagonist, albeit weak, for the HEL cell ADP receptor. Antagonism of the ADP-induced calcium response by ATP was similarly observed in MEG-01 cells, another human megakaryocyte-like cell line, but not in 293 cells, a nonhematopoietic cell line. These studies suggest that HEL cells express an ADP receptor for which ATP and AMP are antagonists. This characteristic of the HEL cell ADP receptor is also displayed by the platelet P2T receptor. Thus, HEL cells appear to be a surrogate source of the platelet ADP receptor.

P in the do Tirofiban And Reopro Give similar Efficacy outcomes Trial (TARGET) treated with abciximab compared with tirofiban had a lower 30-day incidence of the combined end point of death, myocardial infarction, and target vessel revascularization that reflected, in large part, a decrease in the incidence of periprocedural myocardial infarction.1 We have found that the extent of platelet aggregation inhibition was greater from 15 to 60 minutes after abciximab than that after the TARGET study dosage of tirofiban (10 g/kg bolus, followed by a 0.15 g/ kg/min infusion).2–4 To identify a bolus associated with maximal inhibition of platelet aggregation, we used flow cytometry to characterize the inhibitory effects of tirofiban in blood from patients with acute coronary syndromes. In subsequent studies, the average extent of inhibition of turbidometric aggregation in response to 20 M of adenosine diphosphate (ADP) was found to be 90% during the first hour when a bolus of 25 g/kg was combined with the same infusion (0.15 g/kg/min for 18 to 24 hours) employed in the TARGET study.5 In this report, we describe the concentrations of tirofiban in blood and their correlation with the inhibition of turbidometric aggregation in response to 20 M of ADP. • • • Protocols were approved by the institutional review boards of the participating institutions. All patients provided written informed consent. Eligible patients included those with an acute coronary syndrome (ischemic symptoms plus either 0.5 mm of ST-segment depression on electrocardiogram or increased troponin or creatine kinase-MB) in whom a percutaneous coronary intervention was clinically mandated and performed. Exclusion criteria included treatment with antiplatelet agents other than aspirin in the previous 14 days, thrombolytic therapy within 24 hours, renal insufficiency (creatinine 2.5 mg/dl), and contraindication to treatment with a glycoprotein IIb/IIIa inhibitor. To perform in vitro studies, blood was obtained from patients with acute coronary syndromes before coronary intervention or treatment with an antiplatelet agent other than aspirin. After anticoagulation with corn trypsin inhibitor, a specific inhibitor of coagulation factor XIIa,6 the blood was spiked with 0, 50, 100, or 150 ng/ml of tirofiban. After incubation for 15 minutes at room temperature, the capacity to bind fibrinogen in response to 1 M of ADP was determined with the use of flow cytometry. The concentration of tirofiban in blood and its associated inhibition of platelet function were assessed in a preliminary study in which patients were treated with a 10 g/kg bolus2 and in a subsequent study in which patients were treated in a sequential, open-label, nonrandomized fashion with a 20 or 25 g/kg bolus.5 Tirofiban was administered before the start of the coronary intervention, and each bolus was combined with an 18to 24-hour infusion of 0.15 g/kg/min. Patients were treated with aspirin (325 mg before the procedure and daily) and unfractionated heparin (target activated clotting time 250 seconds). Clopidogrel (300 mg initially and then 75 mg daily) was begun after the coronary intervention ( 1 hour). The concentration of tirofiban was determined by radioimmunoassay after 5, 15, 30, 45, 60, 120, and 360 minutes.7 Platelet function was assessed by light transmission aggregometry (Bio Data Corp., Helena Laboratories, Horsham, Pennsylvania), and flow cytometry before treatment and after 15, 30, 45, 60, and 120 minutes, and an additional assessment was made after 5 minutes with flow cytometry. Blood for aggregometry was anticoagulated with D-Phe-Pro-Argchloromethyl ketone (PPACK, 38 M) to avoid effects of citrate on inhibitory properties of tirofiban.8 Blood for flow cytometry was anticoagulated with corn trypsin inhibitor. Maximal turbidometric ex vivo aggregation after 4 minutes was assessed in plateletrich plasma in response to 20 M of ADP (Chronolog, Havertown, Pennsylvania). Assessment of the capacity of platelets to bind fibrinogen was performed as previously described in detail.9 Assay and fixation were performed at each site for flow cytometry. Samples were analyzed at the University of Vermont within 30 hours. As previously described,5 the primary objective was to identify a bolus dose of tirofiban that achieved a mean inhibition of platelet aggregation of 90% plus a lower bound of the 95% confidence interval of inhibition of 85% from 15 to 45 minutes after onset of treatment. The slope of the association between selected concentrations of tirofiban and the extent of From the University of Vermont, Burlington, Vermont; University of Pennsylvania Medical Center, Philadelphia, Pennsylvania; Baylor College of Medicine, Houston, Texas; Prairie Cardiovascular, Springfield, Illinois; and Merck & Company, Inc., West Point, Pennsylvania. This report was supported by Merck & Company, Inc., West Point, Pennsylvania. Dr. Schneider’s address is: University of Vermont, 208 South Park Drive, Suite 2, Colchester, Vermont 05446. E-mail: djschnei@zoo.uvm.edu. Manuscript received August 7, 2002; revised manuscript received and accepted September 24, 2002.

Erythropoietin (EPO) is an important molecule in the erythropoiesis and various forms of EPO have been marketed in managing anemia in humans. Long acting EPOs for less frequent dosing have been generated either by increasing the number of glycosylation sites of the EPO molecule or by linking it to a polyethylene glycol (PEG). We have generated recombinant human EPO (rhEPO) using glycoengineered Pichia pastoris strains and evaluated the pharmacokinetics (PK) in rats of this molecule linked to a 40 kDa PEG (PEGylated rhEPO), in relation to its glycosylation patterns. As expected, the PEGylated rhEPO exhibited a significant improvement in half‐life of serum when compared with the non‐PEGylated version. Interestingly, the PK properties of the PEGylated rhEPO molecule were also significantly influenced by the glycosylation profile. Specifically, PEGylated rhEPO with a significantly higher sialic acid content in the biantennary structure (high A2) exhibited lower systemic clearance and higher systemic exposure than those with a lower sialic acid content (low A2) following either intravenous or subcutaneous administrations. These results suggest that A2 content may be one of the important criteria for release in manufacturing PEGylated rhEPO to ensure consistent PK.

The mechanism underlying subcutaneous absorption of macromolecules and factors that can influence this process were studied in rats using PEGylated erythropoietins (EPOs) as model compounds. Using a thoracic lymph duct cannulation (LDC) model, we showed that PEGylated EPO was absorbed from the subcutaneous injection site mainly via the lymphatic system in rats, which is similar to previous reports in sheep. After subcutaneous administration, the serum exposure was reduced by ∼70% in LDC animals compared with that in the control animals, and most of the systemically available dose was recovered in the lymph. In both LDC and intact rats, the total radioactivity recoveries in excreta after subcutaneous administration were high (70-80%), indicating that catabolism, not poor absorption, was the main cause for the observed low bioavailability (30-40%). Moreover, catabolism of PEGylated EPO was found with both rat subcutaneous tissue homogenate and lymph node cell suspensions, and a significant amount of dose-related breakdown fragments was found in the lymph of LDC rats. In addition, the bioavailability of PEGylated EPOs was shown to be 2- to 4-fold lower in "fat rats," indicating that physiologic features pertinent to lymphatic transport can have a profound impact on subcutaneous absorption. Limited studies in dogs also suggested similar subcutaneous absorption mechanisms. Collectively, our results suggest that the lymphatic absorption mechanism for macromolecules is probably conserved among commonly used preclinical species, e.g., rats and dogs, and that mechanistic understanding of the subcutaneous absorption mechanism and associated determinants should be helpful in biologic drug discovery and development.

Background: Dalotuzumab (MK 0646), an anti-IGF1R antibody intended for cancer therapy, has progressed to Phase III clinical trials. To evaluate pharmacokinetic properties, we developed and compared two ELISAs to measure dalotuzumab in human serum and validated the second method following regulatory guidelines for ligand-binding assays. Results: After an IGF1R-mediated capture step, dalotuzumab was detected by either an antihuman IgGFc- or by an antihuman IgG1-specific antibody. The assay range was 20 to 2000 ng/ml with mean inter-day accuracy of controls ranging from 97 to 108% (method A) and 83 to 97% (method B), respectively. Mean assay precision was ≤20% CV both intra- and inter-day. Other parameters that were validated included dilution linearity, stability, interferences and incurred sample reanalysis. In addition, application of both assay formats to clinical sample analysis was demonstrated establishing time–concentration curves. Conclusion: As the methods rely on commercial reagents, they may be applicable to other anti-IGF1R antibodies and facilitate the development of new therapeutics.

To assess the role of cis-acting elements within the smooth muscle alpha-actin gene in smooth muscle cells (SMC), we transfected chicken smooth muscle alpha-actin promoter-chloramphenicol acetyltransferase gene fusion plasmids into SMC derived from rat and chicken aortas. In marked contrast to effects in chicken skeletal myoblasts and fibroblasts, p122CAT (positions −122 to +19), containing two conserved CArG elements, elicited a modest increase in chloramphenicol acetyltransferase reporter activity in chicken SMC. Addition of upstream sequences between −122 and −151 (p151CAT) increased activity in adult chicken SMC. Addition of sequence between positions −151 and −257 (p257CAT) resulted in a 7-fold increase in chloramphenicol acetyltransferase activity over that of p151CAT in rat SMC, but not in chicken SMC. A genomic clone encoding the rat smooth muscle alpha-actin gene was isolated, and the 5'-flanking region was partially characterized. Comparison of primary sequence between rat and chicken promoters showed a conserved E box motif at position −214 in the chicken gene and at position −213 in the rat gene. Results of these studies demonstrate that regions upstream of the conserved CArG elements exert potent regulatory effects on transcription and that SMC require different cis-acting elements than other cell types to transcriptionally regulate this gene.

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

Cleavage of amyloid precursor protein (APP) by the beta- and gamma-secretases generates the amino and carboxy termini, respectively, of the A beta amyloidogenic peptides A beta40 and A beta42--the major constituents of the amyloid plaques in the brain parenchyma of Alzheimer's disease patients. There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of gamma-secretase activity: mutations in PS1 and PS2 that are associated with early-onset familial Alzheimer's disease increase the production of A beta42 (refs 4-6), the more amyloidogenic peptide; gamma-secretase activity is reduced in neuronal cultures derived from PS1-deficient mouse embryos; and directed mutagenesis of two conserved aspartates in transmembrane segments of PS1 inactivates the ability of gamma-secretase to catalyse processing of APP within its transmembrane domain. It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmembrane aspartyl protease or a gamma-secretase cofactor, or helps to colocalize gamma-secretase and APP. Here we report photoaffinity labelling of PS1 (and PS2) by potent gamma-secretase inhibitors that were designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may contain the active site of gamma-secretase. Interestingly, the intact, single-chain form of wild-type PS1 is not labelled by an active-site-directed photoaffinity probe, suggesting that intact wild-type PS1 may be an aspartyl protease zymogen.

γ-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two Aβ peptide isoforms, Aβ40 and Aβ42. Here we report the detergent solubilization and partial characterization of γ-secretase. The activity of solubilized γ-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the β-secretase cleavage site. Cleavage of C100Flag by γ-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Aβ40 or Aβ42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Aβ40 and Aβ42 termini. Recovery of catalytically competent, soluble γ-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized γ-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Aβ40 and Aβ42 in mammalian cells. Upon gel exclusion chromatography, solubilized γ-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 × 10 6 . Anti-PS1 antibody immunoprecipitates γ-secretase activity from the solubilized γ-secretase preparation. These data suggest that γ-secretase activity is catalyzed by a PS1-containing macromolecular complex.

The subcellular distributions of the endogenous eukaryotic translation initiation factor, eIF-5A, and Rev, a protein of the human immunodeficiency virus proposed to interact with eIF-5A, were studied in COS-7 cells treated with inhibitors of RNA or protein synthesis. We have previously shown that transiently expressed Rev is localized in the nucleolus, whereas eIF-5A is primarily in the cytoplasm. The subcellular localization of Rev was not affected by treatment with protein synthesis inhibitors (cycloheximide, CHX, 10 micrograms/ml; puromycin, 10 micrograms/ml), although its location changed from predominantly the nucleolus to the cytoplasm after treatment with RNA synthesis inhibitors (actinomycin D, 4 micrograms/ml, and 5,6-dichloro-1 beta-D-ribofuranosylbenzimidazole, DRB; 0.1 mM), as previously reported. In contrast, none of the RNA synthesis inhibitors (alpha-amanitin, 10 micrograms/ml; actinomycin D, 4 micrograms/ml, and DRB, 0.1 mM) caused any significant changes in the subcellular distribution pattern of eIF-5A. However, treatment with puromycin, a protein synthesis inhibitor known to dissociate ribosomes, dramatically altered the subcellular distribution pattern of eIF-5A in 30% of the cell population. In these cells, the staining of eIF-5A was changed from an endoplasmic reticulum (ER) net work-like perinuclear structure to a patched dotted pattern dispersed throughout the cytoplasm. This change was not observed in the same cells stained for calnexin, an ER resident protein, nor in cells treated with CHX, which freezes the ribosomes to block protein synthesis. Our data suggest that eIF-5A does not shuttle between the nucleus and cytoplasm in the same way as Rev. Our findings are consistent with our previous conclusion that eIF-5A is associated with the ER through ribosomes and support a role for eIF-5A in protein synthesis.

To gain insight into the role of the eukaryotic translation initiation factor, eIF-5A, we investigated the subcellular distribution of this protein in several cultured cell types and at different stages of the cell cycle using a highly potent monospecific polyclonal antibody to eIF-5A. Studies using indirect immunofluorescence and confocal microscopy in conjunction with subcellular fractionation demonstrate that eIF-5A is primarily localized in the cytoplasm of cells. This cytoplasmic location of eIF-5A is not significantly altered in different stages of the cell cycle and the subcellular distribution pattern of eIF-5A is not changed by viral oncogene transformation. Cell fractionation experiments identified two populations of eIF-5A in the cytoplasm, a soluble fraction and a fraction bound to internal membranes. By double immunofluorescence staining with an antibody against calnexin, a resident protein of the endoplasmic reticulum (ER), we demonstrate that the membrane-bound fraction of eIF-5A colocalizes with the ER and not with the cytoskeleton. Expression of Rev, a regulatory protein of human immunodeficiency virus type 1 (HIV-1), does not alter the subcellular distribution of endogenous eIF-5A in these cells. eIF-5A is detected in all tissues and cells examined including extracts prepared from Xenopus oocytes. Our results indicate that eIF-5A is a ubiquitous cytoplasmic protein and suggest that a site of eIF-5A function is likely to be in association with the ER.

The Escherichia coli 2.2 kilobase dnaZX region contains one 1929 nucleotide reading frame which directs the synthesis of two protein products involved in DNA polymerization. The larger consists of 643 amino acids in a deduced 71,114 dalton chain which could be the tau subunit of DNA polymerase III. The smaller, the DNA polymerase III gamma subunit, is encoded by the same reading frame as the larger. The dnaZX sequence contains a region homologous to ATP binding sites, suggesting that these replication factors are adenine nucleotide binding proteins.