Synthesis and Use of a Phosphonate Amidine to Generate an Anti-Phosphoarginine-Specific Antibody.

Protein arginine phosphorylation is a post-translational modification (PTM) that is important for bacterial growth and virulence. Despite its biological relevance, the intrinsic acid lability of phosphoarginine (pArg) has impaired studies of this novel PTM. Herein, we report for the first time the development of phosphonate amidines and sulfonate amidines as isosteres of pArg and then use these mimics as haptens to develop the first high-affinity sequence independent anti-pArg specific antibody. Employing this anti-pArg antibody, we further showed that arginine phosphorylation is induced in Bacillus subtilis during oxidative stress. Overall, we expect this antibody to see widespread use in analyzing the biological significance of arginine phosphorylation. Additionally, the chemistry reported here will facilitate the generation of pArg mimetics as highly potent inhibitors of the enzymes that catalyze arginine phosphorylation/dephosphorylation.

Validation and implementation of drug-dependent antibody assays in clinical trials for safety monitoring of patients dosed with roxifiban, an orally bioavailable glycoprotein IIb/IIIa antagonist.

Thrombocytopenia exposes patients to increased bleeding risk. This serious adverse event was observed with a frequency of approximately 2% in early clinical trials with the potent, orally bioavailable glycoprotein (GP) IIb/IIIa receptor antagonist roxifiban. We previously reported that drug-dependent antibodies (DDAbs) to GP IIb/IIIa are the main cause of thrombocytopenia with roxifiban. Two ELISA assays for detection of free DDAbs (in citrate plasma) and total DDAbs (in EDTA plasma to elute platelet bound DDAbs) were developed and analytically validated. These tests served two purposes during the clinical development program, to pre-screen patients for pre-existing antibodies and monitor patients for increasing antibody titers as a surrogate for eminent thrombocytopenia. The free DDAb assay showed inter and intra-assay precision of 5-12 and 12-14% CV, respectively. The total DDAb assay showed a precision of 5-10 and 4-12% CV, respectively. Three cycles of freeze-thaw did not significantly alter DDAb values in citrate plasma, EDTA plasma or extraction solution. The clinical qualifications of the two assays were conducted in two phase II clinical trials in coronary arterial disease (CAD) patients dosed with roxifiban. Both assays have demonstrated clinical sensitivity of nearly 99-100% and clinical specificity of nearly 95%.

Clinical trial of a leucotriene B4 receptor antagonist, BIIL 284, in patients with rheumatoid arthritis.

BACKGROUND: Several clinical and experimental lines of evidence suggest that leucotriene B4 (LTB4), an arachidonic acid derivative with potent proinflammatory properties, plays a key role in the pathophysiology of rheumatoid arthritis (RA). OBJECTIVE: To evaluate the efficacy and safety of BIIL 284, an oral long-acting LTB4 receptor antagonist, as monotherapy for the treatment of patients with active RA. METHODS: This was a multi-centre, randomised, double-blind, placebo-controlled trial of patients with active RA of 3 months' duration. A total of 342 patients were randomised to receive 5 mg, 25 mg or 75 mg of BIIL 284 or placebo. The primary end point was the percentage of patients achieving an American College of Rheumatology (ACR) 20. RESULTS: Although a higher percentage of ACR 20 responders was observed in the groups treated with 25 mg and 75 mg of BIIL 284 compared with those treated with placebo, no statistically significant differences were found between any of the three active treatment groups compared with the placebo group with regard to the primary or secondary end points. All trial treatments were safe and well tolerated. CONCLUSIONS: This clinical trial demonstrates that treatment of patients with active RA with a potent oral long-acting LTB4 receptor antagonist produced only modest improvements in disease activity. The results of this trial support the conclusion that LTB4 is not a major contributor to the inflammatory process in RA.

Vaccination with the recombinant allergen ProDer p 1 complexed with the cationic lipid DiC14-amidine prevents allergic responses to house dust mite.

The present study evaluated the prophylactic potential of ProDer p 1, the recombinant precursor form of the major mite allergen Der p 1, combined with the cationic lipid diC14-amidine in a murine model of house dust mite allergy. Naive mice vaccinated with the amidine/allergen complex developed a Th1-biased immune response characterized by the absence of specific IgE, the production of specific IgG2a, and the presence of IFN-gamma in splenocyte cultures. In contrast, ProDer p 1 adjuvanted with alum induced typical strictly Th2-biased allergic responses with strong IgG1 and IgE titers and IL-5 secretion. Removal of negatively charged sialic acids in ProDer p 1 or increasing the ionic strength reduced the binding of ProDer p 1 to the cationic liposomes and resulted in a decrease of the allergen immunogenicity, suggesting that complexation is required for triggering an optimal immune response. Finally, prophylactic vaccination with ProDer p 1-diC14-amidine reduced drastically the production of specific IgE and airway eosinophilia following subsequent immunization with Der p 1-alum and challenge with aerosolized house dust mite extracts. In conclusion, recombinant ProDer p 1 complexed with diC14-amidine could represent an efficient prophylactic vaccine against house dust mite allergy.

Prospective testing for drug-dependent antibodies reduces the incidence of thrombocytopenia observed with the small molecule glycoprotein IIb/IIIa antagonist roxifiban: implications for the etiology of thrombocytopenia.

Thrombocytopenia is a relatively common side effect observed during glycoprotein (GP) IIb/IIIa antagonist therapy. With the oral antagonist roxifiban, we observed thrombocytopenia, defined as 50% reduction of platelets over predose values or below 90 000/microL (9 x 10(10)/L), with a frequency of 2% (8 of 386). Thrombocytopenia occurred either early (days 2 to 4) or delayed (days 11 to 16). No additional cases were observed with up to 6 months of treatment. Retrospective analysis provided evidence for drug-dependent antibodies (DDABs) to GP IIb/IIIa in 5 of 6 subjects, suggestive of an immune etiology of thrombocytopenia. The hypothesis that excluding patients based on positive DDAB reaction would reduce the frequency of thrombocytopenia was tested. Patients were screened for DDABs during the study qualification period and, overall, 3.9% of the patients were excluded based on pre-existing DDAB concentrations above a statistically defined medical decision limit. An additional 2.6% were excluded based on therapy-related antibody production during the first 2 weeks. With antibody testing, 0.2% of patients (2 of 1044) developed immune-mediated thrombocytopenia. One case developed a rapidly increasing antibody concentration and presented with thrombocytopenia despite discontinuation of roxifiban therapy. The second case was related to a false-negative test result. The frequency of thrombocytopenia was statistically significantly reduced from 2% to 0.2% (P =.0007) comparing nonscreened and screened patients. Testing for DDABs can reduce the frequency of thrombocytopenia in patients treated with roxifiban and, by analogy, other GP IIb/IIIa antagonists. Thus, DDAB testing may be employed to increase the safety of GP IIb/IIIa antagonists.

Human immunodeficiency virus type-1-specific immune responses induced by DNA vaccination are greatly enhanced by mannan-coated diC14-amidine.

Use of mannan-coated N-t-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine (diC14-amidine) as an adjuvant for a DNA vaccine encoding glycoprotein 160 of human immunodeficiency virus type-1 (HIV-1) enhanced the antigen-specific immune responses. The role of interferon-gamma (IFN-gamma) and interleukin-12 in the mechanism of adjuvant action was also evaluated. Coating of diC14-amidine with mannan significantly augmented the HIV-specific delayed-type hypersensitivity reaction induced by the immunogenic DNA. HIV-1-specific cytotoxic T lymphocyte activity was also markedly enhanced by the mannan-diC14-amidine cocktail. An immunomodulatory effect of this cocktail was inhibited by treatment with anti-IFN-gamma monoclonal antibody in vivo, which suggests that IFN-gamma plays an important role in inducing cell-mediated immunity by the DNA vaccine containing this adjuvant. The results of both antigen-specific immunoglobulin isotype analysis and cytokine measurement showed that the immunogenic DNA incorporated into mannan-coated diC14-amidine elicits Th1-biased immune responses.

Suppression of the antibody response by a formamidine pesticide: dependence on the route of exposure.

These studies investigated the effects of exposure to chlordimeform (CDM), a formamidine pesticide, on selected in vivo immune parameters in the random bred CD-1 mouse. Further studies were done on the effects of this compound on the in vitro PFC response in C57BL/6 mice. Acute and 14-d exposure to CDM via the i.p. route resulted in a decrease in IgM antibody-forming (plaque-forming) cells (PFC) directed at the sheep red blood cell (sRBC) antigen when measured 4 d after i.p. immunization. This suppression was seen at doses as low as 20 mg/kg . d for 14 d. These same doses of CDM did not result in any alteration of cell-mediated immunity as measured by the delayed hypersensitivity response (DHR) to both keyhole limpet hemocyanin (KLH) and sRBC. Lymphocyte blastogenesis was increased in spleen cells from mice exposed to 40 mg/kg . d CDM in response to media alone, concanavalin A (Con A), and lipopolysaccharide (LPS). The in vitro PFC response by C57BL/6 mice was utilized to determine if CDM could suppress the antibody response due to a direct effect on the immune cells. CDM suppressed the in vitro PFC response only at concentrations that were directly cytolytic. A direct cytolytic effect was considered unlikely following exposure in the whole animal, since the suppression of the antibody response occurred in the absence of any effects on spleen cell number or spleen weight. To determine if route of exposure was a factor in the suppressive effects of CDM, 14-d studies were conducted administering CDM orally at doses up to 120 mg/kg . d. Both the CD-1 and C57BL/6 mouse were used to verify that a strain difference was not a factor. There was no effect on either the d 4 or d 5 antibody response, even though 43% of the mice exposed to 120 mg/kg died from the acute toxicity that can characterize this chemical. From an operational standpoint, these results indicate that the route of exposure of a compound relative to the route of administration of an antigen is an important consideration when determining the effects of that compound on an immune response. From an environmental standpoint, these results indicate that relatively high doses of chlordimeform do not result in consistent immunotoxicity as determined by the assays utilized.

Properties of conjugated protein immunogens which selectively stimulate delayed-type hypersensitivity.

If bovine serum albumin (BSA) is covalently conjugated with dodecanoic acid, its ability to stimulate delayed-type hypersensitivity (DTH) is enhanced whereas its ability to stimulate antibody production is suppressed. Conjugates of BSA prepared with negatively charged succinyl groups and positively charged amidine groups were unable to stimulate any detectable DTH to BSA even though their ability to bind anti-BSA antibody in a radioimmunoassay was similar to that of dodecanoyl-BSA which stimulated strong, sustained DTH. By analyzing a series of conjugates of BSA with fatty acids of varying chain length, we found that enhancement of the ability of a preparation to stimulate DTH to BSA could be separated from the suppression of its ability to stimulate antibody formation. The ability of a preparation to stimulate DTH correlated with the covalent conjugation of a large number of hydrophobic groups whereas the suppression of the ability to stimulate antibody formation correlated with covering of antibody-combining sites as measured in a radioimmunoassay.