Preparation of polyclonal antibodies for nateglinide (NTG) and development of a sensitive chemiluminescent immunoassay to detect NTG in tablets and serum.

In this study, we prepared polyclonal antibodies against anti-diabetic drug nateglinide (NTG), and established a sensitive chemiluminescent immunoassay (CLIA) to detect NTG in tablets and serum. Two kinds of immunogens were synthesized using ethylcarbodiimide (EDC)/hydroxysuccinimide (NHS) and carbonyldiimidazole (CDI)/4-dimethylaminopyridine (DMAP) as coupling reagents respectively. When activated by EDC/NHS, more molecules of NTG coupled with carrier protein in immunogens. A horseradish peroxidase (HRP)-luminol-H2O2 system with p-iodophenol enhancement was applied in the CLIA analysis. The antibodies in EDC/NHS group showed higher titer, sensitivity and wider detection linear range than those in CDI/DMAP group, and were chosen for next studies. The developed CLIA assay exhibited good selectivity towards NTG among structually similar analogs. The method could detect as low as 0.35 ng mL(-1) NTG in buffer, 2.1 ng mL(-1) NTG in serum and 0.84 ng mL(-1) NTG in tablets. The CLIA method provided consistent results with HPLC method (r=0.9986) in determination of NTG from 5.0 to 400 µg mL(-1). The CLIA method could detect 78 samples in one assay, and the samples need only dilution in pretreatment. As a summary, this research offers a sensitive assay for high-throughout screening of NTG in formulation control and pharmacokinetic studies.

The efficacy and safety of maraviroc addition to a stable antiretroviral regimen in subjects with suppressed plasma HIV-RNA is not influenced by age.

There are few data about the immunovirological efficacy, safety/tolerability, and durability of maraviroc (MVC) addition to aging patients on suppressive antiretroviral therapy (cART) and undetectable viral load (<50 copies/ml). The aging population is underrepresented in most HIV clinical trials. This study included 80 patients aged ≥50 years and 161 aged <50 years and showed that after 48 weeks of treatment, there was no between-group differences in the median increase of CD4(+) T cells or the virological suppression rate. Safety and tolerability were also comparable. In multivariable analysis, the effect of age was not modified and was independent of the response to MVC. An immunological recovery of ≥100 CD4(+) T cells was significantly less common in those with a longer HIV history (≥15 years) (OR 0.43; p=0.016) or having <200/mm(3) CD4(+) T cells at MVC initiation (OR 0.27; p=0.004). Meanwhile, achieving a CD4/CD8 ratio ≥0.5 at week 48 was less likely in those with CD4(+) T cell counts <200 at MVC initiation (OR 0.09; p<0.0001) or with a previous AIDS event (OR 0.43; p=0.028). In summary, the immunovirological efficacy, safety/tolerability, and durability of MVC addition in patients virologically suppressed were independent of the patient's age at treatment onset.

Immunologic effectiveness of maraviroc- and raltegravir-containing regimens (R+M+) versus raltegravir-based regimens that do not include maraviroc (R+M-).

OBJECTIVE: To compare the immunologic effectiveness of raltegravir-maraviroc (R+M+)-based regimens with raltegravir-based regimens that do not include maraviroc (R+M-) in treatment-experienced patients in clinical practice. METHODS: We conducted a retrospective study of treatment-experienced HIV-infected adults receiving either R+M+- or R+M--based therapy. Longitudinal CD4 counts were analyzed using a linear mixed model. RESULTS: One hundred and fifty-six patients were included in the analysis, of whom 32 were receiving R+M+ and 124 R+M-. Mean baseline CD4 counts in patients on R+M+ and R+M- were 463.8 and 442.3 cells/mm(3), respectively (P = .67). In multivariable mixed models, a baseline viral load ≥50 copies/mL was significantly associated with CD4 change during follow-up (P < .0001). No difference between R+M+ and R+M- was observed during follow-up (P = .81). CONCLUSION: CD4 cell recovery was similar among patients receiving either R+M+- or R+M--based therapy during a 24-month period of follow-up.

The atypical cannabinoid O-1602 protects against experimental colitis and inhibits neutrophil recruitment.

BACKGROUND: Cannabinoids are known to reduce intestinal inflammation. Atypical cannabinoids produce pharmacological effects via unidentified targets. We were interested in whether the atypical cannabinoid O-1602, reportedly an agonist of the putative cannabinoid receptor GPR55, reduces disease severity of dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid (TNBS)-induced colitis in C57BL/6N and CD1 mice. METHODS: DSS (2.5% and 4%) was supplied in drinking water for 1 week while TNBS (4 mg) was applied as a single intrarectal bolus. RESULTS: Both treatments caused severe colitis. Injection of O-1602 (5 mg/kg intraperitoneally) significantly reduced macroscopic and histological colitis scores, and myeloperoxidase activity. The protective effect was still present in cannabinoid receptor 1 (CB1) and 2 (CB2) double knockout mice and mice lacking the GPR55 gene. To investigate a potential mechanism underlying the protection by O-1602 we performed neutrophil chemotactic assays. O-1602 concentration-dependently inhibited migration of murine neutrophils to keratinocyte-derived chemokine (KC), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the N-formyl-peptide receptor ligand WKYMVm. The inhibitory effect of O-1602 was preserved in neutrophils from CB1/CB2 double knockout and GPR55 knockout mice. No differences were seen in locomotor activity between O-1602-treated and control mice, indicating lack of central sedation by this compound. CONCLUSIONS: Our data demonstrate that O-1602 is protective against experimentally induced colitis and inhibits neutrophil recruitment independently of CB1, CB2, and GPR55 receptors. Thus, atypical cannabinoids represent a novel class of therapeutics that may be useful for the treatment of inflammatory bowel diseases.

Synergistic inhibition of R5 HIV-1 by maraviroc and CCR5 antibody HGS004 in primary cells: implications for treatment and prevention.

CCR5 blockers inhibit CCR5-tropic (R5) HIV-1, including strains resistant to other antiretrovirals. We demonstrate that the CCR5 antibody HGS004 and the CCR5 antagonist maraviroc have potent antiviral synergy against R5 HIV-1, translating into dose reductions of more than 10-fold for maraviroc and more than 150-fold for HGS004. These data, together with the high barrier of resistance to HGS004, suggest that combinations of maraviroc and HGS004 could provide effective preventive and therapeutic strategies against R5 HIV-1.

The Aspergillus fumigatus toxin fumagillin suppresses the immune response of Galleria mellonella larvae by inhibiting the action of haemocytes.

Larvae of Galleria mellonella are widely used to evaluate microbial virulence and to assess the in vivo efficacy of antimicrobial agents. The aim of this work was to examine the ability of an Aspergillus fumigatus toxin, fumagillin, to suppress the immune response of larvae. Administration of fumagillin to larvae increased their susceptibility to subsequent infection with A. fumigatus conidia (P = 0.0052). It was demonstrated that a dose of 2 µg fumagillin ml⁻¹ reduced the ability of insect immune cells (haemocytes) to kill opsonized cells of Candida albicans (P = 0.039) and to phagocytose A. fumigatus conidia (P = 0.016). Fumagillin reduced the oxygen uptake of haemocytes and decreased the translocation of a p47 protein which is homologous to p47(phox), a protein essential for the formation of a functional NADPH oxidase complex required for superoxide production. In addition, toxin-treated haemocytes showed reduced levels of degranulation as measured by the release of a protein showing reactivity to an anti-myeloperoxidase antibody (P<0.049) that was subsequently identified by liquid chromatography-MS analysis as prophenoloxidase. This work demonstrates that fumagillin suppresses the immune response of G. mellonella larvae by inhibiting the action of haemocytes and thus renders the larvae susceptible to infection. During growth of the fungus in the larvae, this toxin, along with others, may facilitate growth by suppressing the cellular immune response.

In vivo effects of a new immunosuppressive sigma ligand, SR 31747, on mouse thymus.

SR 31747 is a new sigma ligand which has immunosuppressive properties. The immunopharmacology of SR 31747 was investigated in vivo by studying its effects on the thymuses of C3H mice. The action of SR 31747 was compared with the reference drugs cyclosporin-A and dexamethasone on the basis of several parameters which were: the thymus weight; the number of thymocytes per organ; the percentages of mature CD4+ or CD8+ thymocytes and of immature CD4+ CD8+ thymocytes. SR 31747 slightly but significantly decreased the thymus weight at the dose of 50 mg/kg whereas the number of thymocytes per organ was significantly decreased from 6.25 mg/kg to the 50 mg/kg dose. It had rather no effect on the percentages of immature and mature subsets. These data led to the conclusion that the effects of SR 31747 on the thymuses of C3H mice were close to those obtained with cyclosporin-A and different from those obtained with dexamethasone.

Immunopharmacological profile of SR 31747: in vitro and in vivo studies on humoral and cellular responses.

In our preceding paper, we demonstrated that both human and rat lymphocytes possess saturable high-affinity binding sites for the new sigma ligand SR 31747. Here we investigate the potential activity of this ligand on immune responses. In vitro, our study shows that SR 31747 exerts a concentration- and time-dependent inhibition of proliferative response to mitogens on mouse and human lymphocytes without affecting cell viability. This suppressive effect elicited by SR 31747 occurs over a concentration range which correlates with the pharmacological profile of the molecule in binding assays, strongly suggesting that SR 31747 acts through a receptor-mediated process. We showed that the SR 31747 effect, which was observed on purified T lymphocytes, affects a late event in the activation process which occurs after the G1 during the S phase of the cell cycle. Interestingly, no anti-proliferative effect was observed in a variety of tumor cell lines, supporting a specific effect limited to normal immune cells. In vivo, in mice, treatment with SR 31747 prevented both graft-versus-host disease and delayed-type hypersensitivity granuloma formation, while antibody response to sheep red blood cells was not affected. These results strongly suggest that the sigma-related receptor recognized by SR 31747 is very likely coupled to a biological function of lymphocytes.

Antibodies against (1R,2R)-cyclohexanediamineplatinum(II)-DNA adduct recognize the conformational differences of isomeric analogues of cyclohexanediamine.

Antibodies reactive to (1R,2R)-cyclohexanediamineplatinum(II)-DNA ((1R,2R)-cyclohexanediamine: 1R,2R-dach) adducts were elicited by immunization of rabbit with calf thymus DNA modified by Pt(1R,2R-dach)Cl2 at a ratio of bound platinum per nucleotide ((D/N)b) of 0.0335. In an enzyme-linked immunosorbent assay (ELISA), the binding of specific antibodies to Pt(1R,2R-dach)-DNA adduct (60 microliters of 1.235 x 10(-7) M Pt in each wells) on the assay plate was competitively inhibited by Pt(1R,2R-dach)-DNA adduct ((D/N)b = 0.0653) in the solution. Almost equal inhibition was observed with Pt(1S,2S-dach)-DNA ((D/N)b = 0.0412), an optical isomer of 1R,2R-dach. Pt(1R,2S-dach)-DNA ((D/N)b = 0.0371) and Pt(1R,3S-dach)-DNA ((D/N)b = 0.0281) in which the cyclohexane ring is stereochemically perpendicular to the platinum chelate plane, also inhibited antibody binding, but these adducts gave only incomplete inhibition at higher Pt-DNA adduct concentrations. Although Pt(1R,2R-dach)-d(GpG) and Pt(1R,2R-dach)(NH3)2 inhibited antibody binding, the affinity of the antibody for Pt(1R,2R-dach)(NH3)2 was lower than with Pt(1R,2R-dach)-DNA, and the inhibition behavior of Pt(1R,2R-dach)-d(GpG) was biphasic, i.e., at the lower concentration the inhibition curve was consistent with that of Pt(1R,2R-dach)-DNA, but at the higher concentration it shifted to that of Pt(1R,2R-dach)(NH3)2. The affinity of the antibody for cis-DDP was markedly lower than with Pt(1R,2R-dach)(NH3)2. These facts suggest that the antibodies may bind to the substituents (the platinum and its surroundings) of the various Pt complexes rather than the DNA structure altered by platinum binding.

Skin sensitization with the new reagents NOPYE (4-nitro-1-cyclohexyl-3-ethoxy-2-oxo-3-pyrroline) and NOPY-L-phenylalanine.

The aim of this study was to investigate the compound 4-nitro-1-cyclohexyl-3-ethoxy-2-oxo-3-pyrroline (NOPYE) and some related compounds for skin sensitization in guinea pigs, as the first step in a search for more effective skin sensitizers for immunotherapy of cutaneous tumors. In guinea pigs, NOPYE and NOPYE-L-alanine produce far milder delayed hypersensitivity reactions than DNCB. Both NOPYE and DNCB fail to act as adjuvants for skin sensitization to tuberculin purified protein derivative (PPD) and ovalbumin (OV). This suggests an explanation for the lack of effectiveness of DNCB in immunotherapy of metastases: DNCB may be relatively ineffective as an adjuvant for production of specific antitumor immunity. Such adjuvant activity may be essential if the action of the immunotherapeutic reagent is not to be confined to its site of application but is to be effective at the site of distant metastases.