Antiviral activity in vitro of two preparations of the herbal medicinal product Sinupret® against viruses causing respiratory infections.

Sinupret(®), a herbal medicinal product made from Gentian root, Primula flower, Elder flower, Sorrel herb, and Verbena herb is frequently used in the treatment of acute and chronic rhinosinusitis and respiratory viral infections such as common cold. To date little is known about its potential antiviral activity. Therefore experiments have been performed to measure the antiviral activity of Sinupret(®) oral drops (hereinafter referred to as "oral drops") and Sinupret(®) dry extract (hereinafter referred to as "dry extract"), in vitro against a broad panel of both enveloped and non-enveloped human pathogenic RNA and DNA viruses known to cause infections of the upper respiratory tract: influenza A, Chile 1/83 (H1N1) virus (FluA), Porcine Influenza A/California/07/2009 (H1N1) virus (pFluA), parainfluenza type 3 virus (Para 3), respiratory syncytial virus, strain Long (RSV), human rhinovirus B subtype 14 (HRV 14), coxsackievirus subtype A9 (CA9), and adenovirus C subtype 5 (Adeno 5). Concentration-dependent antiviral activity (EC(50) between 13.8 and 124.8 µg/ml) of Sinupret(®) was observed against RNA as well as DNA viruses independent of a viral envelope. Remarkable antiviral activity was shown against Adeno 5, HRV 14 and RSV in which dry extract was significantly superior to oral drops. This could be ascertained with different assays as plaque-reduction assays in plaque forming units (PFU), the analyses of a cytopathogenic effect (CPE) and with enzyme immunoassays (ELISA) to determine the amount of newly synthesised virus. Our results demonstrate that Sinupret(®) shows a broad spectrum of antiviral activity in vitro against viruses commonly known to cause respiratory infections.

A recombinant anti-ICAM-1 Fab fragment is as effective as the complete IgG antibody in treatment of burns in rabbits.

The purpose of this study was to examine the efficiency of an Anti-ICAM-1-Fab fragment in the treatment of burns. Blocking ICAM-1 using murine IgG with the purpose to prevent further damage to the zone of stasis has proven its effectiveness in animal models as well as in humans. The use of murine Antibodies has some disadvantages including allergic reactions and complement activation. For the first time we examined an industry produced Fab-fragment blocking ICAM-1 and compared it to the corresponding IgG antibody. We showed in a standardised rabbit burn model that an Fab-fragment is capable of blocking ICAM-1 and therefore improving the reaction of the skin to trauma in the zone of stasis.

Efficacy of the fully human monoclonal antibody MOR102 (#5) against intercellular adhesion molecule 1 in the psoriasis-severe combined immunodeficient mouse model.

BACKGROUND: Psoriasis is considered as a chronic immune-mediated disease characterized by inflammation and proliferation of the epidermis. OBJECTIVES: Targeting intercellular adhesion molecule 1 (ICAM-1) is an attractive therapeutic option as this molecule is critically involved in leucocyte adhesion and extravasation as well as in lymphocyte activation. METHODS: We have selected the fully human monoclonal antibody MOR102 (#5) against ICAM-1 from the Human Combinatorial Antibody Library (HuCAL). This antibody, as human IgG4 [corrected] was tested for its ability to interfere with lymphocyte activation and adhesion in vitro as well as for its antipsoriatic efficacy in vivo using the psoriasis-severe combined immunodeficient (SCID) mouse model. RESULTS: The antibody demonstrated efficient inhibition of lymphocyte adhesion to ICAM-1 in vitro, with an IC(50) of approximately 0.4 microg mL(-1) (3 nmol L(-1)). In addition, MOR102 (#5) reduced lymphocyte proliferation in mixed lymphocyte cultures by approximately 50%. The in vivo efficacy of MOR102 (#5) was tested on grafts derived from lesional skin of patients with chronic plaque-stage psoriasis transplanted on to SCID mice. Intraperitoneal injection of 10 mg kg(-1) of MOR102 (#5) antibody every alternate day over a period of 4 weeks resulted in reconstitution of orthokeratotic differentiation and a significant (P < 0.05) reduction in epidermal thickness as well as marked reduction in the inflammatory infiltrate. Therapeutic activity may be related to the targeting of ICAM-1 on keratinocytes and thus preventing efficient activation of local T cells. CONCLUSIONS: Based on the efficacy of the fully human monoclonal antibody MOR102 (#5) shown in vitro as well as in vivo in the psoriasis-SCID mouse model, initiation of clinical studies is indicated.

Studies comparing in vivo:in vitro metabolism of three pharmaceutical compounds in rat, dog, monkey, and human using cryopreserved hepatocytes, microsomes, and collagen gel immobilized hepatocyte cultures.

The in vivo metabolism of three pharmaceutical compounds, EMD68843, EMD96785, and EMD128130, was compared in fresh and cryopreserved hepatocyte (CPH) suspensions and microsomes from rat, dog, monkey, and human livers and fresh human and rat hepatocyte collagen gel immobilized cultures (GICs). Half of the major in vivo metabolites was produced by phase 1 (hydroxylation, oxidation, hydrolysis, N-dealkylation) and half by phase 2 metabolism (mostly glucuronidation but also sulfation and glycine conjugation). The identity and percentage of phase 1 and 2 metabolites from each compound produced in hepatocytes compared well with that in each species in vivo. Glucuronidation was more extensive in GICs than in CPHs. In contrast, CPHs but not GICs, produced sulfate metabolites. Microsomes (supplemented with NADPH only) produced most of the phase 1 but no phase 2 metabolites. Metabolism in CPHs was the same as in fresh hepatocyte suspensions. Discrete species differences in metabolism were detected by CPHs and microsomes. Cytochrome P450 and glucuronosyl S-transferase contents of CPHs did not account for species differences in the percentage of phase 1 and 2 metabolites or the rate of disappearance of the parent compounds in these cells. These data show a good correlation between major metabolites formed in vivo and in vitro. CPHs and GICs, unlike microsomes, carried out sequential phase 1 and 2 metabolism. Each in vitro system has its own advantages, however, for short-term metabolism studies CPHs may be more useful since they are readily available, easier and quicker to prepare than GICs, and have more comprehensive enzyme systems than microsomes.

In vivo evaluation of epidermal growth factor (EGF) receptor density on human tumor xenografts using radiolabeled EGF and anti-(EGF receptor) mAb 425.

In order to study the potential of non-invasive scintigraphic evaluation of the epidermal growth factor (EGF) receptor status in vivo, the biokinetics and tumor binding of 125I-EGF and anti-(EGF receptor) mAb 425 were investigated in nude mice bearing human tumor xenografts with different EGF-receptor densities as determined by a radioreceptor assay. The results demonstrated a tumor uptake for both substances depending on the receptor level. The EGF receptor status, however, was reflected slightly better by the binding of EGF to tumor tissue compared to the mAb. The rapid blood clearance of EGF with a plasma half-life of less than 1 min led to a tumor-to-blood ratio of approximately 3 within 6 h after injection in tumors with a high receptor expression. A similar ratio for the mAb was not obtained before day 6 after injection. The absolute concentration of EGF, however, was low compared to the mAb. Therefore, it can be concluded that the EGF receptor status as a target for (radio)immunotherapy can be evaluated in vivo with EGF labeled with a short-life positron-emitting radionuclide or with monoclonal antibodies to the EGF receptor or their fragments.

Pharmacokinetics of reshaped MAb 425 in three animal species.

The murine monoclonal antibody (MAb) 425 (mMAb 425) directed against the human EGFR (epidermal growth factor receptor) was reshaped (rMAb 425) in order to improve its therapeutical potential in humans. The pharmacokinetic properties of [125I]-mMAb and [125I]-rMAb 425 were compared in three animal species. Whereas the clearance curves of both antibodies decreased biphasically in rats and nude mice bearing human mammary carcinoma, a monophasic decline was observed in Cynomolgus monkeys. Plasma elimination half-lives of murine and reshaped MAb 425 were similar, short in the monkey (26 h for mMAb 425 and 31 h for rMAb 425) and long in rats (240 h for mMAb 425 and 225 h for rMAb 425). In xenografted nude mice however, the half-life of mMAb 425 (203 h) was about twice as long as that of rMAb 425 (124 h). The half-lives of intact rMAb 425 in the three species obtained by ELISA differed at most by a factor of two from those obtained by radioactivity measurements. Biodistribution studies of [125I]-rMAb 425 revealed a tumor/blood ratio of 1.2 on d 1 and 5.1 on d 18, respectively. Fifty-four and thirty-eight percent of the radioactive dose were excreted with urine in nude mice (within 12 d) and rats (within 11 d), respectively. Specific localization of [125I]-rMAb 425 in human mammary carcinoma xenografted to nude mice was demonstrated.

Distribution of humanized MAb 425 (EMD 62,000) in rats and specific localization in tumor-bearing nude mice.

The murine MAb 425 (IgG2a) directed against human epidermal growth factor receptor is considered to have therapeutic potential in glioma patients. In order to circumvent immune response in clinical use, the MAb 425 was humanized by CDR-grafting (IgG1). We have studied the distribution of reshaped MAb 425 (EMD 62,000) in Wistar rats and the specific localization in female nude mice bearing human mamma carcinoma xenografts. The 125I-labelled MAb 425 was administered intravenously in a single dose (1 mg/kg) using unspecific human IgG1 antibody as control. The biodistribution was investigated both quantitatively and by whole-body autoradiography. The autoradiographs showed a selective uptake of radioactivity by the tumour tissue. 15 days after administration, radioactivity was bound exclusively to the tumour. Similar results were obtained with the murine monoclonal antibody. Quantitative studies exhibited a tumour-blood ratio of about 5. The study demonstrates that the humanized MAb 425 is selectively localized in human mamma carcinoma xenografted to athymic mice.

Mitogenicity of anti-Thy-1 monoclonal antibodies attributable to an Fc-dependent mechanism.

We analyzed the mechanism by which certain anti-Thy-1 monoclonal antibodies (mAb) activate T cells directly without additional stimuli. Using a panel of rat anti-Thy-1 antibodies which included more than 30 IgG2c mAb, we found that only the IgG2c isotype was able to induce a strong proliferative response in both resting T cells and a T cell lymphoma, suggesting that this form of T cell activation is isotype restricted and might be a consequence of a unique physico-chemical property of the IgG2c heavy chain. Results from surface distribution studies of Thy-1 molecules, following specific interactions with anti-Thy-1 antibodies of different isotypes, again showed that only IgG2c mAb formed Thy-1 aggregates of high valence on the surface of a T cell lymphoma, and such clustering always evoked a biological response. This led us to propose that IgG2c mAb have the inherent tendency to self-associate, probably through homophilic Fc-Fc contacts, and that this feature renders anti-Thy-1 mAb mitogenic. To prove this, we set up cross-inhibition studies with randomly selected mitogenic (IgG2c) and nonmitogenic (IgG2b) anti-Thy-1 mAb. The results clearly demonstrated that IgG2c antibodies enhance their own binding, analogous to the new form of antibody binding that was recently demonstrated between murine IgG3 mAb and a multivalent antigen. Confirmation of this was also provided by IgG2c-derived F(ab')2 fragments, which were unable to cause proliferation. Furthermore, masking the Fc part of cell-bound IgG2c mAb with a monomeric and thus non-aggregating IgG-binding protein A-derived fragment cancelled their mitogenic ability. Finally, induction of T cell proliferation appeared to be independent of cross-linking via Fc gamma R. The results support a model in which noncovalent intermolecular homophilic contacts of the Fc regions of the IgG2c isotype bring about effective aggregation of Thy-1 molecules, thereby stimulating the mitotic apparatus of the cell.

Protease histochemistry in normal and uremic rats.

In rats, ARF caused protease changes which could be detected histochemically. The organs which clearly responded were the extraorbital gland, thymus and lung (ARF-sensitive organs); the others did not show a clear-cut response (ARF-insensitive organs). In the affected organs activities of plasma membrane-associated and lysosomal proteases were either increased or decreased. The response of the proteases in the extraorbital gland is considered to be specific whereas this in the thymus and lung is not. However, ARF has not a protease-specific effect which can be shown when further groups of enzymes are investigated using histochemical means.

Enzyme cytochemistry of rat organs after uremia with special reference to proteases.

Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed an increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and beta-N-acetyl-D-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and gamma-glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.