Repeated dose intramuscular injection of the CIMAvax-EGF vaccine in Sprague Dawley rats induces local and systemic toxicity.

CIMAvax-EGF consists of a human recombinant epidermal growth factor (EGF), coupled to P64k, a recombinant carrier protein from N. meningitis, and Montanide ISA 51 as adjuvant. The vaccine immunization induces a specific antibody production, inhibiting the EGF/EGF-R interaction through EGF deprivation. The objective of this study was to assess the CIMAvax-EGF toxicity in Sprague Dawley rats after intramuscular administration of repeated doses (6 months) and at the same time to determine if rat is a relevant species for studying CIMAvax-EGF vaccine. Rats were randomly distributed into four groups: control, Montanide ISA 51, treated with 1× and 15× of human total dose of the antigen. Animals were immunized weekly during 9 weeks, plus 9 immunizations every 14 days. Rats were inspected daily for clinical signs. Body weight, food consumption, and rectal temperature were measured during the administration of doses. Blood samples were collected for hematological, serum biochemical determinations and EGF titles at the beginning, three months and at the end of experimentation. Gross necropsy and histological examination of tissues were performed on animals at the end of the assay. Vaccine provoked the apparition of antibodies against EGF in the rats, demonstrating rat species relevance in these studies. Body weight gain, food and water consumption were not affected. CIMAvax-EGF and Montanide ISA 51 produced local damage at the administration site, showing multiple cysts and granulomas. Both vaccine-treated groups showed neutrophil elevation, besides an AST increase probably related to the damage at the administration site. Rectal temperature was found to be significantly higher in 15× treated group after immunizations, probably induced by the inflammatory process at the injection site. In summary, the clinical pathology findings together with the body temperature results, appear to be caused by the inflammatory reaction at the administration site of the vaccine, mainly mediated by the oil-based adjuvant Montanide ISA 51, probably enhanced by the immunological properties of the antigen. This study showed evidences that intramuscular administration during 26 weeks of CIMAvax-EGF at doses up to 15× human total dose is well tolerated in rats and it has a clinical importance since this long lasting study in relevant species allows to treat cancer patients with tumors during long periods with relative weight safety margin.

Comparative analysis of binding affinities to epidermal growth factor receptor of monoclonal antibodies nimotuzumab and cetuximab using different experimental animal models.

Although pharmaco/toxicological studies have always been conducted in pharmacologically relevant species in which the test material is pharmacologically active, the very specificity of many biopharmaceuticals could present challenges in the identification of a relevant species for pharmaco/toxicological studies. Alternative approaches may improve the predictive value of preclinical assessments of species-specific biopharmaceuticals. This could lead to improved decision-making, reduce the number of experimental animals by eliminating non-relevant studies, and decrease the time and cost involved in the drug development process. As an alternative to utilizing traditional animal models, this study investigated the activity of human EGF and the anti-EGF receptor monoclonal antibodies nimotuzumab and cetuximab using the placenta microsomal fraction of different experimental animals. Ligand-receptor binding curves were obtained from the different experimental animal models, and binding constants were calculated based on the Scatchard plots. The constants for human and monkey EGF receptor expressed on the placental extract showed a K(a)<10(-8)M, while rabbits, mice and rats showed a K(a)>10(-8)M. The K(a) values obtained from animal placentas show that Macaca fascicularis and Cercopitecus aethiops monkeys are relevant species for studying the pharmaco/toxicological properties of nimotuzumab and cetuximab.

Effects of a mixture of fatty acids from sugar cane (Saccharum officinarum L.) wax oil in two models of inflammation: zymosan-induced arthritis and mice tail test of psoriasis.

A mixture of fatty acids obtained from sugar cane (Saccharum officinarum L.) wax oil (FAM), in which the main constituents are palmitic, oleic, linoleic, and linolenic acids, was evaluated in two models of inflammation: zymosan-induced arthritis and in the tail test for psoriasis, both on mice. In the first model, FAM significantly reduced zymozan-induced increase of beta glucuronidase (DE(50) 90+/-7 mg/kg). Histopathological studies showed inhibition in cellular infiltration and reduction of synovial hyperplasia and synovitis, whereas in the second test, histopathological and ultrastructural studies showed that topical application of FAM induced orthokeratosis with the presence of keratohyalin granules in the previously parakeratotic adult mouse tail, and without effects on epidermal thickness. The ED(50) of FAM in this model was 155+/-10 mg. The results of our studies showed that topical application of FAM exerts an important anti-inflammatory activity in both tests without evidence of irritant effects. The anti-inflamatory effects exerted by FAM may be due to its inhibitory effects on arachidonic acid metabolism. To our knowledge, this is the first report on the anti-inflammatory effect of sugar cane by-products in experimental models of arthritis and psoriasis.

Further studies on a mixture of fatty acids from sugar cane (Saccharum officinarum) wax oil in animal models of hypersensitivity.

A mixture of fatty acids obtained from sugar cane wax oil, the main components of which are palmitic, oleic, linoleic and linolenic acids, was evaluated topically in two experimental models of hypersensitivity: the ear swelling response to ovalbumin in sensitized mice (ED50 edema: 0.63 +/- 0.06 mg/ear, ED50 myeloperoxidase: 0.56 +/- 0.04 mg/ear, ED50 degranulated cells: 0.70 +/- 0,08 mg/ear) and oxazolone-induced contact hypersensitivity in mice (ED50 edema: 1.63 +/- 0.26 mg/ear, ED50 myeloperoxidase: 1.50 +/- 0.28 mg/ear, ED50 degranulated cells: 1.69 +/- 0.08 mg/ear). Also, the effect of this mixture was studied on the chemotaxis induced by fmlp (ED50: 25 +/- 3 microg/mL). The mixture showed anti-inflammatory activity in both in vivo models of allergy and in the chemotaxis test. Therefore, these results provide evidence about the potential usefulness of the mixture of fatty acids from sugar cane wax oil in cutaneous inflammatory and allergic disorders.

Effect of an EGF-cancer vaccine on wound healing and inflammation models.

BACKGROUND: Epidermal growth factor (EGF) and its receptor (EGF-R) are attractive targets for cancer immunotherapy. Tolerance has been broken with an EGF-vaccine and antibodies against EGF have been produced in animals and in cancer patients. EGF also plays an important role in the inflammation stage of wound healing. Because this therapeutic approach may be of importance after surgery procedures in cancer patients, we decided to investigate the possible role of the EGF-vaccine in the croton-oil-induced ear edema and in the wound healing experimental animal models. MATERIALS AND METHODS: Mice were immunized with an EGF-vaccine by intramuscular injections and serum titers against EGF were measured through ELISA techniques. Control animals received saline. RESULTS: Immunized mice produced antibodies against EGF while no antibody titers could be measured in control animals. Croton oil applied to the inner ear surface of EGF-vaccine treated mice caused a 61.3% lower ear punch weight and a 60.2% lower myeloperoxidase activity than control mice. In the EGF-vaccine treated animals, planimetry measurements and histological analysis did not led to significant impairment in tissue repair. CONCLUSIONS: The EGF-vaccination in mice decreased the normal croton-oil-induced inflammation response, without apparent impairment in tissue healing.

C-phycocyanin: a biliprotein with antioxidant, anti-inflammatory and neuroprotective effects.

Phycocyanin (Pc) is a phycobiliprotein that has been recently reported to exhibit a variety of pharmacological properties. In this regard, antioxidant, anti-inflammatory, neuroprotective and hepatoprotective effects have been experimentally attributed to Pc. When it was evaluated as an antioxidant in vitro, it was able to scavenge alkoxyl, hydroxyl and peroxyl radicals and to react with peroxinitrite (ONOO(-);) and hypochlorous acid (HOCl). Pc also inhibits microsomal lipid peroxidation induced by Fe(+2)-ascorbic acid or the free radical initiator 2,2' azobis (2-amidinopropane) hydrochloride (AAPH). Furthermore, it reduces carbon tetrachloride (CCl(4))-induced lipid peroxidation in vivo. Pc has been evaluated in twelve experimental models of inflammation and exerted anti-inflammatory effects in a dose-dependent fashion in all of these. Thus, Pc reduced edema, histamine (Hi) release, myeloperoxidase (MPO) activity and the levels of prostaglandin (PGE(2)) and leukotriene (LTB(4)) in the inflamed tissues. These anti-inflammatory effects of Pc can be due to its scavenging properties toward oxygen reactive species (ROS) and its inhibitory effects on cyclooxygenase 2 (COX-2) activity and on Hi release from mast cells. Pc also reduced the levels of tumor necrosis factor (TNF-alpha) in the blood serum of mice treated with endotoxin and it showed neuroprotective effects in rat cerebellar granule cell cultures and in kainate-induced brain injury in rats.

Anti-inflammatory and analgesic effects of a mixture of fatty acids isolated and purified from sugar cane wax oil.

The anti-inflammatory and analgesic effects of FAM, a defined mixture of fatty acids isolated from sugar cane (Saccharum officinarum L.), was evaluated. Oral administration of this mixture showed anti-inflammatory activity in the cotton pellet granuloma assay and in the carrageenin-induced pleurisy test, both in rats, as well as in the peritoneal capillary permeability test in mice. In addition, FAM showed analgesic properties in the hot-plate model and in the acetic acid-induced writhings test, both in mice. In conclusion, these results provide evidence on the potential usefulness of the mixture of fatty acids from sugar cane wax oil in inflammatory disorders.

Role of histamine in the inhibitory effects of phycocyanin in experimental models of allergic inflammatory response.

It has recently been reported that phycocyanin, a biliprotein found in the blue-green microalgae Spirulina, exerts anti-inflammatory effects in some animal models of inflammation. Taking into account these findings, we decided to elucidate whether phycocyanin might exert also inhibitory effects in the induced allergic inflammatory response and on histamine release from isolated rat mast cells. In in vivo experiments, phycocyanin (100, 200 and 300mg/kg post-orally (p.o.)) was administered 1 h before the challenge with 1 microg of ovalbumin (OA) in the ear of mice previously sensitized with OA. One hour later, myeloperoxidase activity and ear edema were assessed. Phycocyanin significantly reduced both parameters. In separate experiments, phycocyanin (100 and 200 mg/kg p.o.) also reduced the blue spot area induced by intradermal injections of histamine, and the histamine releaser compound 48/80 in rat skin. In concordance with the former results, phycocyanin also significantly reduced histamine release induced by compound 48/80 from isolated peritoneal rat mast cells. The inhibitory effects of phycocyanin were dose dependent. Taken together, our results suggest that inhibition of allergic inflammatory response by phycocyanin is mediated, at least in part, by inhibition of histamine release from mast cells.

Inhibition of rat lipoprotein lipid peroxidation by the oral administration of D003, a mixture of very long-chain saturated fatty acids.

Previous results have demonstrated that policosanol, a mixture of aliphatic primary alcohols isolated and purified from sugar cane wax, whose main component is octacosanol, inhibited lipid peroxidation in experimental models and human beings. D003 is a defined mixture of very long-chain saturated fatty acids, also isolated and purified from sugar cane wax, whose main component is octacosanoic acid followed by traicontanoic, dotriacontanoic, and tetracontanoic acids. Since very long-chain fatty acids are structurally related to their corresponding alcohols, we investigated the effect of oral treatment with D003 (0.5, 5, 50, and 100 mg/kg) over 4 weeks in reducing the susceptibility of rat lipoprotein to oxidative modification. The combined rat lipoprotein fraction VLDL + LDL was subjected to several oxidation systems, including those containing metal ions (CuSO4), those having the capacity to generate free radicals 2,2-azobis-2-amidinopropane hydrochloride (AAPH), and a more physiological system (resident macrophages). D003 (5, 50, and 100 mg/kg) significantly inhibited copper-mediated conjugated-diene generation in a concentration-dependent manner. D003 increased lag phase by 53.1, 115.3, and 119.3%, respectively, and decreased the rate of conjugate-diene generation by 16.6, 21.5, and 19.6%, respectively. D003 also inhibited azo-compound initiated and macrophage-mediated lipid peroxidation as judged by the significant decrease in thiobarbituric acid reactive substance (TBARS) generation. In all the systems the maximum effect was attained at 50 mg/kg. There was also a parallel attenuation in the reduction of lysine amino groups and a significant reduction of carbonyl content after oxidation of lipoprotein samples. Taken together, the present results indicate that oral administration of D003 protects lipoprotein fractions against lipid peroxidation in the lipid as well in the protein moiety.

Effects of phycocyanin extract on prostaglandin E2 levels in mouse ear inflammation test.

Recently it was demonstrated that phycocyanin, a biliprotein isolated from microalgae Spirulina, exerts anti-inflammatory activity in several animal models of inflammation. In this report, the effects of phycocyamin on prostaglandin E2 (PGE2) concentrations and phospholipase A2 (PLA2) activity were determined in arachidonic acid (AA) and 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced mouse ear oedema, respectively. Phycocyanin (50-200 mg/kg p.o.) inhibited in a dose-dependent manner PGE2 levels in mouse ear treated with AA. Also, phycocyanin (100-400 mg/kg p.o.) moderately reduced PLA2 activity in TPA-induced mouse ear inflammation test. In this model triamcinolone (10 mg/kg p.o.) used as reference drug exerted a remarkable inhibitory effect on PLA2 activity. These results provide the first evidence that the anti-inflammatory effects of phycocyanin may result, at least partially, from inhibition of PGE2 production and a moderate inhibition of PLA2 activity.

Topical anti-inflammatory activity of human recombinant epidermal growth factor.

The topical anti-inflammatory activity of epidermal growth factor (EGF) was evaluated in inflammation models induced by 12-Otetradecanoylphorbol-13-acetate, croton oil and arachidonic acid. When EGF (1.5, 3 or 6 microg/ear) was coapplied with each inflammatory agent, there was a dose-related decrease in inflammation as assessed by ear punch weights, myeloperoxidase activity as well as by histopathological studies. The precise anti-inflammatory action of EGF is yet unclear, but we believe that interference with arachidonic acid metabolism may play an important role.

Further studies on anti-inflammatory activity of phycocyanin in some animal models of inflammation.

OBJECTIVE: To examine the effects of C-phycocyanin, a pigment found in blue-green algae which acts as an antioxidant in vitro and in vivo, in different animal models of inflammation. MATERIAL: Male Sprague Dawley rats and OF1 mice were used. TREATMENTS: Oedema was induced by: a) AA (0.5 mg/ear) or TPA (4 microg/ear) in the mouse ear b) carrageenan injection (0.1 mL of 1% suspension) in the rat paw (+/-adrenalectomy) and c) cotton pellet implantation in the rat axilla. Phycocyanin (50-300mg/kg, p.o.) or indomethacin (1 mg/ear or 3-10mg/kg, p.o.) as control were tested in the four animal models. METHODS: Measurement of the increase in the weight (mg) of 6 mm ear punch biopsies from treated ears were made in comparison to control ears, together with myeloperoxidase (MPO) activity as an index of neutrophil infiltration. The increase in the paw thickness (mm) was measured with a dial caliper. Cotton pellet was implanted and seven days afterwards the granuloma was removed and the dry weight was determined. Acute toxicity was studied in mice and rats. Statistics were performed using one-way analysis of variance with the Duncan Multirange test. RESULTS: Phycocyanin reduced significantly (p < 0.05) and in a dose-dependent manner ear oedema induced by AA and TPA in mice as well as carrageenan-induced rat paw oedema (both in intact and adrenalectomized animals). In the TPA test, phycocyanin also reduced MPO content. Phycocyanin also exerted an inhibitory effect in the cotton pellet granuloma test. In the acute toxicity test in rats and mice, even at the highest dose tested (3000 mg/kg, p.o.), no toxicity was found. CONCLUSIONS: Phycocyanin shows anti-inflammatory activity in four experimental models of inflammation. Its antioxidative and oxygen free radical scavenging properties may contribute, at least in part, to its anti-inflammatory activity.

Antioxidant and anti-inflammatory properties of C-phycocyanin from blue-green algae.

OBJECTIVE: Phycocyanin is a pigment found in blue-green algae which contains open chain tetrapyrroles with possible scavenging properties. We have studied its antioxidant properties. MATERIALS AND METHODS: Phycocyanin was evaluated as a putative antioxidant in vitro by using: a) luminol-enhanced chemiluminescence (LCL) generated by three different radical species (O2-, OH., RO.) and by zymosan activated human polymorphonuclear leukocytes (PMNLs), b) deoxyribose assay and c) inhibition of liver microsomal lipid peroxidation induced by Fe+2-ascorbic acid. The antioxidant activity was also assayed in vivo in glucose oxidase (GO)-induced inflammation in mouse paw. RESULTS: The results indicated that phycocyanin is able to scavenge OH. (IC50 = 0.91 mg/mL) and RO. (IC50 = 76 microg/mL) radicals, with activity equivalent to 0.125 mg/mL of dimethyl sulphoxide (DMSO) and 0.038 microg/mL of trolox, specific scavengers of those radicals respectively. In the deoxyribose assay the second-order rate constant was 3.56 x 10(11) M(-1) S(-1), similar to that obtained for some non-steroidal anti-inflammatory drugs. Phycocyanin also inhibits liver microsomal lipid peroxidation (IC50 = 12 mg/mL), the CL response of PMNLs (p < 0.05) as well as the edema index in GO-induced inflammation in mouse paw (p < 0.05). CONCLUSIONS: To our knowledge this is the first report of the antioxidant and anti-inflammatory properties of c-phycocyanin.

Antipsoriatic, anti-inflammatory, and analgesic effects of an extract of red propolis.

AIM: To study the antipsoriatic, anti-inflammatory, and analgesic effects of ethanolic extract of red propolis. METHODS AND RESULTS: This extract induced the formation of granular layer in the mouse tail test used as a model of psoriasis. Propolis 50 mg.kg-1 i.g. showed anti-inflammatory activity in the cotton-pellet granuloma assay in rats, in croton oil-induced edema in mice at a dose of 25% (2.5 microL), and in the peritoneal capillary permeability test in mice at a dose of 10 mg.kg-1. The extract (25 mg.kg-1 i.g.) showed analgesic effect in the model of acetic acid-induced writhings, whereas 40 mg.kg-1 was effective in the hot plate test in mice. CONCLUSION: Anti-inflammatory, analgesic, and antipsoriatric properties of Cuban red propolis were evident.