Repeated dose (14 days) rat intramuscular toxicology study of Her1 vaccine.

Our goal was to assess the toxicity of two strengths (200 and 400 µg) of HER1 cancer vaccine (Center of Molecular Immunology, Cuba), presented in two different formulations, in Sprague Dawley rats after repeated intramuscular administration (14 days). Four groups (5 animals/sex) were established: Control, Placebo (adjuvant), and two Treated groups receiving a dose representing ten times of human total dose (10×), 28.6 and 57.1 µg/kg. Clinical observations, body weight and rectal temperature were measured during the study. Clinical pathology analysis was performed, besides gross necropsy and histological examination of tissues on animals at the end of the assay. The assay ended with a 100% survival. Injection site damage, with the presence of cysts and granulomas, was observed in adjuvant and vaccine treated groups, with most severe cases predominating at higher strength. Administration of Placebo and Her1 vaccine induced increase in polymorphonuclear cells, with relative lymphopenia conditioned by primary neutrophilia. In summary, results suggest that Her1 immunization was capable of inducing an inflammatory effect at the injection site, leading to systemic alterations, more significant at higher strength (400 µg, 57.1 µg/kg), probably affected by the immunizations' schedule used. The vaccine was shown to be well tolerated without any obvious signs of systemic toxicity, with findings largely attributable to the adjuvant used.

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.

Effect of the maternofetal and milk transfer of the anti-epidermal growth factor receptor monoclonal antibody 7A7 in mice.

OBJECTIVES: To study the maternofetal and milk transfer of an anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb) and its effects on conceptus, we administered to pregnant and lactating dams the murine anti-EGFR MAb 7A7 in an autologus model. STUDY DESIGN: For determining the embryo-fetal toxicity, 7A7 at gestational days (GD) 6, 8, 12 and 14 were intravenously administered. Clinical signs and body weights were recorded. On GD 18 pregnant mice were euthanized and the alive and dead fetuses were examined. For measuring the maternofetal transfer mice were dosed on GD 14 with (125)I-7A7, after 24 h, mice were euthanized and main maternal organs and fetuses were counted separately for radioactivity. For studying the MAb transferred throughout the milk, lactating dams were intravenously dosed on lactation day (LD) 2 with (125)I-7A7. Blood samples were obtained from dams at different times post. One lactating pup from each dam was also euthanized at different times, and their blood and gastric milk were removed and the radioactivities measured. RESULTS: The administration of the 7A7 did not elicit toxicity to adult pregnant mice nevertheless; there was evidence of embryo-fetal toxicity in the 7A7 group characterized by a decrease in litters' body weights and head deformities. The maternofetal transfer of (125)I-7A7 antibody on GD 15 was only of a 4%. CONCLUSION: Results suggest that 7A7 crosses placenta and it is transferred in a superior quantity to pups through the milk and that anti-EGFR MAbs have a potential toxic effect to fetuses.

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.

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.

Multiple dose toxicity study of the humanized anti-epidermal growth factor receptor monoclonal antibody h-R3 intravenously administered to Cercopithecus aethiops sabaeus monkeys.

The h-R3 is a humanized growth factor receptor monoclonal antibody (mAb) in development for the treatment of head and neck tumours in which malignant cells overexpress the Epidermal Growth Factor receptor. The present study was designed to evaluate the toxicity of repeated intravenous doses of the h-R3 mAb in a relevant species demonstrated by the avidin-biotin-peroxidase immunohistochemical (IHC) technique in skin biopsy samples from three Cercopithecus aethiops sabaeus monkeys (green monkeys). Additionally, 18 green monkeys were daily intravenously treated during 14 consecutive days. Monkeys were distributed into three experimental groups with three animals of each sex in each group. Group I received saline solution and served as control group; group II received 2.85 mg/kg of h-R3 mAb; and group III received 11.4 mg/kg of the h-R3 mAb. During the study there were no deaths, neither pathological clinical signs, or variations in the corporal weight curve. The electroneurophysiological and sanguine chemistry results did not evidence alterations related to the assay substance. Areas of haematomas, haemorrhages and inflammation, probably related with the administration procedure, were observed at the administration zones of all animals; this fact could also explain the increase in the neutrophil count of all animals at the end of the study. The electrocardiography study showed that in the 14 days of the study one female monkey, from the higher dose group, shifted its cardiac axis from +60 degrees to + 120 degrees; this finding could be interpreted as a right ventricular elongation due to the relative high daily administered volume. It is concluded that doses up to 11.4 mg/kg of h-R3, intravenously administered during 14 consecutive days to Cercopithecus aethiops sabaeus monkeys do not produce considerable toxic effects in the studied system.

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.

Radiotoxicity of h-R3 monoclonal antibody labeled with 188Re administered intracerebrally in rats.

Brain tumors are often incurable despite current aggressive treatment modalities. Regional intracerebral administration of labeled monoclonal antibodies (Mabs) can maximize the radioisotope and Mab concentration to tumor sites while reducing systemic toxicity. h-R3 is a humanized antiepidermal growth factor receptor Mab that successfully targets the epidermal growth factor receptor, which is overexpressed in glioblastomas. We studied the acute local and systemic toxicity effects of intraventricular 188Re-h-R3 in rats. Forty rats were distributed into four groups with five animals of each sex in each group. A single 5 -microl dose (2.5 microl into the left and 2.5 microl into the right lateral ventricles) of neutral solution containing 50 microg of h- R3 labeled with 49.5 +/- 1.7,284 +/- 13.7 or 579 +/- 23.7 muCi of 188Re were stereotactically administered to each animal. Control animals received vehicle alone. Each animal was observed twice daily for detection of toxicity signs. Body weights were recorded on days 0, 7 and 14. Blood samples for analysis of hematological and clinical chemistry parameters were taken on days 0 and 14. Necropsy and histopathological studies were carried out after completion of the study. All animals, but one, remained clinically stable. Toxicities included local radionecrosis, discrete increase in ALAT and creatinine blood values at higher dose level. We concluded that a single intraventricular administration of relatively large doses of 188Re-h-R3 is tolerable and causes minimal local and systemic toxicity effects in rats. Nevertheless, further studies are necessary to discard learning and behavioral problems.

Radiotoxicity of h-R3 monoclonal antibody labeled with 188Re administered intracerebrally in rats

Brain tumors are often incurable despite current aggressive treatment modalities. Regional intracerebral administration of labeled monoclonal antibodies (Mabs) can maximize the radioisotope and Mab concentration to tumor sites while reducing systemic toxicity. h-R3 is a humanized antiepidermal growth factor receptor Mab that successfully targets the epidermal growth factor receptor, which is overexpressed in glioblastomas. We studied the acute local and systemic toxicity effects of intraventricular 188Re-h-R3 in rats. Forty rats were distributed into four groups with five animals of each sex in each group. A single 5- µl into the right lateral ventricles) of neutral solution containing 50 µg of h-R3 labeled with 49.5ñ1.7, 284ñ13.7 ir 579ñ23.7 µCi of 188Re were stereotactically administered to each animal. Control animals received vehicle alone. Each animal was observed twice daily for detection of toxicity signs. Body weights were recorded on days 0,7 and 14. Blood samples for analysis of hematological and clinical chemistry parameters were taken on days 0 and 14. Necropsy and histopathological studies were carried out after completion of the study. All animals, but one, remained clinically stable. Toxicities included local radionecrosis, discrete increase in ALAT and creatinine blood values at higher dose level. We concluded that a single intraventricular administration of relatively large doses of 188Re-h-R3 is tolerable and causes minimal local and systemic toxicity effects in rats. Never-theless, further studies are necessary to discard learning and behavioral problems(AU)

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.

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.

Cerebral dural arteriovenous fistulas: percutaneous transvenous embolization.

PURPOSE: To evaluate angiographic and clinical results in patients with a dural arteriovenous fistula (AVF) who underwent percutaneous transvenous embolization. MATERIALS AND METHODS: Retrospective chart analysis and radiologic studies were performed in 24 patients (aged 20-87 years) with a dural AVF treated with percutaneous transvenous embolization. Lesions were located in the transverse and/or sigmoid or superior sagittal sinus. Clinical follow-up was 3-44 (mean, 10.8) months. RESULTS: After percutaneous transvenous embolization of 24 dural AVFs, there was complete occlusion in 17 patients, important flow reduction in three, and moderate flow reduction in four. Twenty patients were clinically cured, 17 with complete occlusion and three with important flow reduction. In patients with moderate flow reduction, clinical improvement was good in two and moderate in one. One patient remained clinically unchanged. A transient complication was seen in one patient, and a permanent complication was seen in one patient. One patient, whose preexisting clinical status was poor, died. During long-term follow-up, the condition of two patients worsened. CONCLUSION: Percutaneous transvenous embolization appears to be effective in the treatment of dural AVFs. More experience is needed to evaluate long-term results.

Passive cutaneous anaphylaxis in diabetic rats.

The effect of diabetic condition on heterologous passive cutaneous anaphylaxis (PCA) reaction was studied. In alloxan diabetic rats, PCA reaction was inhibited by 68.2% of control values. The skin hyposensitivity in diabetic rats could not be attributed to a lower quantity of skin mast cells; it is suggested that alterations in the endothelial cells of the microcirculatory blood vessels decrease permeability to biologically active mast cell factors.

Is bronchial asthma a pancreatic disease?

Bronchial asthma and diabetes mellitus seldom occur in the same patient. The exact mechanism of this mutual exclusion is still unknown and its elucidation can make clear the physiopathology of both diseases. Clinical and experimental evidences suggest that insulin is a proinflammatory hormone and glucagon an antiinflammatory and a bronchodilator one. We hypothesize that the relationship between plasma insulin and glucagon may play an important role in bronchial asthma.

Hyperresponsiveness to bronchoconstrictor agents in experimental animals treated with terbutaline and its effect on pancreatic beta cells.

Attempts have been made to obtain further experimental evidence in the development of hyperresponsiveness measured as mortality rate in terbutaline-treated animals after the administration of carbachol in rats and ovoalbumin in sensitized guinea pigs. The dose of terbutaline chosen was approximately the dose an asthmatic patient might use in an attack, and its effect on pancreatic insulin synthesis was studied in rats since it has been suggested that insulin is a pro-inflammatory hormone. Our results show that prolonged treatment with terbutaline increases the mortality from bronchoconstrictor stimuli. Increased levels of pancreatic insulin synthesis were also observed by immunocytochemical study carried out on pancreas from terbutaline-treated rats.

Retrospective histopathology of endocrine pancreas in bronchial asthma.

A retrospective histopathological, immunocytochemical and morphometric study was done on samples of pancreas of 16 asthmatic dead humans in comparison with 6 dead control subjects, by other causes not related with allergic or endocrine diseases. A ribbon-like type of islet was the only histological feature found with the exception of one case of chronic pancreatitis. A significative increase of insulin islets (p < 0.05) was observed in the asthmatics patients while no differences in calculated area, perimeter and diameter of pancreatic islets were recorded. A probable hyperplasia of beta-cells could be related to the onset of the asthmatic crisis based on the pro-inflammatory stimulation of insulin or the action of anti-asthmatic treatments in the acute attack.

Diabetes-induced rat hyposensitivity to compound 48/80.

Rat mortality and contractile responses of isolated tracheas to compound 48/80 from rats made diabetic 4 days before by a single intravenous injection of alloxan and from diabetic rats that had been treated with insulin 6 h before were compared with control animals. Diabetic animals and tracheal segments from diabetic rats were significantly less responsive to compound 48/80 than control and insulin-treated diabetic animals. On the other hand, diabetic animals have a lower quantity of peritoneal mast cells than control rats, and insulin restored the normal quantity of cells in diabetic animals. These data indicate that diabetes elicits an hyposensitivity to compound 48/80, possibly related to a diabetes-induced decrease in the mast cell count.

Pharmacological screening of plant decoctions commonly used in Cuban folk medicine.

Pharmacological screening for cardiotonic, hypotensive or bronchodilator activities was performed on 50 plant species depending on their traditional medical use by the Cuban population. Eleven of these species may have promise as therapeutic agents, but additional preclinical pharmacological and toxicological studies are needed to establish their therapeutic effectiveness and safety in human disease.