RESUMO
AIMS: Recent reports in the literature have suggested that glucagon-like peptide-1 (GLP-1)-based therapies may lead to increased risk of pancreatic pathology leading to chronic pancreatic injury and pancreatic neoplasia. Extensive non-clinical and clinical safety testing was conducted to support the global development of exenatide twice daily, exenatide once weekly and saxagliptin. Our aim was to integrate these non-clinical data obtained with both mechanisms of GLP-1-based drugs to provide complementary data regarding the potential for drug-induced pancreatic safety signals. METHODS: More than 70 regulated non-clinical toxicology studies in rodents and non-rodents were conducted in accordance with International Conference on Harmonisation and US Food and Drug Administration guidance documents, current industry standards, animal welfare regulations and in compliance with Good Laboratory Practice regulations. Treatment duration was up to 2 years in rodents and up to 12 months in non-rodents using high doses representing large multiples of human exposures (up to 130× for exenatide and 2200× for saxagliptin). Comprehensive pancreas assessments involved more than 2400 pancreata from animals exposed to exenatide and over 1700 pancreata from animals exposed to saxagliptin. RESULTS: Neither exenatide nor saxagliptin treatment resulted in drug-related microscopic changes indicative of acute or chronic adverse effects (including neoplasia) in the endocrine or exocrine pancreas, at doses far exceeding the maximum human systemic exposures. CONCLUSIONS: These data substantially add to the weight of evidence supporting the lack of non-clinical drug-induced pancreatic safety signals in animals exposed to GLP-1-based therapies.
Assuntos
Adamantano/análogos & derivados , Diabetes Mellitus Experimental/tratamento farmacológico , Dipeptídeos/farmacologia , Peptídeo 1 Semelhante ao Glucagon/agonistas , Pâncreas/patologia , Peptídeos/farmacologia , Peçonhas/farmacologia , Adamantano/farmacologia , Animais , Cães , Exenatida , Haplorrinos , Injeções Subcutâneas , Dose Máxima Tolerável , Camundongos , Tamanho do Órgão , Pâncreas/efeitos dos fármacos , RatosRESUMO
The purpose of the present study was to establish a dose-response relationship for thioacetamide (TA), where tissue regeneration as well as liver injury were two simultaneous but opposing responses. Male Sprague-Dawley rats were injected intraperitioneally with a 12-fold dose range of TA, and both liver injury and tissue repair were measured. Liver injury was assessed by serum enzyme elevations. Serum alanine aminotransferase (ALT) elevation did not show any dose response over a 12-fold dose range up to 24 hr. A dramatic ALT elevation was evident after 24 hr and only for the highest dose (600 mg/kg). Tissue regeneration response was measured by 3H-thymidine (3H-T) incorporation into hepatocellular DNA and by proliferating cell nuclear antigen (PCNA) procedure during a time course (6, 12, 24, 36, 48, 72, and 96 hr). Tissue regeneration, as indicated by 3H-T incorporation, peaked at 36 hr after administration of a low dose of TA (50 mg/kg). With increasing doses, a greater but delayed stimulation of cell division was observed until a threshold was reached (300 mg/kg). Above the tissue repair threshold (600 mg/kg), because stimulated tissue repair as revealed by 3H-T incorporation in hepatonuclear DNA was significantly delayed and attenuated, injury assessed by serum enzyme elevations was remarkably accelerated, indicating unrestrained progression of injury leading to animal death. These findings suggest that, in addition to the magnitude of tissue repair response, the time at which this occurs is critical in restraining the progression of injury, thereby determining the ultimate outcome of toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Alanina Transaminase/sangue , DNA/biossíntese , Regeneração Hepática , Fígado/efeitos dos fármacos , Tioacetamida/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Injeções Intraperitoneais , Fígado/enzimologia , Fígado/patologia , Masculino , Necrose/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Fase S , Tioacetamida/toxicidade , Fatores de TempoRESUMO
In an earlier study we established that timely and adequate tissue repair response following the administration of a six-fold dose-range of thioacetamide (TA; 50, 150, and 300 mg/kg) prevented progression of injury and led to recovery and animal survival. Delayed and attenuated repair response after the 600 mg/kg TA dose resulted in a marked progression of injury and 100% lethality. The objective of the present study was to further scrutinize this concept in an experimental protocol in which we hypothesized that a selective ablation of the tissue repair response should lead to lethality from the nonlethal, moderately toxic doses of 150 and 300 mg/kg TA. In this study we investigated the effect of the antimitotic agent colchicine (CLC, 1 mg/kg) on the outcome of TA hepatotoxicity. Male Sprague-Dawley rats (175-225 g) were injected intraperitoneally (ip) with 150 and 300 mg/kg TA. We assessed liver injury by serum enzyme elevations and histopathology. Tissue regeneration response was measured by 3H-thymidine incorporation into hepatonuclear DNA and by proliferating cell nuclear antigen (PCNA) assay. S-Phase stimulation, as indicated by 3H-thymidine incorporation, was noted at 36 and 48 hr following the administration of 150 mg/kg TA, whereas with the 300 mg/kg TA S-phase stimulation was elicited at 48 hr following treatment. Therefore, two doses of CLC (30 hr and 42 hr, 1 mg/kg, ip) were administered to the 150 mg/kg treated group while a single dose of CLC (42 hr, 1 mg/kg, ip) was administered to the 300 mg/kg group. CLC treatment resulted in 100% lethality in both groups. Thus, CLC administration converted nonlethal doses into lethal doses. The 150 mg/kg TA dose was then chosen to further investigate the underlying mechanism. Rats treated with TA alone recovered from injury by 36-48 hr while CLC treatment resulted in a progression of injury as indicated by serum enzyme elevation and histopathology. Tissue repair, as evidenced by 3H-thymidine incorporation and PCNA studies explained this dichotomy. Antimitotic intervention with CLC resulted in a significantly diminished repair response leading to unrestrained progression of injury and lethality even from nonlethal doses. This model demonstrates the critical role of tissue repair response in determining the final outcome of toxicity.
Assuntos
Antineoplásicos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Colchicina/toxicidade , Regeneração Hepática/efeitos dos fármacos , Tioacetamida/toxicidade , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Trichloroethylene (TCE), a widely used organic solvent and degreasing agent, is regarded as a hepatotoxicant. The objective of the present studies was to investigate whether the extent and timeliness of tissue repair has a determining influence on the ultimate outcome of hepatotoxicity. Male Sprague-Dawley rats (200-250 g) were injected with a 10-fold dose range of TCE and hepatotoxicity and tissue repair were studied during a time course of 0 to 96 h. Light microscopic changes as evaluated by H&E-stained liver sections revealed a dose-dependent necrosis of hepatic cells. Maximum liver cell necrosis was observed at 48 h after the TCE administration. However, liver injury as assessed by plasma sorbitol dehydrogenase (SDH) showed a dose response over a 10-fold dose range only at 6 h, whereas alanine aminotransferase (ALT) did not show a dose response at any of the time points studied. A low dose of TCE (250 mg/kg) showed an increase in SDH at all time points up to 96 h without peak levels, whereas higher doses showed peak only at 6 h. At later time points SDH declined but remained above normal. In vitro addition of trichloroacetic acid, a metabolite of TCE to plasma, decreased the activities of SDH and ALT indicating that metabolites formed during TCE toxicity may interfere with plasma enzyme activities in vivo. This indicates that the lack of dose-related increase in SDH and ALT activities may be because of interference by the TCE metabolite. Tissue regeneration response as measured by [3H]thymidine incorporation into hepatocellular nuclear DNA was stimulated maximally at 24 h after 500 mg/kg TCE administration. A higher dose of TCE led to a delay and diminishment in [3H]thymidine incorporation. At a low dose of TCE (250 mg/kg) [3H]thymidine incorporation peaked at 48 h and this could be attributed to very low or minimal injury caused by this dose. With higher doses tissue repair was delayed and attenuated allowing for unrestrained progression of liver injury. These results support the concept that the toxicity and repair are opposing responses and that a dose-related increase in tissue repair represents a dynamic, quantifiable compensatory mechanism.
Assuntos
Fígado/efeitos dos fármacos , Tricloroetileno/toxicidade , Alanina Transaminase/sangue , Animais , Núcleo Celular/metabolismo , Relação Dose-Resposta a Droga , L-Iditol 2-Desidrogenase/sangue , Fígado/patologia , Fígado/fisiologia , Regeneração Hepática , Masculino , Ratos , Ratos Sprague-Dawley , Timidina/metabolismo , Ácido TricloroacéticoRESUMO
Recent studies indicate that the rate and extent of tissue repair, elicited as an endogenous response to toxic insult, are critical determinants in the ultimate outcome of hepatic injury. Therefore, the objective of this study was to develop a dose-response relationship for CCl4 measuring liver injury and tissue repair as two simultaneous but opposing responses. Male Sprague-Dawley rats were injected with a 40-fold dose range of CCl4 (0.1-4 ml/kg i.p.) in corn oil vehicle. Liver injury was assessed by serum enzyme elevations and histopathology, and tissue repair was measured by [3H]thymidine incorporation into hepatonuclear DNA and proliferating cell nuclear antigen immunohistochemistry over a time course of 0 to 96 h. Stimulation of cell division, evident even after a subtoxic dose of CCl4, increased in a dose-dependent manner until a threshold (2 ml/kg) was reached. Doses above this threshold yielded no further increase in tissue repair. Instead, tissue repair response was significantly delayed and diminished. Injury was markedly accelerated above the threshold indicating an unrestrained progression of injury. Although 4 ml CCl4/kg consistently caused 80% lethality by 48 h, tissue repair response in the 20% surviving rats was increased by about 5-fold, aptly demonstrating the critical role of tissue repair in overcoming injury and enabling these animals to survive. This study suggests that, in addition to the extent of tissue repair, the time of onset of tissue repair also determines the extent of hepatic injury and inter-individual differences in the magnitude of tissue repair may contribute significantly to inter-individual differences in susceptibility to toxic chemicals. Thus, while dose-related and prompt stimulation of tissue regeneration leads to recovery, delayed and attenuated repair response, occurring at higher doses, leads to progression of injury and animal mortality. Such dose-response relationships may lead to a better understanding of the underlying cellular mechanisms of injury inflicted by chemical toxicants and aid in fine-tuning risk assessment.
Assuntos
Tetracloreto de Carbono/toxicidade , Fígado/efeitos dos fármacos , Alanina Transaminase/sangue , Animais , Tetracloreto de Carbono/administração & dosagem , Divisão Celular/efeitos dos fármacos , Colchicina/farmacologia , DNA/metabolismo , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Injeções Intraperitoneais , Marcação por Isótopo , L-Iditol 2-Desidrogenase/sangue , Fígado/enzimologia , Fígado/patologia , Masculino , Intoxicação/mortalidade , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-Dawley , Medição de Risco , Fase S/efeitos dos fármacos , Timidina/metabolismoRESUMO
Smokeless tobacco usage is a growing public health concern in the United States. Lesions of the oral cavity have been clearly linked to smokeless tobacco use. The objective of this study was to determine the biochemical effects of smokeless tobacco extract (STE) exposure upon hamster cheek pouch cell (HCPC-1) cultures. HCPC-1 cells were exposed to a 5 -fold dose-range of STE (0.5, 1.0 and 2.5%) over a time-course of 24-96 h. Following each exposure we measured various biochemical parameters of cell proliferation and cell death. Cell viability, cell cycle progression and S-phase DNA synthesis were measured as markers of cell proliferation. We measured lactate dehydrogenase leakage as a marker of cell membrane damage and cell death due to necrosis. No significant alterations were observed in cell cycle progression and cell proliferation as a result of exposure to STE. LDH measured colorimetrically indicated no significant effect with the lower doses(0.5, 1.0 and 2.5% STE). Apoptosis measured as the A0 peak and by the TUNEL procedure revealed that STE caused significant rates of apoptosis. Maximal apoptosis was noted between 48-96 h. In order to probe the mechanism further we measured the levels of nitrites as an indicator of nitric oxide (NO) in the media. NO levels were significantly elevated at the doses that caused an induction of apoptosis. The results from this study indicate that STE causes a dose-dependent induction of apoptosis and that this is mediated by nitric oxide.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Óxido Nítrico/fisiologia , Plantas Tóxicas , Tabaco sem Fumaça/química , Tabaco sem Fumaça/toxicidade , Animais , Linhagem Celular , Cricetinae , Ciclo-Oxigenase 2 , Dinoprostona/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Isoenzimas , L-Lactato Desidrogenase/metabolismo , Proteínas de Membrana , Óxido Nítrico Sintase/metabolismo , Extratos Vegetais/química , Extratos Vegetais/toxicidade , Prostaglandina-Endoperóxido Sintases/metabolismoRESUMO
In an earlier study it was reported that a single low dose of thioacetamide (TA, 50 mg/kg) administered 36 h prior to challenge with a high dose of 400 mg/kg offers protection from lethality of high dose (Mangipudy et al., Pharmacol. Toxicol. 77, 1995). The mechanism underlying this protection was found to be preplaced hepatocellular division and tissue repair that peaked by 36 h following the low-dose treatment. In a separate study using the dose-response paradigm, it was established that the rate and the extent of the tissue repair response following infliction of injury after acute exposure has a critical bearing on the ultimate outcome of toxicity (Mangipudy et al., Environ. Health Perspect. 103, 1995). The objective of this study was to investigate the cell proliferation dynamics after repeated exposure to TA (50 mg/kg i.p.). Male Sprague-Dawley rats (200-225 g) were administered TA at intervals of 96 h. Liver injury and tissue repair were studied over a time course following each treatment. Tissue repair was estimated by S-phase DNA synthesis measuring 3H-thymidine incorporation into hepatonuclear DNA while liver injury was estimated by serum alanine aminotransferase activity. After the first dose of 50 mg/kg, peak S-phase DNA synthesis was observed at 36 h. This returned to control values by 96 h at which time the rats are known to overcome liver injury. A second dose of TA (repeated dose 1, RD1) resulted in peak S-phase DNA synthesis 12 h later at 48 h. Following the third dose (RD2) a dramatic increase in S-phase DNA synthesis was noted from as early as 12 h. Much higher peak was observed at 72 h. Interestingly, following the fourth dose (RD3) S-phase stimulation did not occur. Instead, a significant latency was observed for cells in the S-phase DNA synthesis even at time points as late as 144 h following the treatment. Liver injury on the other hand exhibited no significant differences between repetitions until RD2. However, injury was sustained in the rats treated with the fourth dose (RD3) while it returned to control levels in the earlier three instances. Sustained prolongation of liver injury after the fourth dose is presumably because tissue repair was not operational. Thus repeated exposure to TA causes a significant increase in tissue repair response although it is temporally delayed until a threshold is reached. Repetition beyond the threshold results in a marked attenuation of the repair response. These findings suggest that toxicodynamics of cell proliferation are altered after repeated exposure.
Assuntos
Carcinógenos/toxicidade , DNA/biossíntese , Fígado/efeitos dos fármacos , Tioacetamida/toxicidade , Alanina Transaminase/sangue , Animais , Carcinógenos/administração & dosagem , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Injeções Intraperitoneais , Fígado/enzimologia , Fígado/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Fase S , Tioacetamida/administração & dosagem , Timidina/metabolismo , Fatores de TempoRESUMO
Low doses of thioacetamide stimulate cell division and tissue repair in the liver. The objective of this study was to develop an autoprotection model for thioacetamide and investigate if a low dose of thioacetamide (50 mg/kg orally) protects against lethality of a subsequently administered lethal dose (400 mg/kg orally) of the same compound. The extent of cell division was investigated to test if autoprotection results from augmented tissue repair and recovery from injury rather than decreased injury itself. After a single administration of the protective dose of thioacetamide, hepatocellular nuclear DNA synthesis as measured by 3H-thymidine incorporation into hepatocellular nuclear DNA peaked at 36 hr indicating maximum level of S-phase stimulation. Pretreatment with the antimitotic colchicine abolished autoprotection and this was associated with a significantly decreased 3H-thymidine incorporation. Preadministration of the protective dose of thioacetamide did not result in an altered infliction of injury from the subsequently administered lethal dose. Colchicine intervention in the autoprotected group resulted in injury that followed a pattern similar to the group that received the high dose alone, ultimately resulting in animal death. These findings suggest that cell division stimulated by the protective low dose of thioacetamide is the critical mechanism in thioacetamide autoprotection.
Assuntos
Carcinógenos/farmacologia , Regeneração Hepática/efeitos dos fármacos , Fígado/citologia , Microssomos Hepáticos/efeitos dos fármacos , Tioacetamida/farmacologia , Administração Oral , Animais , Carcinógenos/administração & dosagem , Carcinógenos/toxicidade , Divisão Celular/efeitos dos fármacos , Colchicina/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , DNA/biossíntese , DNA/genética , Relação Dose-Resposta a Droga , Supressores da Gota/toxicidade , Marcação por Isótopo , Fígado/efeitos dos fármacos , Fígado/lesões , Masculino , Microssomos Hepáticos/enzimologia , Ratos , Ratos Sprague-Dawley , Tioacetamida/administração & dosagem , Tioacetamida/toxicidade , Timidina/metabolismo , TrítioRESUMO
Thioacetamide (TA) is a well-known hepatotoxicant. It has been reported that an obligate intermediate of TA binds to proteins with the formation of acetylimidolysine derivatives that are responsible for TA-induced hepatotoxic effects. TA has also been reported to cause chemically induced cell death via both apoptosis and necrosis. The objective of this study was 2-fold: first, to investigate the effect of TA exposure on protein charge modifications in the rat liver and second, to study the role of these molecular correlates in the regulation of cell death. Male Sprague-Dawley rats (200-225 g, 7-8 weeks old) were divided into four major groups and treated intraperitoneally with a 12-fold dose range of TA (50, 150, 300, and 600 mg TA/kg) dissolved in water. Using whole liver extracts, alterations in the hepatic protein pattern following treatment with the 12-fold dose range of TA were studied using high-resolution, two-dimensional polyacrylamide gel electrophoresis and computerized image analysis. The results indicate that charge modification was clearly evident as early as 2 hr with the lowest dose of 50 mg TA/kg. At this dose and time endoplasmic reticulum proteins, calreticulin, grp78, and ER6O exhibited acidic charge variants. The effect of TA became more prominent with dose and time. Generally the elevation of charge modification indices (CMI) by TA appeared to reach a peak between 4 and 6 hr and then while CMI either leveled off or declined in the lower two doses of 50 and 150 mg TA/kg, it continued to remain elevated with the higher doses of 300 and 600 mg TA/kg. This dichotomy in the elevation of CMI is in close correspondence to the pattern of cell death observed with a similar dose range of TA, where lower doses (50 and 150 mg TA/kg) predominantly cause cell death via apoptosis while higher doses cause cell death via necrosis. Delayed charge modification was observed with the cytosolic hsc70s with the 300 and 600 mg TA/kg treatments, indicating that the reactive metabolite(s) slowly leak out into the cytosol from the endoplasmic reticulum. There were no alterations in the mitochondrial proteins hsp60 and grp75, suggesting that TA has no effect on the mitochondrion, its effects primarily being confined to the endoplasmic reticulum. The concept of looking at these proteins as biomarkers of tissue injury has validity. These changes may be indicators of bioactivation and adduct formation and also may be signaling events in the regulation of the mode of cell death.
Assuntos
Carcinógenos/toxicidade , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/efeitos dos fármacos , Fígado/química , Fígado/efeitos dos fármacos , Tioacetamida/toxicidade , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Eletroforese em Gel Bidimensional , Processamento de Imagem Assistida por Computador , Fígado/patologia , Masculino , Chaperonas Moleculares/química , Prótons , Ratos , Ratos Sprague-DawleyRESUMO
Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that causes massive centrilobular hepatic necrosis at high doses, leading to death. The objectives of this study were to test our working hypothesis that preplaced cell division and hepatic tissue repair by prior thioacetamide (TA) administration provides protection against APAP-induced lethality and to investigate the underlying mechanism. Male Sprague-Dawley rats were treated with a low dose of TA (50 mg/kg, intraperitoneally [i.p.]) before challenge with a 90% lethal dose (1,800 mg/kg, i.p.) of APAP. This protocol resulted in a 100% protection against the lethal effect of APAP. Because TA caused a 23% decrease of hepatic microsomal cytochromes P-450, the possibility that TA protection may be caused by decreased bioactivation of APAP was examined. A 30% decrease in cytochromes P-450 induced by cobalt chloride failed to provide protection against APAP lethality. Time course of serum enzyme elevations (alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase) indicated that actual infliction of liver injury by APAP peaked between 12 to 24 hours after the administration of APAP, whereas the ultimate outcome of that injury depended on the biological events thereafter. Although liver injury progressed in rats receiving only APAP, it regressed in rats pretreated with TA. Acetaminophen t1/2 was not altered in TA-treated rats, indicating that significant changes in APAP disposition and bioactivation are unlikely. Moreover, hepatic glutathione was decreased to a similar extent regardless of TA pretreatment, suggesting that decreased bioactivation of APAP is unlikely to be the mechanism underlying TA protection. [3H]Thymidine incorporation studies confirmed the expected stimulation of S-phase synthesis, and proliferating cell nuclear antigen studies showed a corresponding stimulation of cell division through accelerated cell cycle progression. Intervention with TA-induced cell division by colchicine antimitosis ended the TA protection in the absence of significant changes in the time course of serum enzyme elevations during the inflictive phase of APAP hepatotoxicity. These studies suggest that hepatocyte division and tissue repair induced by TA facilitate sustained hepatic tissue repair after subsequent APAP-induced liver injury, producing recovery from liver injury and protection against APAP lethality.