RESUMO
The first-in-patient (FIP) starting dose for oncology agents should be reasonably safe and provide potential therapeutic benefit to the patient. For late-stage oncology patients, this dose is often based on the ICH S9 guidance, which was developed primarily based on experience with cytotoxic chemotherapeutic agents using the rodent STD10 or non-rodent HNSTD and an appropriate safety factor. With the increase in molecularly targeted chemotherapeutics, it is prudent to re-evaluate how the FIP dose is derived to ensure that the appropriate balance between risk and therapeutic benefit to the patient is achieved. Blinded data on 92 small molecule oncology compounds from 12 pharmaceutical companies who are members of the IQ DruSafe consortium were gathered to investigate if a NOAEL-based starting dose without a safety factor would have been tolerated in the FIP trial and if so, estimating how many dose escalation cohorts could have been reduced. Our analysis suggests that the NOAEL-based alternative starting dose would have been tolerated in most cases evaluated, with an anticipated mean reduction of 2.3 cohorts. Of the 12 cases where the alternative approach resulted in a starting dose that would have exceeded the MTD/RP2D, none of the nonclinical toxicities in these cases were considered irreversible and would be monitorable in all but one instance. Most non-tolerated cases were within two-threefold of the MTD/RP2D, with the clinical AEs considered manageable and mitigated by dose de-escalation. No one method of FIP dose calculation will likely be appropriate for all oncology small molecules and starting dose selection should be performed using a case-by-case approach. However, the NOAEL-based method that does not utilize a safety factor should be considered when appropriate to minimize the number of patients exposed to sub-therapeutic doses of an investigational oncology agent and accelerating development to RP2D.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Nível de Efeito Adverso não Observado , Antineoplásicos/efeitos adversos , Dose Máxima Tolerável , Neoplasias/tratamento farmacológico , Oncologia , Relação Dose-Resposta a DrogaRESUMO
Lysosomes are acidic organelles essential for degradation and cellular homoeostasis and recently lysosomes have been shown as signaling hub to respond to the intra and extracellular changes (e.g. amino acid availability). Compounds including pharmaceutical drugs that are basic and lipophilic will become sequestered inside lysosomes (lysosomotropic). How cells respond to the lysosomal stress associated with lysosomotropism is not well characterized. Our goal is to assess the lysosomal changes and identify the signaling pathways that involve in the lysosomal changes. Eight chemically diverse lysosomotropic drugs from different therapeutic areas were subjected to the evaluation using the human adult retinal pigmented epithelium cell line, ARPE-19. All lysosomotropic drugs tested triggered lysosomal activation demonstrated by increased lysosotracker red (LTR) and lysosensor green staining, increased cathepsin activity, and increased LAMP2 staining. However, tested lysosomotropic drugs also prompted lysosomal dysfunction exemplified by intracellular and extracellular substrate accumulation including phospholipid, SQSTM1/p62, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and opsin. Lysosomal activation observed was likely attributed to lysosomal dysfunction, leading to compensatory responses including nuclear translocation of transcriptional factors TFEB, TFE3 and MITF. The adaptive changes are protective to the cells under lysosomal stress. Mechanistic studies implicate calcium and mTORC1 modulation involvement in the adaptive changes. These results indicate that lysosomotropic compounds could evoke a compensatory lysosomal biogenic response but with the ultimate consequence of lysosomal functional impairment. This work also highlights a pathway of response to lysosomal stress and evidences the role of TFEB, TFE3 and MITF in the stress response.
Assuntos
Adaptação Fisiológica , Lisossomos/efeitos dos fármacos , Linhagem Celular , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/enzimologia , Lisossomos/metabolismo , Lisossomos/fisiologia , Opsinas/metabolismo , Epitélio Pigmentado da Retina/citologia , Proteína Sequestossoma-1/metabolismoRESUMO
PURPOSE: Palbociclib (PD-0332991) is the first selective cyclin-dependent kinase (CDK) 4/6 inhibitor approved for metastatic breast cancer. Hematologic effects, especially neutropenia, are dose-limiting adverse events for palbociclib in humans. EXPERIMENTAL DESIGN: Reversible hematologic effects and bone marrow hypocellularity have been identified in toxicology studies in rats and dogs after palbociclib treatment. To understand the mechanism by which the hematologic toxicity occurs, and to further differentiate it from the myelotoxicity caused by cytotoxic chemotherapeutic agents, anin vitroassay using human bone marrow mononuclear cells (hBMNC) was utilized. RESULTS: This work demonstrated that palbociclib-induced bone marrow suppression occurred through cell-cycle arrest, with no apoptosis at clinically relevant concentrations, was not lineage-specific, and was reversible upon palbociclib withdrawal. In contrast, treatment with chemotherapeutic agents (paclitaxel and doxorubicin) resulted in DNA damage and apoptotic cell death in hBMNCs. In the presence or absence of the antiestrogen, palbociclib-treated hBMNCs did not become senescent and resumed proliferation following palbociclib withdrawal, consistent with pharmacologic quiescence. The breast cancer cells, MCF-7, conversely, became senescent following palbociclib or antiestrogen treatment with additive effects in combination and remained arrested in the presence of antiestrogen. CONCLUSIONS: Palbociclib causes reversible bone marrow suppression, clearly differentiating it from apoptotic cell death caused by cytotoxic chemotherapeutic agents. This study also distinguished the cell-cycle arresting action of palbociclib on normal bone marrow cells from the senescent effects observed in breast cancer cells. These results shed light on the mechanism and support risk management of palbociclib-induced bone marrow toxicity in the clinic.
Assuntos
Antineoplásicos/farmacologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Hematopoese/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Contagem de Células Sanguíneas , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Cães , Sinergismo Farmacológico , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Fulvestranto , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Modelos AnimaisRESUMO
PURPOSE: Taprenepag isopropyl is an EP2 receptor agonist that is in development for the treatment of glaucoma. Iritis, photophobia, and increased corneal thickness observed in a Phase 2 clinical trial with taprenepag isopropyl were not previously observed in topical ocular toxicity studies in rabbits and dogs. In vivo studies using cynomolgus monkeys and in vitro models were used to elucidate the mechanisms underlying these ocular events. METHODS: Monkeys were dosed daily for 28 days in 1 eye with taprenepag and in the other with vehicle control. Complete ophthalmic examinations were performed at baseline and weekly thereafter. Serial sections of eyes were examined histopathologically at the end of the study. Recovery after the discontinuation of taprenepag was assessed for 28 days in the monkeys in the high-dose group. In vitro studies evaluated cell viability, paracellular permeability, and cytokine induction with human corneal epithelial or endothelial cell cultures. RESULTS: Monkeys demonstrated a dose-related incidence of iritis and increased corneal thickness that resolved within 28 days of discontinuing taprenepag. There was no evidence in vivo of taprenepag toxicity to the corneal endothelium or epithelium. Cell viability of stratified epithelial cells was primarily affected by excipients and was similar to Xalatan(®). The viability of HCEC-12 cells was not affected by taprenepag at concentrations up to 100 µM. CONCLUSIONS: The lack of in vivo or in vitro endothelial cytotoxicity and the reversibility of the increase in corneal thickness and iritis in the monkey provide confidence to permit further clinical development of taprenepag.
Assuntos
Acetatos/administração & dosagem , Descoberta de Drogas/tendências , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/patologia , Glaucoma/tratamento farmacológico , Receptores de Prostaglandina E Subtipo EP2/agonistas , Sulfonamidas/administração & dosagem , Administração Oftálmica , Administração Tópica , Animais , Bovinos , Células Cultivadas , Cães , Descoberta de Drogas/métodos , Glaucoma/patologia , Humanos , Macaca fascicularis , Masculino , Resultado do TratamentoRESUMO
PURPOSE: Latanoprost is used for the treatment of an increased intraocular pressure (IOP) to prevent the progression of glaucoma. Since the lack of compliance with topical ocular dosing may compromise efficacy, alternate methods of delivery are being sought. A 9-month study was conducted to assess the safety and tolerability of latanoprost-containing subconjunctivally implanted devices. METHODS: Dutch-belted rabbits were implanted subconjunctivally with up to 5 placebo or drug-loaded devices containing from 50 to 180 µg of latanoprost per device. Study assessment consisted of irritation scoring, clinical signs, ophthalmic exams, IOP, electroretinography (ERG), ocular histology of cohorts at 3 and 9 months postimplantation, and systemic exposure to latanoprost acid. RESULTS: The implants were well tolerated, with minimal-to-mild clinical and microscopic ocular findings attributable to either the placebo or drug-loaded devices. Mild conjunctival congestion persisted through week 13 of the study and tended to correlate with the number of devices and presence of drug. Ophthalmic examinations revealed no effects beyond conjunctival surface hyperemia. No effects on the IOP, corneal thickness, or ERG parameters were observed. The lack of changes in the IOP was expected due to the known lack of the IOP-lowering effects in rabbits from latanoprost. Microscopically, implants at the 3-month necropsy were associated with subconjunctival tissue cavities (containing the implants), fibrous encapsulation, and an infiltrate of lymphocytes and macrophages, sometimes as multinucleate cells, into the subconjunctival implant cavity. The drug-containing implants were often associated with inflammatory cell infiltrates, including heterophils (neutrophils), within the implant subconjunctival cavities and adjacent to the implant sites. At the 9-month necropsy, heterophils were no longer common among the inflammatory cell infiltrates; macrophages and lymphocytes persisted; most of the biodegradable implants were fragmented and disintegrating; and fibrovascular proliferation was present within implant luminal remnants. None of the findings were considered adverse. Systemic exposures were above the limit of quantification (0.1 ng/mL) for up to 96 h in the higher-dose groups, consistent with the initial burst phase of compound release. CONCLUSION: Overall, the study supports the safety of the latanoprost-containing subconjunctival device as a means of extended delivery of the antiglaucoma medication. Latanoprost-containing subconjunctival implants were well tolerated by Dutch-belted rabbits for up to 9 months. Such devices may improve patient compliance and serve as a means of extended delivery of antiglaucoma medications.
Assuntos
Anti-Hipertensivos/administração & dosagem , Anti-Hipertensivos/efeitos adversos , Túnica Conjuntiva , Implantes de Medicamento , Prostaglandinas F Sintéticas/administração & dosagem , Prostaglandinas F Sintéticas/efeitos adversos , Animais , Anti-Hipertensivos/farmacocinética , Anti-Hipertensivos/farmacologia , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Túnica Conjuntiva/cirurgia , Eletrorretinografia , Feminino , Pressão Intraocular/efeitos dos fármacos , Latanoprosta , Masculino , Oftalmoscopia , Prostaglandinas F Sintéticas/farmacocinética , Prostaglandinas F Sintéticas/farmacologia , CoelhosRESUMO
The Bcr-abl tyrosine kinase inhibitor imatinib mesylate is the frontline therapy for chronic myeloid leukemia. Imatinib has been reported to cause congestive heart failure and left ventricular contractile dysfunction in patients and cardiomyopathy in rodents, findings proposed to be associated with its pharmacological activity. To investigate the specific role of Abelson oncogene 1 (c-Abl) in imatinib-induced cardiac toxicity, we performed targeted gene inhibition of c-Abl by RNA interference in neonatal cardiomyocytes (NCMs). Suppression of c-Abl did not lead to cytotoxicity or induction of endoplasmic reticulum (ER) stress. To further dis associate c-Abl from imatinib-induced cardiac toxicity, we designed imatinib structural analogs that do not have appreciable c-Abl inhibition in NCMs. The c-Abl inactive analogs induced cytotoxicity and ER stress, at similar or greater potencies and magnitudes as imatinib. Furthermore, combining c-Abl gene silencing with imatinib and analogs treatment did not significantly shift the cytotoxicity dose response curves. Imatinib and analogs were shown to accumulate in lysosomes, likely due to their physicochemical properties, and disrupt autophagy. The toxicity induced by imatinib and analogs can be rescued by bafilomycin A pretreatment, demonstrating the involvement of lysosomal accumulation in cardiac toxicity. The results from our studies strongly suggest that imatinib induces cardiomyocyte dysfunction through disruption of autophagy and induction of ER stress, independent of c-Abl inhibition.
Assuntos
Antineoplásicos/toxicidade , Coração/efeitos dos fármacos , Piperazinas/toxicidade , Proteínas Quinases/metabolismo , Pirimidinas/toxicidade , Animais , Sequência de Bases , Benzamidas , Primers do DNA , Mesilato de Imatinib , Interferência de RNA , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Lysosomes are acidic organelles that are essential for the degradation of old organelles and engulfed microbes. Furthermore, lysosomes play a key role in cell death. Lipophilic or amphiphilic compounds with a basic moiety can become protonated and trapped within lysosomes, causing lysosomal dysfunction. Therefore, high-throughput screens to detect lysosomotropism, the accumulation of compounds in lysosomes, are desirable. Hence, we developed a 96-well format, high content screening assay that measures lysosomotropism and cytotoxicity by quantitative image analysis. Forty drugs, including antidepressants, antipsychotics, antiarrhythmics and anticancer agents, were tested for their effects on lysosomotropism and cytotoxicity in H9c2 cells. The assay correctly identified drugs known to cause lysosomotropism and revealed novel information showing that the anticancer drugs, gefitinib, lapatinib, and dasatinib, caused lysosomotropism. Although structurally and pharmacologically diverse, drugs that were lysosomotropic shared certain physicochemical properties, possessing a ClogP>2 and a basic pKa between 6.5 and 11. In contrast, drugs which did not lie in this physicochemical property space were not lysosomotropic. The assay is a robust, rapid screen that can be used to identify lysosomotropic, as well as, cytotoxic compounds, and can be positioned within a screening paradigm to understand the role of lysosomotropism as a contributor to drug-induced toxicity.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Lisossomos/efeitos dos fármacos , Xenobióticos/toxicidade , Animais , Linhagem Celular , Ratos , Testes de Toxicidade , Xenobióticos/classificaçãoRESUMO
Preservatives are used in ocular medications to prevent microbial contamination. The use of benzalkonium chloride (BAC), the most widely used preservative in ocular medications, has been scrutinized with a number of studies indicating its toxicity to monolayer cultures of corneal and conjunctival epithelial cells. The purpose of this study was to evaluate and compare the toxicity of BAC and other preservatives and common components of ocular formulations on monolayer and stratified air-lifted cultures of Chang conjunctival cells. Air-lifting Chang cells grown on transwell filters increased stratification as assessed by transepithelial electrical resistance and histology. Unlike monolayer cultures in which ocular medications containing BAC caused near complete loss of cell viability, stratified, air-lifted cultures were not affected by the presence of BAC in ocular medications with up to 30-min exposures. Stratification shifted the dose-response curve to the right for benzalkonium chloride, thimerosal, chlorhexidine digluconate, potassium sorbate and EDTA. These results demonstrate that stratification significantly affects cell viability of Chang conjunctival cells in response to preservatives and additives of ophthalmic preparations.
Assuntos
Compostos de Benzalcônio/toxicidade , Túnica Conjuntiva/efeitos dos fármacos , Conservantes Farmacêuticos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Clorexidina/análogos & derivados , Clorexidina/toxicidade , Túnica Conjuntiva/citologia , Relação Dose-Resposta a Droga , Ácido Edético/toxicidade , Soluções Oftálmicas , Ácido Sórbico/toxicidade , Timerosal/toxicidade , Testes de ToxicidadeRESUMO
BACKGROUND: Benzalkonium chloride (BAC) is a common preservative used in ophthalmic solutions. The aim of this study was to compare the cytotoxic effects of BAC-containing ophthalmic solutions with a BAC-free ophthalmic solution using an organotypic 3-dimensional (3-D) corneal epithelial model and to determine the effects of latanoprost ophthalmic solution and its BAC-containing vehicle on corneal thickness in a monkey model. METHODS: The cytotoxicity of commercially available BAC-containing ophthalmic formulations of latanoprost (0.02% BAC) and olopatadine (0.01% BAC) was compared to that of BAC-free travoprost and saline in a corneal organotypic 3-D model using incubation times of 10 and 25 minutes. To compare the extent of differentiation of 3-D corneal cultures to monolayer transformed human corneal epithelial (HCE-T) cell cultures, expression levels (mRNA and protein) of the corneal markers epidermal growth factor receptor, transglutaminase 1 and involucrin were quantified. Finally, latanoprost ophthalmic solution or its vehicle was administered at suprapharmacologic doses (two 30 microL drops twice daily in 1 eye for 1 year) in monkey eyes, and corneal pachymetry was performed at baseline and at weeks 4, 13, 26 and 52. RESULTS: In the 3-D corneal epithelial culture assays, there were no significant differences in cytotoxicity between the BAC-containing latanoprost and olopatadine ophthalmic solutions and BAC-free travoprost ophthalmic solution at either the 10- or 25-minute time points. The 3-D cultures expressed higher levels of corneal epithelial markers than the HCE-T monolayers, indicating a greater degree of differentiation. There were no significant differences between the corneal thickness of monkey eyes treated with latanoprost ophthalmic solution or its vehicle (both containing 0.02% BAC) and untreated eyes. CONCLUSION: The lack of cytotoxicity demonstrated in 3-D corneal cultures and in monkey studies suggests that the levels of BAC contained in ophthalmic solutions are not likely to cause significant direct toxicity to epithelium of otherwise normal corneas.
Assuntos
Compostos de Benzalcônio/efeitos adversos , Epitélio Corneano/efeitos dos fármacos , Soluções Oftálmicas/efeitos adversos , Conservantes Farmacêuticos/efeitos adversos , Animais , Antialérgicos/efeitos adversos , Anti-Hipertensivos/efeitos adversos , Compostos de Benzalcônio/administração & dosagem , Linhagem Celular , Cloprostenol/efeitos adversos , Cloprostenol/análogos & derivados , Dibenzoxepinas/efeitos adversos , Avaliação de Medicamentos , Epitélio Corneano/patologia , Humanos , Latanoprosta , Macaca fascicularis , Modelos Biológicos , Cloridrato de Olopatadina , Soluções Oftálmicas/administração & dosagem , Conservantes Farmacêuticos/administração & dosagem , Prostaglandinas F Sintéticas/efeitos adversos , Fatores de Tempo , TravoprostRESUMO
Cardiovascular disease has recently been suggested to be a significant complication of cancer treatment with several kinase inhibitors. In some cases, the mechanisms leading to cardiotoxicity are postulated to include mitochondrial dysfunction, either as a primary or secondary effect. Detecting direct effects on mitochondrial function, such as uncoupling of oxidative phosphorylation or inhibition of electron transport chain components, as well as identifying targets within the mitochondrial electron transport chain, can be accomplished in vitro. Here, we examined the effects of the tyrosine kinase inhibitor drugs imatinib, dasatinib, sunitinib, and sorafenib on ATP content in H9c2 cells grown under conditions where cells are either glycolytically or aerobically poised. Furthermore, we measured respiratory capacity of isolated rat heart mitochondria in the presence of the four kinase inhibitors and examined their effect on each of the oxidative phosphorylation complexes. Of the four kinase inhibitors examined, only sorafenib directly impaired mitochondrial function at clinically relevant concentrations, potentially contributing to the cytotoxic effect of the drug. For the other three kinase inhibitors lacking direct mitochondrial effects, altered kinase and other signaling pathways, are a more reasonable explanation for potential toxicity.
Assuntos
Benzenossulfonatos/toxicidade , Indóis/toxicidade , Mitocôndrias Cardíacas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Piperazinas/toxicidade , Inibidores de Proteínas Quinases/toxicidade , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/toxicidade , Pirimidinas/toxicidade , Pirróis/toxicidade , Tiazóis/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Benzamidas , Sobrevivência Celular/efeitos dos fármacos , Dasatinibe , Relação Dose-Resposta a Droga , Transporte de Elétrons/efeitos dos fármacos , Galactose/metabolismo , Glucose/metabolismo , Mesilato de Imatinib , Masculino , Mitocôndrias Cardíacas/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Miócitos Cardíacos/enzimologia , Niacinamida/análogos & derivados , Fosforilação Oxidativa/efeitos dos fármacos , Compostos de Fenilureia , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Sorafenibe , SunitinibeRESUMO
PURPOSE: Cyclin-dependent kinases (CDKs) play a significant role in the control of cell-cycle progression and exhibit aberrant regulation in various neoplastic diseases. AG-024322 is a potent inhibitor of CDK1, CDK2, and CDK4 that produces cell-cycle arrest and antitumor activity in preclinical models. This study evaluated the toxicity of AG-024322 when given by intravenous (IV) infusion to cynomolgus monkeys, including reversibility of effects. METHODS: Male and female monkeys received AG-024322 by 30-min IV infusion once daily for 5 days at doses of 2, 6, and 10 mg/kg (24, 72, and 120 mg/m(2), respectively). Controls received vehicle alone which was aqueous 5% dextrose, pH 3.8. Three animals/sex/group were necropsied on day 6, and two animals/sex/group at 6 and 10 mg/kg were necropsied on day 22 (reversal cohort). Doses were based upon the results of a dose range-finding study in monkeys; decreased white blood cells occurred at > or =3 mg/kg and 12 mg/kg produced central nervous system effects and was above the maximum-tolerated dose. RESULTS: No deaths occurred and clinical signs of toxicity, including swelling at the IV administration site, were seen at > or =6 mg/kg. AG-024322 at > or =6 mg/kg produced pancytic bone marrow hypocellularity, lymphoid depletion, and vascular injury at the injection site. Renal tubular degeneration occurred at 10 mg/kg. These changes were either reversible or in a process of repair following the 17-day recovery period. Hematology changes included decreases in reticulocytes and/or granulocytes at > or =6 mg/kg, which were reversible and consistent with changes in the bone marrow. Lymphoid and bone marrow depletion are consistent with pharmacologic inhibition of CDKs by AG-024322 and were expected findings. On day 22, vacuolar degeneration of pancreatic acinar cells with increased serum amylase and lipase levels occurred in one female at 10 mg/kg. Neither sex-related differences in toxicokinetics nor plasma accumulation over 5 days of dosing were seen. Terminal phase overall mean half-life on day 5 ranged from 6.69 to 8.87 h (across dose levels) and was not dose dependent. CONCLUSION: The no-adverse-effect dose of AG-024322 was 2 mg/kg and associated with overall mean plasma AUC(0-24.5) of 2.11 microg h/mL.
Assuntos
Antineoplásicos/toxicidade , Benzimidazóis/toxicidade , Quinases Ciclina-Dependentes/antagonistas & inibidores , Indazóis/toxicidade , Inibidores de Proteínas Quinases/toxicidade , Animais , Anorexia/induzido quimicamente , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Ataxia/induzido quimicamente , Benzimidazóis/administração & dosagem , Benzimidazóis/farmacocinética , Vasos Sanguíneos/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Relação Dose-Resposta a Droga , Feminino , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/patologia , Doenças Hematológicas/induzido quimicamente , Indazóis/administração & dosagem , Indazóis/farmacocinética , Infusões Intravenosas , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Letargia/induzido quimicamente , Macaca fascicularis , Masculino , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Automutilação , Comportamento Estereotipado/efeitos dos fármacos , Vômito/induzido quimicamenteRESUMO
Cyclin-dependent kinases (CDKs) have been pursued for more than a decade for the treatment of cancer. CDK inhibitors are expected to slow the rate of cell division and potentially increase the apoptotic fraction of rapidly dividing cells. Although CDK activity is often increased in tumors, normal dividing tissues are also susceptible to the cytostatic and cytotoxic effects of CDK inhibitor action. Therefore the typical toxicity profile associated with cytotoxic anti-cancer therapy, bone marrow suppression and gastrointestinal toxicity, is expected with CDK inhibitors. Bone marrow toxicity and the ensuing delayed peripheral leukocyte suppression often limit the therapeutic application of cytotoxic anticancer drugs. Here we characterize an unusual bone marrow-independent acute toxicity toward leukocytes from broad spectrum CDK inhibitors in monkeys and rodents. The potential combination of both acute and delayed immunosuppression would likely further restrict the application of these particular compounds. Since the cells targeted were non-proliferating, it was assumed that the toxicity was not driven by the intended pharmacological mechanism thereby facilitating the development of a testing strategy to identify compounds with a reduced potential for acute leukocyte toxicity. This testing strategy resulted in a CDK inhibitor void of bone marrow-independent leukocyte toxicity that is currently undergoing clinical testing.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/toxicidade , Leucócitos Mononucleares/efeitos dos fármacos , Animais , Benzamidas/farmacocinética , Benzamidas/toxicidade , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacocinética , Feminino , Humanos , Injeções Intravenosas , Leucócitos Mononucleares/enzimologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos ICR , Ratos , Ratos Sprague-Dawley , Tiazóis/farmacocinética , Tiazóis/toxicidadeRESUMO
Cyclin-dependent kinases (cdks) play a crucial role in cell cycle regulation and are considered promising targets for cancer therapy. Intravenous administration of AG-012986, a pan-cyclin-dependent kinase inhibitor (cdk(i)), resulted in unexpected retinal and peripheral nerve toxicity in mice. AG-012986 was administered daily to CD-1 or B6C3F1 mice for 5 consecutive days. Mice were euthanized 24 h after the last dose (study day 6) or after a 21-day post-dose period (study day 26). Compound related microscopic findings were seen in the sciatic nerves (axonal degeneration) of both strains and in the retina (retinal degeneration/atrophy) of CD-1 mice only after the post-dose period. Although retinal degeneration/atrophy was not detected by routine histology in mice euthanized on day 6, apoptotic retinal cells were evident at this time using TUNEL assay. To our knowledge retinal or peripheral nerve toxicity secondary to the administration of cdk(i)s has not been previously reported. Although the pathogenesis of these lesions is unclear, the toxicities may reflect the unique profile of cdk inhibition, off-target kinase inhibition or receptor binding, or metabolism/distribution properties of AG-012986. Multi-targeted-inhibitors may interfere with cdks and other kinases involved in a wide range of functions other than cell cycle regulation, which could result in unexpected toxicities that may hinder their clinical applications.
Assuntos
Benzamidas/toxicidade , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/toxicidade , Sistema Nervoso Periférico/efeitos dos fármacos , Retina/efeitos dos fármacos , Tiazóis/toxicidade , Animais , Apoptose/efeitos dos fármacos , Feminino , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Retina/patologia , Doenças Retinianas/induzido quimicamente , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/patologiaRESUMO
BACKGROUND: Matrix metalloproteinases (MMPs) play key roles in remodeling of the extracellular matrix during embryogenesis and fetal development. The objective of this study was to determine the effects of prinomastat, a potent selective MMP inhibitor, on fetal growth and development. METHODS: Prinomastat (25, 100, 250 mg/kg/day, p.o.) was administered to pregnant female Sprague-Dawley rats on gestational days (GD) 6-17. A Cesarian section was carried out on GD 20 and the fetuses were evaluated for viability and skeletal and soft tissue abnormalities. RESULTS: Prinomastat treatment at the 250 mg/kg/day dose produced a decrease in body weight and food consumption in the dams. A dose-dependent increase in post-implantation loss was observed in the 100 and 250 mg/kg/day-dose groups, resulting in only 22% of the dams having viable litters for evaluation at the 250 mg/kg/day dose. Fetal skeletal tissue variations and malformations were present in all prinomastat treated groups and their frequency increased with dose. Variations and malformation in fetal soft tissue were also increased at the 100 and 250 mg/kg/day doses. Prinomastat also interfered with fetal growth of rat embryo cultures in vitro. CONCLUSIONS: These data confirm that MMP inhibition has a profound effect on fetal growth and development in vivo and in vitro.
Assuntos
Inibidores Enzimáticos/toxicidade , Feto/anormalidades , Inibidores de Metaloproteinases de Matriz , Compostos Orgânicos/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Osso e Ossos/anormalidades , Ingestão de Alimentos/efeitos dos fármacos , Técnicas de Cultura Embrionária , Perda do Embrião/induzido quimicamente , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/efeitos dos fármacos , Inibidores Enzimáticos/farmacocinética , Feminino , Feto/efeitos dos fármacos , Feto/enzimologia , Tamanho do Órgão/efeitos dos fármacos , Compostos Orgânicos/farmacocinética , Gravidez , Ratos , Ratos Sprague-Dawley , Útero/anatomia & histologia , Útero/efeitos dos fármacosRESUMO
BACKGROUND: TGM1(transglutaminase 1) is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment. METHODS: In vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes. RESULTS: In vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA) and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the transcriptional activity. CONCLUSIONS: A distal region of the TGM1 gene promoter, containing AP1 and Sp1 binding sites, is evolutionarily conserved and responsible for high level expression in transgenic mice and in transfected keratinocyte cultures.
Assuntos
Expressão Gênica , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Transglutaminases/genética , Animais , Linhagem Celular , Genes Reporter/genética , Humanos , Queratinócitos/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Transglutaminases/metabolismoRESUMO
The use of in vitro systems to predict in vivo responses to chemical agents provides the benefits of requiring fewer animals, reducing variability between samples, requiring less test material, and enabling higher throughput. In the present study rat tissue slices and primary hepatocytes were compared as in vitro systems to predict in vivo changes in gene expression in response to treatment with known liver toxicants or inducers. Five compounds (phenobarbital, carbon tetrachloride, Wy-14,634, alpha-napthylisothiocyanate, and tacrine) were chosen for their established and diverse mechanisms of hepatoxicity or microsomal induction. Expression profiles from male Sprague-Dawley rats or in vitro systems treated for 24 h were measured by DNA oligonucleotide microarrays containing 8700 probe sets. Qualitative comparison of expression revealed a >80% concordance between in vivo liver and both in vitro systems; however, the responsiveness of both in vitro systems to compound-induced changes in gene expression was far less than that of in vivo. Furthermore, both in vitro systems appeared similar in their ability to reproduce compound-induced changes in gene expression observed in vivo.
Assuntos
Perfilação da Expressão Gênica/métodos , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Xenobióticos/toxicidade , Animais , Células Cultivadas , Hepatócitos/metabolismo , Técnicas In Vitro , Fígado/citologia , Fígado/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Valor Preditivo dos Testes , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Xenobióticos/metabolismoRESUMO
The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Over the course of several days confluent 3T3-L1 cells can be converted to adipocytes in the presence of an adipogenic cocktail. Changes in gene expression were measured by DNA microarrays at three time points (24 h, 4 days, and 1 week) during the course of differentiation from preadipocytes to mature adipocytes. Several functional categories of genes were affected by adipocyte conversion. In addition, seven genes were found to be commonly altered by 5-fold or more by adipocyte conversion at all three time points. Lipocalin 2, haptoglobin, serum amyloid A3, stearoyl-CoA desaturase, and 11beta-hydroxysteroid dehydrogenase 1 were induced while actin alpha2 and procollagen VIII alpha1 were suppressed by adipocyte differentiation. Further study of the regulation of these genes and pathways will lead to an increased understanding of the biochemical pathways involved in adipocyte differentiation and possibly to the identification of new therapeutic targets for treatment of obesity and other metabolic diseases.