- Email: [email protected]
Non-clinical development of anticancer drugs: Safety considerations
Abstracts / Toxicology Letters 180S (2008) S32–S246
evaluated for the above mentioned biochemical parameters. Results: The statistical analysis (ANOVA) of the results showed significant changes for GGT (p < 0.001), LDL-cholesterol (p = 0.0013), uric acid (p = 0.001), during the study. The LDL-cholesterol level showed a decreasing tendency, while the uric acid level had an increasing tendency during the study. An increase for the percentage of the patients with CT levels below 200 mg/dL was observed after 6 weeks of therapy. The activity of GGT increased, and the level of the total proteins was relatively stationary, except for a significant reduction after 10 days of therapy. The statistical analysis of the correlation between biochemical parameters evaluated in dynamics, the comorbidities and methadone doses revealed the following significant positive correlations: comorbidities – GGT (p = 0.036); comorbidities – uric acid (p = 0.033); total cholesterol – methadone doses. Conclusions: Monitoring the biochemical parameters at addict patients during the methadone substitution therapy could be useful for the evaluation of the cardio-vascular, hepatic and renal risk factors. doi:10.1016/j.toxlet.2008.06.328 P03 Is an antioxidative capacity of HI-6 oxime a key factor to improve the therapy with irinotecan? Suzana Berend 1,∗ , Ana Lucic Vrdoljak 1 , Bozica Radic 1 , Jasenka Mladinic 1 , Davor Zeljezic 1 , Nevenka Piljac-Zegarac 2 , Marin Kopjar 1 1 Institute for medical research and occupational health, Zagreb, Croatia, 2 Ruder Boskovic Institute, Zagreb, Croatia
New and improved therapy modalities currently employed in the management of malignant diseases significantly contribute to the overall survival rate in cancer patients. Therefore, some compounds although not primarily designed as supportive drugs in chemotherapy are promising candidates for possible clinical use. Our in vitro and in vivo experiments have demonstrated that HI-6 could “protect” cholinesterases against the irinotecan, one of the frequently used antineoplastic drugs, and in that way a part of cholinergic symptoms might be reduced. Interestingly, certain antioxidants may counteract the effects of chemotherapy-induced oxidative stress on the cell cycle and enhance the cytotoxicity of antineoplastic agents. So with the purpose to investigate the levels of oxidative stress and DNA damage (lipid peroxidation, the alkaline comet assay and micronucleus assay) related to treatments with irinotecan and HI-6 given alone or in combination we also focused on the assessment of possible antioxidative capacity of HI-6. In view of the DPPH assay results, HI-6 shows moderate radical scavenging at the tested concentration. Administration of HI-6 alone or with irinotecan diminished the levels of TBARS in rat plasma. Confirmatory results regarding HI-6 efficacy were also obtained by the alkaline comet and micronucleus assay. When given alone, it mostly did not induce significant disturbances in the level of primary DNA damage in somatic cells of treated rats and did not significantly induce the formation of micronuclei. Results obtained in the present study seem to be a proper argument to consider HI-6 as beneficial substance during chemotherapy. doi:10.1016/j.toxlet.2008.06.329
S151
P04 High content secondary screening assay to model NRTI-induced mitochondrial toxicity Clotilde Biscarrat ∗ , Nathalie Compagnone ICDD-SAS Predictive Toxicology Unit, Meyreuil, France Since the introduction of highly active antiretroviral therapy (HAART), HIV infections are manageable clinical entities. Despite this positive impact, the clinical use of HAARTs has led to the recognition of drug-induced mitochondrial toxicity principally attributed to nucleoside reverse transcriptase inhibitors (NRTIs). As the AIDS epidemic continues and as survival with HIV infection is prolonged by the treatment with HAART, long-term side effects of NRTIs may become increasingly common. These include lactic acidosis, hepatic steatosis, neuropathy, myopathy, pancreatitis, and lipodystrophy. Furthermore, lactic acidosis and encephalopathy have been reported in children exposed in-utero and/or postnatally to NRTIs. Because there is not a single mechanism by which the NRTI-induced mitochondrial toxicity evolves, there is currently no relevant model that can assess the mitochondrial toxicity in early preclinical development. We have thus developed an in vitro assay based on the analysis of mitochondrial behavior in live human cells that can model NRTI-induced mitochondrial toxicity. Using mitochondrial behavior allows us to measure in a single outcome the ensemble of mechanisms that participate in the regulation of mitochondrial function. Our assay thus provides a high content secondary screening model to select the safest leads within the most active. In this paper, we present the effects of NRTIs including that of AZT, the most widely studied NRTI and reference molecule in its class, and NNRTIs on mitochondrial behavior. We also show the degree of concordance between the in vitro toxicity indexes deduced from the analysis of the in vitro mitochondrial behavior with the LD50 found in animal studies. doi:10.1016/j.toxlet.2008.06.330 P05 Non-clinical development of anticancer drugs: Safety considerations Paolo Colombo ∗ , Marina Cattoni, Guido Di Gallo, Romana Pulci, Marco Brughera Accelera - Nerviano Medical Sciences Srl, Nerviano, Milano, Italy Considering the high unmet medical need in the field of oncology, it becomes more and more important to ensure a harmonized approach to anticancer drugs in order to make available new therapies to patients with shorter development programs and welldefined study typologies to avoid unnecessary use of resources and laboratory animals. Because of lack of a specific guideline as only regional ones or recommendations are followed in different geographical areas there is no universally accepted approach in the development of oncology drugs. This may result in some freedom to operate, however, many times this lack of harmonization may create problems in designing programs to be accepted worldwide. Furthermore it should also be considered that the design of a pre-clinical program is also depending on the class (e.g. small molecule vs. biologic) and characteristics (e.g. cytotoxic vs. a non-cytotoxic) drugs under investigation. On this basis the international scientific community through the ICH has initiated the process of preparing a guideline (ICHS9) that aims to address all the main points concerning the non-clinical development of anticancer drugs. This guideline should become effective from 2010.
S152
Abstracts / Toxicology Letters 180S (2008) S32–S246
Working for more than 20 years in the pre-clinical development of oncology drugs, Accelera has acquired a wide expertise in this field having successfully developed several compounds. Some specific approaches used to pre-clinically evaluate anticancers are exemplified on the basis of general toxicology, safety pharmacology and other in vitro/in vivo tests. New perspectives and scenarios based on current trends and on the forthcoming ICH guideline are mentioned.
the drug directly to the site of action (i.e. into the joint). In these cases, intra-articular injection into the knee joint is frequently the preferred dose route for investigatory studies. Doses were performed on up to four occasions over a period of 16 days, the route of administration is a possible human therapeutic route. This poster will describe the procedure used to successfully dose rabbits by the intra-articular route. doi:10.1016/j.toxlet.2008.06.333
doi:10.1016/j.toxlet.2008.06.331 P06 Drug NP031112 development: In vitro and in vivo studies predict no phototoxicity
P08 Assessment of colony number and morphology for hematopoietic progenitors by flow cytometry and in vitro culture in macaque species
Nicolas Fabre 1,∗ , Isolde Anglade 1 , Genevieve Arcelin 2 , Conxita de Castellarnau 3 , Petra Giannini 2 , Joan Albert Vericat 1
Hugues Contamin, Nadine Eltchinger, Franc¸oise Phoukham Phothirath, Fabienne Condevaux
Neuropharma SA, Tres Cantos, Spain, 2 RCC Ltd., Fuellinsdorf, Switzerland, 3 Advancell, S.L., Barcelona, Spain
MDS Drug Safety Assessment, L’Arbresle, France
1
Horand ∗ ,
NP031112 is a promising new drug, today under clinical development, for the treatment of neurodegenerative diseases. Since elderly people usually have a thinner skin, it is especially important to evaluate the risk for phototoxicity of the drug candidate, and mainly when the candidate drug possesses an absorption peak at 286 nm. The in vitro 3T3 Neutral red uptake phototoxicity test (OECD guideline no. 432) has been performed. Chlorpromazine was selected as positive control. In this assay, the photoirritation factor is expressed as the ratio between IC50 with and without UV-A exposition. NP031112 treatment resulted in a ratio (1.747) close to the positive limit (2). Then, according to these results, the phototoxicity risk could not be ruled out. It was then decided to conduct a GLP in vivo confirmation study. NP-12 was orally administered to Ico:OFA-Hr/hr hairless rats using a formulation resembling that used in clinical trials. The positive control used, 8-MOP, has been described as giving clear response in the skin of animals. Rats were exposed to UV-A light after a 7-day period of daily treatment with NP031112 and scoring for erythema was performed. The results confirmed that treatment with NP031112 did not induce such an effect and response to the treatment with 8-MOP was considered sufficient to confirm the functionality of the model. In conclusion, NP031112 did not show phototoxic potential in the conditions of the in vitro and in vivo studies performed. This information will be taken in consideration at time of designing further clinical trial protocols in elderly patients.
Repeated evaluation of bone marrow is valuable for the investigation of the effects of drugs on haematopoiesis. Our laboratory previously demonstrated that repeated sampling of bone marrow in cynomolgus and rhesus monkeys caused no major side-effects. The aim of the present study was to address the suitability of a combined battery of assays to detect clinically relevant haematological abnormalities in bone marrow lineages. Standardized clonogenic assays for myeloid, erythroid and megakaryocitic progenitors were assessed for the detection of effects on blood cell precursors. Gross effects (e.g. a reduction in colony numbers) and subtle effects (e.g. altered colony morphology) should be detected. 5 cynomolgus and 5 rhesus monkeys were sampled. The samples were cultured in vitro for 14 days (5% CO2 , >95% humidity, 37 ◦ C). Results were compared following sampling from the humerus, femur and iliac crest. Cell concentrations of 2.5 × 104 to 2.5 × 106 cells/mL were employed in various culture media supplemented with cytokines and growth/differentiation factors. A methylcellulose-based culture (MethoCult, Stem Cell Technologies) was selected for clonogenic progenitors of the erythroid and myeloid lineages. A collagen-based culture (Megacult, Stem Cell Technologies) was selected for the progenitors of the megakaryocytic lineage. Bone marrow differentials (total lymphocytes, erythroid and myeloid cells) were monitored by flow cytometry using anti-CD34, -CD71, -CD45, -CD41a, and CD61 antibodies. Bone marrow progenitor cells were characterized and counted. The proposed array of endpoints also facilitates the assessment of potential stimulatory or inhibitory effects on the bone marrow.
doi:10.1016/j.toxlet.2008.06.332
doi:10.1016/j.toxlet.2008.06.334
P07 Intra-articular dosing in the rabbit
P09 Trainable QSAR model of Ames genotoxicity
Dawn Haida
Remigijus Didziapetris, Kiril Lanevskij, Pranas Japertas ∗
Covance, Harrogate, United Kingdom
Pharma Algorithms, Inc., Vilnius, Lithuania
Intra-articular dosing is a skilled technique on which there appears to be limited literature. Historically, the main area for research using this dose route has been in the treatment of Rheumatoid arthritis. More recently, many of the prospective treatments for this fall into the general “biological” categories (typically proteins and anti-bodies). The modes of action of these tend to be very specific but they may be poorly absorbed and/or distributed, particularly following oral or intravenous administration and hence it may be necessary to administer
This study presents a predictive model of Ames genotoxicity built on a data set of more than 8000 compounds. A baseline fragmental model was developed using binomial PLS regression with multiple bootstrapping. This was followed by an automatic correction of predicted values based on similarity analysis of experimental data for similar compounds in the dataset. The final model produces highly accurate results (less than 5% of mispredictions in the validation set). This novel approach also allows assessing the quality of predictions by means of estimated Reliability Index values. A
evaluated for the above mentioned biochemical parameters. Results: The statistical analysis (ANOVA) of the results showed significant changes for GGT (p < 0.001), LDL-cholesterol (p = 0.0013), uric acid (p = 0.001), during the study. The LDL-cholesterol level showed a decreasing tendency, while the uric acid level had an increasing tendency during the study. An increase for the percentage of the patients with CT levels below 200 mg/dL was observed after 6 weeks of therapy. The activity of GGT increased, and the level of the total proteins was relatively stationary, except for a significant reduction after 10 days of therapy. The statistical analysis of the correlation between biochemical parameters evaluated in dynamics, the comorbidities and methadone doses revealed the following significant positive correlations: comorbidities – GGT (p = 0.036); comorbidities – uric acid (p = 0.033); total cholesterol – methadone doses. Conclusions: Monitoring the biochemical parameters at addict patients during the methadone substitution therapy could be useful for the evaluation of the cardio-vascular, hepatic and renal risk factors. doi:10.1016/j.toxlet.2008.06.328 P03 Is an antioxidative capacity of HI-6 oxime a key factor to improve the therapy with irinotecan? Suzana Berend 1,∗ , Ana Lucic Vrdoljak 1 , Bozica Radic 1 , Jasenka Mladinic 1 , Davor Zeljezic 1 , Nevenka Piljac-Zegarac 2 , Marin Kopjar 1 1 Institute for medical research and occupational health, Zagreb, Croatia, 2 Ruder Boskovic Institute, Zagreb, Croatia
New and improved therapy modalities currently employed in the management of malignant diseases significantly contribute to the overall survival rate in cancer patients. Therefore, some compounds although not primarily designed as supportive drugs in chemotherapy are promising candidates for possible clinical use. Our in vitro and in vivo experiments have demonstrated that HI-6 could “protect” cholinesterases against the irinotecan, one of the frequently used antineoplastic drugs, and in that way a part of cholinergic symptoms might be reduced. Interestingly, certain antioxidants may counteract the effects of chemotherapy-induced oxidative stress on the cell cycle and enhance the cytotoxicity of antineoplastic agents. So with the purpose to investigate the levels of oxidative stress and DNA damage (lipid peroxidation, the alkaline comet assay and micronucleus assay) related to treatments with irinotecan and HI-6 given alone or in combination we also focused on the assessment of possible antioxidative capacity of HI-6. In view of the DPPH assay results, HI-6 shows moderate radical scavenging at the tested concentration. Administration of HI-6 alone or with irinotecan diminished the levels of TBARS in rat plasma. Confirmatory results regarding HI-6 efficacy were also obtained by the alkaline comet and micronucleus assay. When given alone, it mostly did not induce significant disturbances in the level of primary DNA damage in somatic cells of treated rats and did not significantly induce the formation of micronuclei. Results obtained in the present study seem to be a proper argument to consider HI-6 as beneficial substance during chemotherapy. doi:10.1016/j.toxlet.2008.06.329
S151
P04 High content secondary screening assay to model NRTI-induced mitochondrial toxicity Clotilde Biscarrat ∗ , Nathalie Compagnone ICDD-SAS Predictive Toxicology Unit, Meyreuil, France Since the introduction of highly active antiretroviral therapy (HAART), HIV infections are manageable clinical entities. Despite this positive impact, the clinical use of HAARTs has led to the recognition of drug-induced mitochondrial toxicity principally attributed to nucleoside reverse transcriptase inhibitors (NRTIs). As the AIDS epidemic continues and as survival with HIV infection is prolonged by the treatment with HAART, long-term side effects of NRTIs may become increasingly common. These include lactic acidosis, hepatic steatosis, neuropathy, myopathy, pancreatitis, and lipodystrophy. Furthermore, lactic acidosis and encephalopathy have been reported in children exposed in-utero and/or postnatally to NRTIs. Because there is not a single mechanism by which the NRTI-induced mitochondrial toxicity evolves, there is currently no relevant model that can assess the mitochondrial toxicity in early preclinical development. We have thus developed an in vitro assay based on the analysis of mitochondrial behavior in live human cells that can model NRTI-induced mitochondrial toxicity. Using mitochondrial behavior allows us to measure in a single outcome the ensemble of mechanisms that participate in the regulation of mitochondrial function. Our assay thus provides a high content secondary screening model to select the safest leads within the most active. In this paper, we present the effects of NRTIs including that of AZT, the most widely studied NRTI and reference molecule in its class, and NNRTIs on mitochondrial behavior. We also show the degree of concordance between the in vitro toxicity indexes deduced from the analysis of the in vitro mitochondrial behavior with the LD50 found in animal studies. doi:10.1016/j.toxlet.2008.06.330 P05 Non-clinical development of anticancer drugs: Safety considerations Paolo Colombo ∗ , Marina Cattoni, Guido Di Gallo, Romana Pulci, Marco Brughera Accelera - Nerviano Medical Sciences Srl, Nerviano, Milano, Italy Considering the high unmet medical need in the field of oncology, it becomes more and more important to ensure a harmonized approach to anticancer drugs in order to make available new therapies to patients with shorter development programs and welldefined study typologies to avoid unnecessary use of resources and laboratory animals. Because of lack of a specific guideline as only regional ones or recommendations are followed in different geographical areas there is no universally accepted approach in the development of oncology drugs. This may result in some freedom to operate, however, many times this lack of harmonization may create problems in designing programs to be accepted worldwide. Furthermore it should also be considered that the design of a pre-clinical program is also depending on the class (e.g. small molecule vs. biologic) and characteristics (e.g. cytotoxic vs. a non-cytotoxic) drugs under investigation. On this basis the international scientific community through the ICH has initiated the process of preparing a guideline (ICHS9) that aims to address all the main points concerning the non-clinical development of anticancer drugs. This guideline should become effective from 2010.
S152
Abstracts / Toxicology Letters 180S (2008) S32–S246
Working for more than 20 years in the pre-clinical development of oncology drugs, Accelera has acquired a wide expertise in this field having successfully developed several compounds. Some specific approaches used to pre-clinically evaluate anticancers are exemplified on the basis of general toxicology, safety pharmacology and other in vitro/in vivo tests. New perspectives and scenarios based on current trends and on the forthcoming ICH guideline are mentioned.
the drug directly to the site of action (i.e. into the joint). In these cases, intra-articular injection into the knee joint is frequently the preferred dose route for investigatory studies. Doses were performed on up to four occasions over a period of 16 days, the route of administration is a possible human therapeutic route. This poster will describe the procedure used to successfully dose rabbits by the intra-articular route. doi:10.1016/j.toxlet.2008.06.333
doi:10.1016/j.toxlet.2008.06.331 P06 Drug NP031112 development: In vitro and in vivo studies predict no phototoxicity
P08 Assessment of colony number and morphology for hematopoietic progenitors by flow cytometry and in vitro culture in macaque species
Nicolas Fabre 1,∗ , Isolde Anglade 1 , Genevieve Arcelin 2 , Conxita de Castellarnau 3 , Petra Giannini 2 , Joan Albert Vericat 1
Hugues Contamin, Nadine Eltchinger, Franc¸oise Phoukham Phothirath, Fabienne Condevaux
Neuropharma SA, Tres Cantos, Spain, 2 RCC Ltd., Fuellinsdorf, Switzerland, 3 Advancell, S.L., Barcelona, Spain
MDS Drug Safety Assessment, L’Arbresle, France
1
Horand ∗ ,
NP031112 is a promising new drug, today under clinical development, for the treatment of neurodegenerative diseases. Since elderly people usually have a thinner skin, it is especially important to evaluate the risk for phototoxicity of the drug candidate, and mainly when the candidate drug possesses an absorption peak at 286 nm. The in vitro 3T3 Neutral red uptake phototoxicity test (OECD guideline no. 432) has been performed. Chlorpromazine was selected as positive control. In this assay, the photoirritation factor is expressed as the ratio between IC50 with and without UV-A exposition. NP031112 treatment resulted in a ratio (1.747) close to the positive limit (2). Then, according to these results, the phototoxicity risk could not be ruled out. It was then decided to conduct a GLP in vivo confirmation study. NP-12 was orally administered to Ico:OFA-Hr/hr hairless rats using a formulation resembling that used in clinical trials. The positive control used, 8-MOP, has been described as giving clear response in the skin of animals. Rats were exposed to UV-A light after a 7-day period of daily treatment with NP031112 and scoring for erythema was performed. The results confirmed that treatment with NP031112 did not induce such an effect and response to the treatment with 8-MOP was considered sufficient to confirm the functionality of the model. In conclusion, NP031112 did not show phototoxic potential in the conditions of the in vitro and in vivo studies performed. This information will be taken in consideration at time of designing further clinical trial protocols in elderly patients.
Repeated evaluation of bone marrow is valuable for the investigation of the effects of drugs on haematopoiesis. Our laboratory previously demonstrated that repeated sampling of bone marrow in cynomolgus and rhesus monkeys caused no major side-effects. The aim of the present study was to address the suitability of a combined battery of assays to detect clinically relevant haematological abnormalities in bone marrow lineages. Standardized clonogenic assays for myeloid, erythroid and megakaryocitic progenitors were assessed for the detection of effects on blood cell precursors. Gross effects (e.g. a reduction in colony numbers) and subtle effects (e.g. altered colony morphology) should be detected. 5 cynomolgus and 5 rhesus monkeys were sampled. The samples were cultured in vitro for 14 days (5% CO2 , >95% humidity, 37 ◦ C). Results were compared following sampling from the humerus, femur and iliac crest. Cell concentrations of 2.5 × 104 to 2.5 × 106 cells/mL were employed in various culture media supplemented with cytokines and growth/differentiation factors. A methylcellulose-based culture (MethoCult, Stem Cell Technologies) was selected for clonogenic progenitors of the erythroid and myeloid lineages. A collagen-based culture (Megacult, Stem Cell Technologies) was selected for the progenitors of the megakaryocytic lineage. Bone marrow differentials (total lymphocytes, erythroid and myeloid cells) were monitored by flow cytometry using anti-CD34, -CD71, -CD45, -CD41a, and CD61 antibodies. Bone marrow progenitor cells were characterized and counted. The proposed array of endpoints also facilitates the assessment of potential stimulatory or inhibitory effects on the bone marrow.
doi:10.1016/j.toxlet.2008.06.332
doi:10.1016/j.toxlet.2008.06.334
P07 Intra-articular dosing in the rabbit
P09 Trainable QSAR model of Ames genotoxicity
Dawn Haida
Remigijus Didziapetris, Kiril Lanevskij, Pranas Japertas ∗
Covance, Harrogate, United Kingdom
Pharma Algorithms, Inc., Vilnius, Lithuania
Intra-articular dosing is a skilled technique on which there appears to be limited literature. Historically, the main area for research using this dose route has been in the treatment of Rheumatoid arthritis. More recently, many of the prospective treatments for this fall into the general “biological” categories (typically proteins and anti-bodies). The modes of action of these tend to be very specific but they may be poorly absorbed and/or distributed, particularly following oral or intravenous administration and hence it may be necessary to administer
This study presents a predictive model of Ames genotoxicity built on a data set of more than 8000 compounds. A baseline fragmental model was developed using binomial PLS regression with multiple bootstrapping. This was followed by an automatic correction of predicted values based on similarity analysis of experimental data for similar compounds in the dataset. The final model produces highly accurate results (less than 5% of mispredictions in the validation set). This novel approach also allows assessing the quality of predictions by means of estimated Reliability Index values. A